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The ribonuclease enzyme system in rat brain Popow, William Philip 1975

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-THE  RIBONUCLEASE ENZYME SYSTEM IN RAT BRAIN  by  William Philip B.A.., U n i v e r s i t y  Popow  of B r i t i s h  C o l u m b i a 1970  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  t h e Department of Biochemistry  We  accept  required  THE  this  thesis  as c o n f o r m i n g t o t h e  standard  UNIVERSITY OF B R I T I S H August  1975  COLUMBIA  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  for  freely  of  the  requirements  B r i t i s h Columbia, I agree  available  for  t h a t p e r m i s s i o n for e x t e n s i v e copying o f  this  representatives. thesis for  It  of  financial  g a i n s h a l l not  / / o g ^ g ^ - ' ^ ^ y  The U n i v e r s i t y o f B r i t i s h Columbia 2 0 7 5 Wesbrook  Place  Vancouver, Canada V6T 1W5  Date  S  that  this  thesis or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  for  r e f e r e n c e and study.  s c h o l a r l y purposes may be granted by the Head of my Department  by h i s of  agree  fulfilment  the U n i v e r s i t y of  the L i b r a r y s h a l l make it I  in p a r t i a l  /S>-T  be allowed without my  ABSTRACT  A study was made of the enzyme system responsible f o r the catabolism of ribonucleic acid i n r a t brain.  Initial  work with whole brain homogenates and extracts revealed the presence of three ribonucleases (RNases) distinguishable by the pH at which they exhibit optimal a c t i v i t y .  The i d e n t i f i e d  RNases are referred to according to t h e i r pH optima as pH 6.7 RNase, pH 7»8 RNase and pH 9«5 RNase.  Evidence was also  obtained indicating the presence of a protein i n h i b i t o r of the pH 7*8 RNase.  The components of t h i s multi-enzyme-  i n h i b i t o r system were separated and p a r t i a l l y p u r i f i e d by ammonium sulphate f r a c t i o n a t i o n of whole brain extracts followed by DEAE-cellulose column chromatography of the ammonium sulphate p r e c i p i t a b l e f r a c t i o n s . The DEAE-cellulose eluate RNases were characterized with regard to the effect of various reagents upon t h e i r activity.  The i n t r a c e l l u l a r d i s t r i b u t i o n and developmental  p r o f i l e s of the three RNase a c t i v i t i e s and the pH 7«8 RNase i n h i b i t o r a c t i v i t y were also  determined.  ***-»»#«  - i i -  TABLE OF CONTENTS PAGE L i s t of Tables L i s t of Figures Acknowledgement  •• •  I. INTRODUCTION 1.0 General Frame of Reference  ...  1  1.1 Changes i n Brain RNA During Ontogenesis  . . •  5  1.11 Changes i n brain RNA content and composition during postnatal maturation  5  1.12 Changes i n brain RNA content and composition during aging . . . . . .  19  1.2 Turnover of Brain RNA  22  1.3 Changes i n Brain RNA Content and Composition Accompanying Sensory Stimulation, Physiological Challenges and Learning Experience  25  1.4 The Role of the RNase Enzyme System i n RNA Metabolism  33  1.5 Regulation of #Nase A c t i v i t y and RNA Degradation 1.6 I n t r a c e l l u l a r RNases of Non-Neural Mammalian Tissues  • • 36 . . . . . . . .  41  1.61 Acid RNase  41  1.62 pH 9.5 RNase  43  1.63 pH 7.8 RNase 1164 RNase Inhibitor 1.65 Ribosomal RNase 1.66 Nuclear RNases .  . 44 46 .48 49  - iii PAGE 1.7 Changes i n RNase A c t i v i t i e s under Various Physiological and Pathological Conditions •  51  1.8 The RNase Enzyme System of Brain  53  I I . MATERIALS AND METHODS 2.0 Materials  . . . . .  . . . . . . . .  2.01 Experimental animals 2.02 Chemicals  57 57 57  2.1 Methods  58  2.11 Preparation of tissue homogenates and extracts •  58  2.12 Fractionation of isotonic sucrose homogenates by d i f f e r e n t i a l centrifugation  59  2 . 1 3 P u r i f i c a t i o n procedure  .  60  2.131 Ammonium sulphate f r a c t i o n a t i o n . . . 6 0 2.132 DEAE cellulose, column chromatography of ammonium sulphate fractions 2.14 Enzyme assays  61 61  2.141 Determination of RNase a c t i v i t y . . . 61 2.142 Assay f o r deoxribonuclease a c t i v i t y • 6 3 2.143 Assay f o r phosphodiesterase activity • • 63 2.144 Assay f o r pH 7.8 RNase inhibitor activity . 64 I I I . EXPERIMENTAL RESULTS 3.0 Characteristics of the RNase A c t i v i t i e s of Adult Rat Whole Brain Homogenates and Extracts  66  3.01 E f f e c t s of pH and buffer system on the RNase a c t i v i t i e s assayed i n isotonic sucrose homogenates  66  - ivPAGE 3.02  S o l u b i l i z a t i o n of latent RNase a c t i v i t i e s by horaogenization i n Q,lfo T r i t o n X100  68  3 . 0 3 E f f e c t s of pH, buffer system, i o n i c strength, and NaCl on RNase a c t i v i t i e s assayed i n 0,1% T r i t o n X100 extracts . . . 3.04 Evidence i n d i c a t i n g the presence of a protein i n h i b i t o r of pH 7.8 RNase a c t i v i t y i n brain 3.041  71  ?$8  Time-dependent a c t i v a t i o n of pH 7.8 RNase a c t i v i t y i n stored enzyme preparations  78  3.042 I n h i b i t i o n of bovine pancreatic RNase A a c t i v i t y by brain extracts  • 78  A c t i v a t i o n by pCMB of RNase a c t i v i t y assayed at pH 7 * 8 . . . .  • 79  3.043  3.044 E f f e c t of EDTA on RNase a c t i v i t y assayed at pH 7 * 8 . . . . . . . . . . 3.045  Comparison of the e f f e c t s of pCMB on RNase a c t i v i t i e s i n l i v e r and brain •  3 . 0 5 Summary comment on the variables influencing the determination of RNase a c t i v i t i e s i n crude extracts  82  . . . . 86  3.1 Separation and P a r t i a l P u r i f i c a t i o n of the Components of the Multi-Enzyme-Inhibitor System of Adult Rat Whole Brain 6.1$ T r i t o n X100 Extracts 3.11 Ammoniu# sulphate f r a c t i o n a t i o n of G.1% T r i t o n X100 extracts of adult r a t whole brain 3 . 1 2 DEAE-cellulose column chromatography of the 25-858* and the 75-100$ saturated ammonium sulphate fractions . . . . . . . 3,2 Properties of the Three Separated RNase A c t i v i t i e s .  80  87  88  94 100  -  V -  PAGE 3.21 3.22  E f f e c t of pH E f f e c t of NaCl  3.23 3.24 3.25 3.26  E f f e c t of MgCl E f f e c t of EDTA E f f e c t of pCMB. E f f e c t of 3-mercaptoethanol and d i t h i o t h r e i t o l E f f e c t of detergents E f f e c t of storage  3.27 3.28  101 105 2  105 10? 109 109 110 112  3.3 DNase and Phosphodiesterase A c t i v i t i e s of DEAE-cellulose eluate enzyme fractions . . . . 113 3.4 I n t r a c e l l u l a r D i s t r i b u t i o n of RNase A c t i v i t i e s and RNase Inhibitor A c t i v i t y . . . . 116 3»5 Characterization of RNase A c t i v i t i e s i n Separated Subcellular Fractions  129  3,6 Developmental Changes i n RNase A c t i v i t i e s and i n RNase Inhibitor A c t i v i t y i n Rat Whole Brain  139  IV. DISCUSSION 4.0  Regulation of the In Vivo Function of RNases i n brain  148  4.1 Correlation of Developmental Changes i n the Content, Synthesis and Degradation of RNA i n brain • . . . . . . . .  • 158  4.2 Regional Differences i n the Metabolism of RNA and i n the Functional Roles of RNases i n brain , . . . « • . . .  • 163  4.3 Tissue-specific Differences i n RNA Turnover and RNase A c t i v i t i e s i n the Adult Animal  • 167  B„ BIBLIOGRAPHY  173  . .,  *«*«*«*  s  f l -  LIST OF TABLES TABLE  PAGE  I. E f f e c t of buffer concentration on RNase a c t i v i t y i n 0.1% T r i t o n X100 extracts . . . . . . . I I . E f f e c t of pCMB and EDTA on pH 7.8 RNase a c t i v i t y i n 0.1% T r i t o n X100 extracts I I I . pCMB reversal activity •  74 77  of EDTA-inhibited pH 7.8 RNase 81  IV. Enhancement of RNase i n h i b i t o r a c t i v i t y i n the presence of 1 mM EDTA V. Recovery of RNase a c t i v i t i e s i n ammonium sulphate precipitated fractions  82 . . . . 89  VI, E f f e c t of various reagents upon DEAEc e l l u l o s e eluate RNase A c t i v i t i e s . . . . . . . . . 106 VII. E f f e c t of various detergents upon DEAEc e l l u l o s e eluate RNase a c t i v i t i e s  • 111  VIII. DNase a c t i v i t y of DEAE-cellulose eluate enzyme fractions . . . . . . .  . 114  IX. Phosphodiesterase a c t i v i t y of DEAEcellulose eluate enzyme fractions . . . . . . . . .  115  X, I n t r a c e l l u l a r d i s t r i b u t i o n of pH 6.7 RNase a c t i v i t y • • • • •  . . . . . 118  XI. I n t r a c e l l u l a r d i s t r i b u t i o n of pH 9.5 RNase a c t i v i t y  . . . . . . . 120  XII. I n t r a c e l l u l a r d i s t r i b u t i o n of free pH 7,8 RNase a c t i v i t y . . . . . . . . 122 XIII. I n t r a c e l l u l a r d i s t r i b u t i o n of t o t a l pH 7.8 RNase a c t i v i t y  124  XIV. Calculated i n t r a c e l l u l a r d i s t r i b u t i o n of latent, inhibitor-bound pH 7.8 RNase  . 125  XV. I n t r a c e l l u l a r d i s t r i b u t i o n of free RNase inhibitor activity . •  126  *««##*«  - vii-  LIST GF FIGURES FIGURE  PAGE  1. pH p r o f i l e s of RNase a c t i v i t y i n isotonic sucrose homogenates of adult r a t whole brain . . . 67 2. pH p r o f i l e s of RNase a c t i v i t y i n 0.1% T r i t o n X100 extracts of adult r a t whole brain« e f f e c t of various buffers . . . . . . . . . .  72  3. E f f e c t of NaCl on the a c t i v i t y and pH optimum of acid RNase a c t i v i t y i n 0.1% T r i t o n X100 extracts of adult r a t whole brain  75  4. pH dependence of 0.1 mM pCMB-stimulated RNase a c t i v i t y i n 0.1$ T r i t o n X100 extracts of adult r a t whole brain  83  5 . pH-activity p r o f i l e s and e f f e c t of 0,1 mM pCMB on RNase a c t i v i t y i n 0.1% T r i t o n X100 homogenates of adult r a t l i v e r . . . . . . . . . .  83a  6a pH-activity p r o f i l e of 75-100$ saturated ammonium sulphate f r a c t i o n  . . . . 92  16b pH-activity p r o f i l e of 25-55% saturated ammonium sulphate f r a c t i o n  • 93  7. E l u t i o n p r o f i l e of 75-100% saturated ammonium sulphate f r a c t i o n chromatographed on DEAE-cellulose  95  8. E l u t i o n p r o f i l e of 25-55$ saturated ammonium Sulphate f r a c t i o n chromatographed on DEAE-cellulose  97  9a pH-activity p r o f i l e of DEAE-cellulose eluate acid RNase a c t i v i t y .  102  19b pH-activity p r o f i l e of DEAE-cellulose eluate pH 9 . 5 RNase a c t i v i t y  103  9c pH-activity p r o f i l e of DEAE-cellulose eluate pH 7.8 RNase a c t i v i t y  104  8  10. Reactivation of EDTA-inhibited RNase a c t i v i t y by Mg++  pH 9 . 5  108  11a pH-activity p r o f i l e of nuclear f r a c t i o n i n presence of 2.0 M urea or 0.2 mM pCMB . . . . . 132  - viii FIGURE  PAGE  l i b pH-activity p r o f i l e of nuclear f r a c t i o n i n presence of 1 mM EDTA  132  12a pH-activity p r o f i l e of crude mitochondrial f r a c t i o n i n the presence of G.2 mM pCMB . . . .  133  12b pH-activity p r o f i l e of crude mitochondrial f r a c t i o n i n the presence of 1 mM EDTA or 0.5 mM MgCl  133  13. pH-activity p r o f i l e of microsomal f r a c t i o n i n the presence of 2 . 0 M urea  135  14. pH-activity p r o f i l e of cytosol f r a c t i o n  138  2  . . . .  15. Developmental p r o f i l e of pH 6.7 RNase activity _•-  v  139a  16. Developmental p r o f i l e of pH 9*5 RNase a c t i v i t y •  139b  17. Developmental p r o f i l e of pH 7.8 RNase a c t i v i t y .  139c  18. Developmental p r o f i l e RNase i n h i b i t o r a c t i v i t y •  143 :  19. Total RNA-degradative capacity of whole brain as a function of developmental age . . . .  159  - ixACKNOWLEDGEMENTS  I would l i k e to express my immeasurable gratitude to Shan-Ching Sung f o r h i s l i m i t l e s s patience, t r u s t , understanding and encouragement without which t h i s work could not have been sustained or completed, I would l i k e to thank my laboratory colleagues Jen-Fu Chiu, Vijendra Singh, Raja Rosenbluth and Jim Nagy f o r t h e i r companionship,  thoughtfulness and helpfulness.  I also thank a l l the people of the Kinsmen Laboratories of Neurological Sciences and the Health Science Centre Hospital, with whom I have shared so much time and space, f e e l i n g and thought, f o r the p r i v i l e g e of knowing them and through knowing them expanding the l i m i t s of my understanding and appreciation of the inneruworkings of the human brain. F i n a l l y , I thank the many others who intersected my l i f e i n the course of t h i s project f o r the i n s p i r a t i o n they i n s t i l l e d i n me and f o r a l l they have taught me. I t i s only i n collaboration with a l l of the abovementioned people that the ongoing i n vivo processes of my own brain have been sustained and regulated throughout the course of t h i s work. ««#««««  I. INTRODUCTION  1.0 General Frame of Reference 1—7  I t i s generally regarded differences i n macromolecular  ' that t i s s u e - s p e c i f i c  composition are a consequence  of the d i f f e r e n t i a l expression of genome information common to a l l somatic c e l l s .  The problem of formulating neuro-  b i o l o g i c a l phenomena i n molecular terms i s hence e s s e n t i a l l y that of understanding the s p e c i f i c nature, expression and Regulation of the genetic c a p a b i l i t i e s of the nervous  system  and i t s constituent c e l l types. Severe methodological d i f f i c u l t i e s a r i s i n g from the organizational complexity and morphological heterogeneity of the mammalian central nervous system have hindered progress toward t h i s goal and, consequently, the molecular understanding of brain function has lagged behind that of other organs. Biochemical studies of the brain have followed four main avenues of approach.  F i r s t , there have been studies  concerned with elucidating those changes i n molecular comp o s i t i o n which produce or p a r a l l e l morphological changes as c e l l s d i f f e r e n t i a t e toward their mature s t r u c t u r a l and functional states.  Information as to the molecules and  molecular events occurring i n the course of development w i l l permit a more detailed understanding of how they p a r t i c i p a t e  - 2 -  to y i e l d the s p e c i f i c morphology, metabolism, and functional c a p a b i l i t i e s of the f u l l y d i f f e r e n t i a t e d c e l l types of the adult brain. Secondly, considerable e f f o r t has been directed toward achieving an understanding of the molecular composition and metabolism of normal adult brain and c o r r e l a t i n g brains p e c i f i c molecular constituents with the unique morphology and functional c h a r a c t e r i s t i c s of neuronal t i s s u e . Thirdly, attempts have also been made to detect a l t e r a t i o n s i n molecular composition and molecular function accompanying normal adult changes i n neuronal function. Morphological changes i n f u l l y - d i f f e r e n t i a t e d neurons have Q  been demonstrated  to occur i n response to changes i n sensory  input and behavior-modifying challenges from the external environment• An understanding of the molecular events correlated with or responsible f o r such morphological a l t e r a t i o n s w i l l help to elucidate the nature and l i m i t s of i n t e r a c t i o n between sensory experience and i n t r a c e l l u l a r  metabolism;  that i s , how perturbations of intraneuronal metabolism are effected, the capacity of the molecular mechanisms regulating metabolic adaptation and hence the l i m i t s of response a b i l i t y of metabolic processes within f u l l y - d i f f e r e n t i a t e d neuronal c e l l types.  I t i s hoped that such work w i l l eventually  - 3r e s u l t i n the i d e n t i f i c a t i o n of those b r a i n - s p e c i f i c molecules and molecular events which may  mediate the  and storage of sensory information or which may  processing underlie  cognitive and a f f e c t i v e processes and other psychobiological phenomena peculiar to the central nervous system. F i n a l l y , there have been studies of how a l t e r a t i o n s i n the genome can a f f e c t defined functions of the nervous system i n neurological and behavioral mutants; such studies can provide clues concerning  the contributions made by  s i n g l e , i d e n t i f i e d gene products to complex and  integrated  brain function and behavior. It has become clear from such studies that both the comparatively 1  gross molecular changes which occur throughout 3  development and the more subtle neurochemical correlates of experientally- and behaviorally-related neural a c t i v i t y t J  9-l6  i n the adult b r a i n  7  e n t a i l quantitative and q u a l i t a t i v e  changes i n the protein composition of c e l l s . These changes i n c e l l u l a r protein composition  may  occur within a p a r t i c u l a r c e l l , i n a population of c e l l s linked to one another s t r u c t u r a l l y and f u n c t i o n a l l y , and i n even more complex systems.  Proteins by t h e i r intervention  as enzymes and membrane constituents influence a l l other molecular constituents of the c e l l .  Hence, such changes 17 i n protein composition could be expected ' to correspond  - 4to the establishment of new  functional connections between  neurons, a l t e r a t i o n s i n the properties of c e r t a i n neurons, families of neurons, and i n t h e i r steady-state 1ft  synaptic function  mode of  .? 1  "  *  Because ribonucleic acid i s the primary gene product and any s p e c i f i c information manifested at t h i s l e v e l of genome expression i s subsequently transferred to proteins through the mechanism®©.? t r a n s l a t i o n (protein synthesis) y i e l d the t i s s u e - and c e l l type-specific metabolism,  to  mo»-  phology and functional c a p a b i l i t i e s of an organ, i t seems l i k e l y that a l t e r a t i o n s i n c e l l u l a r protein composition be secondary to changes i n de novo RNA  synthesis and  may  RNA  turnover. Changes i n c e l l u l a r protein patterns thus a r i s e as a consequence of modulations i n gene  expression--either  d i r e c t l y through (1) changes i n the rate of t r a n s c r i p t i o n of c o n s t i t u t i v e l y expressed genetic  information,  or through  (2) q u a l i t a t i v e s h i f t s i n the read-out of the r e v e r s i b l y expressible pool of genetic information  ( i . e . , through induc-  t i o n or repression)i or more i n d i r e c t l y by (3) post-transcript i o n a l mechanisms regulating gene expression (modulation of RNA  turnover, RNA  t r a n s l a t i o n and protein turnover).  It i s  t h i s t h i r d general category of regulation of c e l l u l a r protein  - 5 -  composition which i s the domain of concern s p e c i f i c a l l y dealt with i n the present t h e s i s . P r i o r to discussing the role of the enzyme system c h i e f l y responsible f o r the post-transcriptional metabolism of RNA  and the post-transcriptional control of c e l l u l a r  RNA  c o n t e n t , i t w i l l be useful to review the information a v a i l able to date on changes i n content, composition and turnover of RNA  i n the infant and adult central nervous  system.  1.1 Changes i n Brain RNA During Ontogenesis 1.11 Changes i n brain RNA content and composition during postnatal maturation In the r a t , t o t a l RNA  content of whole brain increases  up to the 15th to 18th day postnatally. • 3» ^ 24 2 5 22  the nuclear and transfer RNA  content  '  J  2  2  Subsequently,  of whole brain r e -  mains r e l a t i v e l y constant throughout adulthood whereas the 24 26 microsomal and ribosomal RNA  content declines.  '  There  also occurs a change i n the base composition--an increase i n adenine and a commensurate decrease i n guanine—of whole brain 26  microsomal RNA  as the brain matures.  In the cerebral cortex of the r a t , the increase i n a l l classes of RNA  ceases at 18 days and i s followed by a  40?S decrease i n polysomal RNA by the time the animals reach  - 6 -  adulthood (200-250 g. body weight)/**  This decline i n  polysomal RNA content per cerebrum i s accompanied by an increase i n the pyri^aidine and a decrease i n the purine content of t h i s RNA f r a c t i o n , whereas the base compositions of nuclear RNA of cerebral cortex of both young and adult animals remains the same. Such developmental changes i n the base composition of RNA isolated from the microsomal and polysomal fractions of brain are most l i k e l y accounted f o r i n terms of (1) s h i f t s i n the base sequence and base compositions of s p e c i f i c mRNA molecules associated with ribosomes at d i f f e r e n t stages of development, and/or (2) an age-dependent increase i n the mRNA/rRNA r a t i o i n these f r a c t i o n s . The a b i l i t y of RNA molecules homologous i n base sequence to compete i n hybridization to complementary base sequences of DNA has been u t i l i z e d to demonstrate alterations i n the kind of genome information transcribed at d i f f e r e n t •' 27 31 32 stages of ontogenesis. * -j:c@Eoasei;elblal< have studied the % J  a b i l i t y of t o t a l RNA isolated from d i f f e r e n t regions of the adult mouse and human brain to compete with t o t a l RNA from the same brain regions of f e t a l or infant mouse and human brain hybridized to unique sequence DNA.  They found that  i n both mouse and human brain there occurs with development  - 7 -  a net increase i n the variety of transcribed RNA.  Moreover,  the greater t r a n s c r i p t i o n a l d i v e r s i t y evident i n adult as compared to f e t a l brain i s attributable to d i v e r s i f i c a t i o n of RNA i n the cerebral cortex since the number of d i f f e r e n t kinds of RNA molecules present i n other regions of the adult brain was s i m i l a r to that of whole f e t a l brain.  Twenty per  cent of the maximal unique sequence genome information was expressed i n whole adult brain compared to 6$ f o r other organs i n the human, and 12$ as compared to 6% i n the mouse. 28  Similar competition  hybridization studies  have indicated  that i n mouse l i v e r the variety of mRNA molecules declines during development.  Bondy and R o b e r t s  33  synthesized demonstrated  that during maturation chromatin prepared from whole r a t brain as well as s p e c i f i c brain regions exhibits a decline i n i t s capacity to function as a template f o r E. c o l i RNA polymerasecatalyzed RNA synthesis i n v i t r o , i n d i c a t i n g that  during  development there occurs a progressive  template-limiting  decline i n the rate of t r a n s c r i p t i o n .  Chromatin from brain  was found to support the incorporation of more l a b e l into RNA than hepatic chromatin.  The decline i n template a c t i v i t y of  chromatin was correlated with a concurrent decline i n the non-histone protein content of chromatin.  Such a develop-  mental drop-out i n non-histone chromosomal proteins associated with DNA has also been observed by Kurtz and S i n e x ^ 3  - 8-  i n mouse brain and may be relevant to the explanation of the r e s u l t s of Grouse et a l .  3 2  which indicate that chromatin  from adult brain has more regions of i t s DNA derepressed and available f o r t r a n s c r i p t i o n .  Bondy and R o b e r t s  33  found that  whereas the t o t a l mRNA transcribed from whole brain chromatin was lower i n the adult than i n the newborn r a t s , the mRNA f r a c t i o n of the t o t a l RNA transcribed was proportionally higher i n the adult than i n the i n f a n t .  Thus, although the  t o t a l amount of mRNA-like RNA i n rat brain also decreases during development,  J  the decrease i n t o t a l rRNA i s s t i l l  greater and r e s u l t s i n a net increase i n the mRNA/rRNA r a t i o . Bondy and Roberts ** also found that nuclear IfM* from 3  both whole brain and l i v e r of adult rats hybridized to r a t t o t a l DNA to a greater extent (by about 20%) than cytoplasmic RNA.  Both nuclear and cytoplasmic RNA from brain hybridized  to a twofold greater extent than nuclear and cytoplasmic RNA from l i v e r .  The authors suggest: that i n the adult a larger  proportion of RNA synthesis i s directed towards mRNA product i o n i n brain as compared to l i v e r . unequivocally  Such an inferrence i s not  supported by the data since i n t h i s case the  hybridization of RNA transcribed from single copy genes i s not c l e a r l y distinguished from that of RNA which i s the product of r e p e t i t i v e genes.  Also, nuclei contain r a p i d l y  synthesized RNA molecules which are not transferred to the  - 9c y t o p l a s m ^ ' ^ and t h i s type of RNA presumably does not 3  function as mRNA. This conclusion i s , nevertheless, supported by a considerable body of other evidence. composition  Comparison of the base  of RNA isolated from various s u b c e l l u l a r f r a c t i o n s  of brain and l i v e r indicate that the adult brain contains more RNA which i s DNA-like i n i t s base composition  than l i v e r . ' ' 3  By t h i s c r i t e r i o n brain has a greater nuclear heterodisperse RNA and mRNA content than l i v e r . Zomzely et a l . * * have also demonstrated that i n the 3  adult a larger proportion of RNA synthesis i n vivo r e s u l t s i n mRNA i n brain as compared to other organs i n the r a t . Whereas i n brain the number of polysomes i s r e l a t i v e l y high at b i r t h and declines during development, ^ i n l i v e r they 3  are r e l a t i v e l y few i n number at b i r t h and increase with development.  Concurrent with the decrease i n polysomal RNA  and i n the t o t a l number of polysomes, as the brain matures there occurs a decrease i n the number of membrane-bound polyki 25 4l somes and a decrease i n the size and s t a b i l i t y of J  i s o l a t e d polysomes. I t remains uncertain to what extent the smaller polysome size class d i s t r i b u t i o n i n the adult may represent (1) a developmental s h i f t i n the size of mRNA molecules associated with r i b o s o m e s ^ 3  ,i+2  or (2) breakdown of polysomes during t h e i r  10 -  isolation"*  3  which may r e f l e c t age-related differences  between the RNase a c t i v i t y l e v e l s of the post-mitochond r i a l supernatant from which polysomes are prepared.  The  proportion of polysomes t o free ribosomes i n brain has also been shown to be affected by sensory stimulation and to be dependent on the functional state of the tissue  (see  section 1 . 3 ) . 24  i4  Adams  found that the rate of  C-orotic a c i d incor-  poration i n vivo into nuclear RNA i s the same f o r both the adult and 4-day old r a t cerebral cortex.  However, there i s  a l a g period of about 6 0 minutes i n the young animals which i s not present i n adults, during which l a b e l l i n g of the microsomal and ribosomal f r a c t i o n s of the cytoplasm proceeds only slowly.  *  J V  Sharma and Singh  observed a s i m i l a r l a g i n  the rate of transfer of l a b e l from the nucleus to RNA i n the cytoplasm i n newborn r a t whole brain s l i c e s incubated i n vitro.  Mandel*'-' i n studying developing chick brain observed 1  that newly synthesized RNA remains within the nucleus of chick embryo neuroblasts and spongioblasts u n t i l about 15 . days of embryonic l i f e .  The nature of the nucleocytoplasmic  b a r r i e r responsible f o r t h i s phenomenon i s unknown. has shown that the entry of polyribosomal  Murthy ^ 3  RNA into the cyto-  plasm follows the temporal sequence) mRNA, 18S RNA, 28S RNA.  - 11  I t i s not known to what extent the r a t e - l i m i t i n g step cont r i b u t i n g to t h i s difference i s due to the rate of RNA  conversion  to f u n c t i o n a l l y mature RNA,  precursor  the rate of  RNA  assembly into ribosomal subunits and informosomes or the rate of transport across the nuclear envelope* Comparison of i n vivo rates of RNA  synthesis i n the  brain or s p e c i f i c brain regions of animals of d i f f e r e n t ages i s complicated by the f a c t that i n t r a c r a n i a l routes of administering the l a b e l l e d precursor have been demonstrated to r e s u l t i n uneven d i s t r i b u t i o n of the l a b e l i n d i f f e r e n t brain regions.  Also, such studies have not controlled f o r  age-  dependent changes i n the rate of uptake of labial into the c e l l s nor f o r differences i n i n t r a c e l l u l a r precursor  pool  size. Guroff et a l . ^ reported that the i n v i t r o rate of 3  H-uridine  incorporation/mg DNA  was  3-fold greater i n  cerebral c o r t i c a l s l i c e s than i n cerebellar s l i c e s of the IB-day old r a t . of RNA  Between age 10 days and 4 months, the rate  synthesis/mg DNA  decreases 6-fold i n the cortex  10-fold i n the cerebellum, so that the rate of RNA sis/mg DNA  and  synthe-  i s about 6-fold higher i n the adult cortex as 46  compared to the adult cerebellum. that the rate of RNA  Guroff also reported  synthesis/mg DNA  was  2-fold higher  i n s l i c e s of l i v e r than i n s l i c e s of cerebral cortex of the adult r a t .  - 12 -  Sung**' found that the i n v i t r o rate of ^ C - u r i d i n e incorporation/mg  DNA i n t i s s u e s l i c e s incubated f o r one  hour was s l i g h t l y higher i n the cerebellum than i n the cortex of 2-day old r a t s .  By age 10 days, the rate of RNA  synthesis/mg DNA had decreased 2.2-fold i n the cerebellum, whereas the cortex exhibited a 1.3-fold increase r e l a t i v e to the rate of RNA synthesis at age 2 days. 48 32 Johnson studied the i n v i t r o incorporation of PJ  orthophosphate into RNA of mouse whol brain c e l l suspensions during a 2-hour incubation period and also found a rapid post-natal decline i n the rate of RNA synthesis up to age 7 to 9 days. Several workers have measured the rate of RNA synthesis i n i s o l a t e d n u c l e i incubated  i n v i t r o under conditions which  p r e f e r e n t i a l l y detect either RNA polymerase A- or RNA polymerase B- catalyzed RNA synthesis. Bbndy and Waelsch^'^  0  studied the incorporation ofi  ^C-UTP and "^C-ATP into RNA of l i v e r and brain c e l l n u c l e i incubated f o r 3 minutes i o n i c s t r e n g t h — c o n d i t i o n s determined to be optimal f o r RNA pftlyraerase B- catalyzed RNA synthesis.  The rate of RNA  synthesis/mg DNA under these assay conditions was consistently 2-fold higher for whole brain as compared to l i v e r of rats at a l l ages between 8 days and 4 months.  Moreover, the capacity  - 13 -  of n u c l e i f o r RNA polymerase B- catalyzed RNA  synthesis  increased almost proportionally i n both organs during t h i s time period r e s u l t i n g i n a net increase of 20% f o r brain and 30% for liver.  These workers also studied the regional d i s -  t r i b u t i o n of RNA polymerase B- catalyzed RNA synthesis i n the adult brains of the rabbit and s q u i r r e l monkey.  Nuclei  prepared from the cerebral cortex, caudate and hippocampus were found to exhibit 2-fold higher RNA synthesizing a c t i v i t y per mg DNA than those from the thalamus-hypothalamus,  corpus  callosum, pons-medulla and cerebellum. Gfiugfrida et a l . ^ studied the RNA synthesizing 1  a c t i v i t y of c e l l n u c l e i prepared from the cerebral cortex,, cerebellum and brainstem of 5 » 10, and 18-day o l d r a t s . U t i l i z i n g assay conditions which p r e f e r e n t i a l l y discriminate between the rates of RNA synthesis catalyzed by RNA polymerase A and RNA polymerase B they found that under both conditions, nuclei i s o l a t e d from a l l three brain regions of older rats exhibited s u b s t a n t i a l l y lower rates of %-UTP and H-GTP 3  incorporation than those derived from the 5-day old r a t s .  In  5-day old animals, nuclei from the cerebellum had a 1 . 4 - f o l d higher rate of RNA polymerase B- catalyzed RNA  synthesis  than n u c l e i from both the cerebral cortex and brainstem, and underwent a 3 . 3 - f o l d decrease to become 2-fold lower than the a c t i v i t y i n the cerebral cortex and brainstem by 18 days.  By  - 14 -  18 days the RNA  polymerase B activity/rag DNA  had  declined  i n the cerebral cortex and brainstem to 7 5 % and 86%  respec-  t i v e l y of t h e i r 5-day old l e v e l .  catalyzed  RNA  synthesis/mg DNA,  i o n i c strength, was  RNA  polymerase A-  assayed i n the presence of M g  ++  and  low  s l i g h t l y higher i n the cortex and brain-  stem than i n the cerebellum of 5-day old animals and underwent a 2 . 5 - f o l d decrease i n a l l three brain regions between 5 and 18 days. Thus, there i s a predominant amount of evidence i n d i cating that on a per unit DNA  and hence per nucleus basis,  there occurs a decline i n the rate of t o t a l c e l l u l a r synthesis as well as RNA  RNA  polymerase A- and B- catalyzed  RNA  synthesis during development i n the whole brain as well as i n s p e c i f i c brain regions.  On the basis of the consistent body  of evidence i t can be inferred that the demand f o r mRNA and rRNA, as indicated by the rate of RNA during c e l l division,  synthesis, i s highest  and c e l l d i f f e r e n t i a t i o n and,  subsequently,  declines to lower l e v e l s as the brain matures and attains i t s adult number of c e l l s by age 18 to 2 0 days. However, the apparently  anomalous r e s u l t s of Bondy and  Waelsh*^'-* are not to be disregarded. 0  These workers have  reported f i n d i n g an increase i n the rate of RNA catalyzed RNA  polymerase B-  synthesis during maturation i n a l l regions of  the rabbit brain, i n the whole brain of the rat and i n the l i v e r of both rabbit and r a t .  -  The  15 -  d i s c r e p a n c y between the r e s u l t s o f d i f f e r e n t  workers may be r e l a t e d t o d i f f e r e n c e s i n t h e assay t i o n s f o r RNA polymerase B a c t i v i t y .  condi-  Whereas Bondy and  WaelsJbh's data were obtained with an i n c u b a t i o n p e r i o d o f 3 minutes, G i u f f r i d a e t a l . ^ have found t h a t the Mn++/ 1  (NH_y)2 S O k - s t i m u l a t e d r e a c t i o n i s l i n e a r f o r K 5 minutes a f t e r an i n i t i a l l a g p e r i o d o f a t l e a s t 5 minutes.  This,  however, does n o t o f f e r an obvious e x p l a n a t i o n o f the apparent d i s c r e p a n c y ,  s i n c e Barondes-'-' i n a study o f Mn**  /(NHk) 2 S0k - s t i m u l a t e d RNA s y n t h e i s by "aggregate enzyme" d u r i n g a s i m i l a r l y s h o r t i n c u b a t i o n p e r i o d o f 5 minutes found a 2 - f o l d lower r a t e o f  C-CTP i n c o r p o r a t i o n i n t o RNA  (per u n i t DNA) i n the whore b r a i n of 9-month o l d as compared to  12-day o l d r a t s . • A l s o , i n 9-month o l d r a t s , t h i s enzyme  p r e p a r a t i o n from b r a i n was only 37$ as a c t i v e as t h a t from liver. The  e x p l a n a t i o n f o r why the r a t e o f RNA s y n t h e s i s has  been r e p o r t e d t o be lower i n b r a i n as compared t o l i v e r d u r i n g l o n g i n c u b a t i o n p e r i o d s i s not l i k e l y t o be due t o a more r a p i d d e g r a d a t i o n  o f newly s y n t h e s i z e d RNA-^  i n brain  as compared with l i v e r i n view o f the data r e p o r t e d by Dutton and Mahler.  J  These workers found t h a t i n £dult r a t s the  r a t e o f RNA polymerase A- c a t a l y z e d RNA s y n t h e s i s (Mg++ and low i o n i c s t r e n g t h ) o f n u c l e i prepared  from t h e c e r e b r a l  - 16 -  cortex was  only 2% of that of l i v e r n u c l e i .  that the incorporation of %-CTP 50% by preincubation  into RNA  They also found  was  enhanced 2 5 to  of brain nuclei at 3 7 ° (see s i m i l a r 46  observation by Guroff  f o r brain s l i c e s ) whereas preincuba-  t i o n of l i v e r n u c l e i f o r 3° minutes completely i n h i b i t e d t h e i r capacity to synthesize RNA.-^ that the RNA nuclear RNA  synthetic capacity was  These workers concluded more stable and  the  more protected from nucleases i n brain nuclei  than i n l i v e r n u c l e i of adult animals. Since r e s u l t s following b r i e f incubation periods probably more r e l i a b l e measures of the i n i t i a l rate of  are RNA  synthesis and minimally affected by the rate of degradation of the newly synthesized product, ' one would expect Bondy and Waelsch's data to be a more accurate measure of the i n vivo rate of RNA  synthesis than r e s u l t s obtained with l i n g e r  incubation periods where the rate of degradation of the newly .synthesized RNA  becomes an important  p a r t i c u l a r l y i f : t h e rate of nuclear RNA  consideration,  turnover varies with  age or shows tissue s p e c i f i c differences.  However, the  d i f f e r e n t i a l s e n s i t i v i t y of brain and l i v e r nuclei to preincubation might also explain the discrepancy i n r e s u l t s . I f the duration between preparation of n u c l e i and assay of nuclear RNA  synthesizing a c t i v i t y was  long i n Bondy's case,  then the l i v e r nuclei should be p r e f e r e n t i a l l y i n h i b i t e d r e s u l t i n g i n lower a c t i v i t i e s than brain n u c l e i ; but t h i s  - 17 -  b y Dutton and Mahler of the  presupposes that the observations  d i f f e r e n t i a l s t a b i l i t y of the polymerase A a c t i v i t y of brain and l i v e r n u c l e i i s also v a l i d f o r t h e i r polymerase B a c t i vity—and  t h i s would not account f o r an increase i n poly-  merase B a c t i v i t y i n brain during maturation.  In any case,  the resolution of t h i s apparent discrepancy must await more comprehensive data on the enzyme systems responsible f o r RNA  i  metabolism, p a r t i c u l a r l y the rate of degradation of nuclear RNA  i n brain as a function of  age.  A l l the aforementioned data consistently agrees that by age 18 days when net increase i n organ content of DNA 22 2?  c e l l u l a r content of RNA  24  has ceased"" •  c e l l s of the  cerebral cortex have at least a 2-fold higher rate of synthesis  and  RNA  (6-fold higher with tissue s l i c e s ) than c e l l s of  the cerebellum and most other regions of the brain (Bondy and CQ  Waelsch's data-' l e v e l s of RNA  suggest that caudate and hippocampus have  polymerase B- catalyzed RNA  synthesis  activity  comparable to that of cortex), and hence presumably a proport i o n a l l y higher rate of RNA  turnover.  The functional s i g n i -  ficance of t h i s regional difference i n RNA i n v i t r o i s not known.  metabolism measured  However, the data of G i u f f r i d a et al.-*  indicate that the difference i s predominantly due to a 2-fold higher rate of RNA  polymerase B- catalyzed RNA  the cerebral cortex since the l e v e l of RNA  synthesis i n  polymerase A-  - 18 -  catalyzed RNA synthesis i s nearly equal i n cerebral cortex, cerebellum and brainstem.  Since RNA polymerase B i s thought^'  to catalyze the synthesis of messenger-like RNA while RNA polymerase A synthesizes rRNA, t h i s suggests a higher rate of synthesis and u t i l i z a t i o n of mRNA i n the c e l l s of the adult cerebral cortex (and possibly caudate and hippocampus) i n comparison to other brain regions. I t i s not known to what extent the high RNA polymerase B a c t i v i t y of adult cerebral cortex i s due to the higher proportion of neuronal n u c l e i (25%) i n t h i s region of the brain or whther i t i s due to a high RNA synthesis a c t i v i t y s p e c i f i c to the neurons and/or non-neuronal c e l l types of the neocortex. G u i f f r i d a et a l . " *  1  have demonstrated that within a  given region of the brain d i f f e r e n t classes of n u c l e i are found having differences of at l e a s t 2-fold i n t h e i r RNA polymerase a c t i v i t i e s .  Neuronal n u c l e i were found to be more  active than oligodendroglial or a s t r o c y t i c nuclei.51»59.60 Yamagami et a l . - ^ have studied the composition of r a t brain nuclear RNA at various stages of development and found that as the brain matures the high molecular weight components decrease.  Their r e s u l t s indicate that during the  maturation of the brain, both mRNA and rRNA are decreased  - 19 -  s i g n i f i c a n t l y i n the nuclei a f t e r the t h i r t i e t h day and, subsequently, t h i s decrease continues u n t i l the nuclear RNA i s predominantly composed of small molecular weight RNA 74 molecules i n the adult brain.'  No evidence has yet  appeared i n d i c a t i n g to what extent these r e s u l t s represent a p r e f e r e n t i a l decrease i n the synthesis of high molecular weight RNA species  and/or to increased nuclear RNA degra-  d a t i o n ^ i n the adult. These developmental changes i n c e l l u l a r RNA correspond 4l 1  to a decline i n the rate of protein synthesis  and would be  expected to be accompanied by a q u a l i t a t i v e s h i f t i n the mRNA species as the genetic program synthesizing mRNA coding f o r proteins required for c e l l d i v i s i o n switches sequentially to the synthesis of mRNA species coding f o r the synthesis of proteins required f o r c e l l d i f f e r e n t i a t i o n , c e l l growth and finally,  maintenance functions of the mature c e l l .  Thus at  early stages of development brain RNA c h a r a c t e r i s t i c s are compatible with the rapid rate of protein synthesis observed. There i s a higher requirement f o r rRNA i n the rapidly dividing cells. 1.12  Changes i n brain RNA content and composition during aging  Atrophy and weight loss of the brain are the most common features of aging.  This i s accompanied by morphological  changes i n neuronal perikarya, neuropil, g l i a l elements and  -  20 -  blood vessels which are believed to be responsible f o r the 62  concurrent deterioration of function.  I t i s not known to  what extent the changes i n morphology of aging brain r e present g e n e t i c a l l y coded, time-dependent changes i n the c e l l or r e s u l t from exposure to harmful environment.  Such factors  as errors introduced into protein synthesis, chronic ischemia, slow virus i n f e c t i o n , n u t r i t i o n a l d e f i c i e n c i e s , i n t o x i c a t i o n s , and f a i l u r e of auto-oxidation are among those to be seriously considered.  L i t t l e i s known about the molecular  underlying "normal" c e l l senescence.  processes  I t i s hoped that i n -  formation as to the content, composition,  synthesis and turn-  over of RNA i n brain during aging w i l l y i e l d insight i n t o the molecular events responsible f o r or contributing to the complex processes of c e l l degeneration and c e l l death. According to Hollander and Barrows^ whole brain RNA/ 3  DNA r a t i o i n the C57 BL/6J mouse and Wistar rat does not 64 change during aging.  Chaconas and Finch  found a s l i g h t  decrease i n the RNA/DNA r a t i o of the striatum, but l i t t l e or no decline i n other regions of the C57 BL/6J mouse brain. Hyden^-* studying i n d i v i d u a l anterior horn s p i n a l neurons of humans found a progressive decline i n the t o t a l RNA content of these c e l l s a f t e r s i x t y years.  R i n g b o r g ^ has also  reported a similar decline i n the t o t a l RNA content of single pyramidal c e l l s of rat hippocampus.  In the l a t t e r , the amount  of RNA per c e l l increases from 24 pg i n newborn rats to 110 pg  - 21 f o r mature r a t s and, subsequently, drops t o 53 pg i n very old rats.  I n the very o l d r a t s , the G+C/A+U r a t i o o f the  RNA o f these c e l l s r i s e s from 1.66  i n mature r a t s t o  1.95.  Thus, i n d i v i d u a l neurons i n the CA^ l a y e r o f the hippocampus of 36-month o l d r a t s c o n t a i n about h a l f the RNA found i n these c e l l s i n 2-month o l d r a t s .  Wulff and F r e s h m a n ^  u s i n g a microspectrophotometric technique f o r q u a n t i t a t i n g the t o t a l RNA content o f s i n g l e c e l l s found a s l i g h t RNA l o s s i n s p i n a l motoneurons and c e r e b e l l a r P u r k i n j e c e l l s o f aged r a t s , but found no change i n the RNA content o f neurons of the s u p r a o p t i c nucleus o f the hypothalmus. • I t i s , perhaps,  "  s i g n i f i c a n t that increases i n c e l l u -  l a r RNA content have not been r e p o r t e d t o occur with a g i n g . The r e l a t i o n s h i p o f those decreases i n c e l l u l a r RNA which have been demonstrated  t o the concomitant  development o f  d e f i c i t s i n i n t e g r a t e d b r a i n f u n c t i o n and behavior remains to be e l u c i d a t e d . I t must be noted t h a t upon such b a s a l l i f e - c y c l e changes i n c e l l u l a r RNA content a r e superimposed  short-  l a s t i n g , r e v e r s i b l e f l u c t u a t i o n s i n RNA content t h a t r e s u l t from i n c r e a s e s or decreases i n f u n c t i o n a l demand.  -  1.2  Turnover of Brain  22  RNA  In adult r a t , the h a l f - l i f e of both the RNA proteins i n brain ribosoraes was In 35-day old r a t  P  and  found to be 12 to 18 days.^*^  0  brain rRNA has a h a l f - l i f e of 6 to 7  d a y s . • ^ Both the RNA  and protein components of ribosomes 72  within each age group turn over synchronously.'  This  age-  dependent decrease i n the turnover of ribosomes appears to be peculiar to the brain, since i n a comparative study of other rat tissues Menzies et a l . ' '  3  found no age-dependent  differences i n the turnover of rRNA i n l i v e r , spleen, or i n t e s t i n a l mucosa.  lung  The h a l f - i i l e of adult rat l i v e r rRNA  has been found to be about 5 to 7 d a y s . ^ • ^  These figures  indicate a s i g n i f i c a n t t i s s u e - s p e c i f i c difference i n the turnover rates of rRNA i n the l i v e r and brain of adult rats. In the adult animals brain rRNA turns over at about h a l f the rate of l i v e r rRNA.  Since rRNA constitutes nearly 90%  the t o t a l c e l l u l a r BNA^  of  i t s turnover rate would be expected  to have a decisive bearing on such processes as chromatolysis following axonal s e c t i o n . ^  A half-ikife of at least 6 days  i s compatible with estimates of h i s t o l o g i c a l methods of the rate of chromatolysis.''''  However, the disappearance of  N i s s l staining a f t e r severe anoxia and possibly also a f t e r intense chronic stimulation and s t r e s s , * * " ^ i s too rapid 1 0  to be accounted f o r by f a i l u r e of RNA  1 0  synthesis i n conjunction  -  23 -  w i t h a normal r a t e o f rRNA t u r n o v e r .  Rapid disappearance o f  N i s s l substance may hence i n v o l v e a c t i v a t i o n o f r i b o n u c l e a s e s present i n the c y t o s o l o r a s s o c i a t e d with  ribosomes.  No a g e - a s s o c i a t e d d i f f e r e n c e has been observed i n the t u r n o v e r o f tRNA.  T h i s c l a s s o f RNA e x h i b i t s a double expo-  n e n t i a l t u r n o v e r p a t t e r n ; one component has a h a l f - l i f e o f 5 t o 8 days, s i m i l a r t o t h a t o f l i v e r tRNA ^' ^ and the 7  second component 1 3 t o 1 6 d a y s .  7 0 , 7 2  * ® 7  7  I t i s not known t o  what extent these r e s u l t s r e f l e c t d i f f e r e n t i a l t u r n o v e r o f d i f f e r e n t components o f tRNA p o p u l a t i o n w i t h i n a s s i n g l e  cell  type o r t o d i f f e r e n t i a l turnover o f the t o t a l tRNA p o p u l a t i o n w i t h i n d i f f e r e n t c e l l types such as neurons  and g l i a .  M i t o c h o n d r i a l RNA o f r a t b r a i n has been shown t o have a h a l f - l i f e o f 1 1 . 6 and 1 0 days i n young and a d u l t  animals  respectively. Data on the t u r n o v e r r a t e s o f mRNA i n r a t b r a i n c a t e s the presence o f a t l e a s t two p o p u l a t i o n s o f mRNA.  indiA  more l a b i l e f r a c t i o n r e p r e s e n t i n g most o f the mRNA t u r n s over w i t h a h a l f - l i f e o f l e s s than  k  hours,79-83  a n (  _ another  s m a l l e r p o p u l a t i o n o f mRNA molecules t u r n s over with a mean  Qh. h a l f - l i f e of 1 0 t o 12 hours. rat cerebral cortex  J  Reports t h a t polysomes from  and whole b r a i n  d u r i n g maturation presumably  decrease i n s t a b i l i t y  due t o an enhanced  susceptibility  t o d e g r a d a t i o n o f the mRNA i n the polysomes would suggest a h i g h e r r a t e o f mRNA t u r n o v e r i n the a d u l t ; however, d i r e c t  - 24 -  data on developmental  changes i n the turnover rates of  mRNA i s lacking. There i s l i t t l e available data on RNA n u c l e i of brain c e l l s .  turnover i n the  Bondy^® studied the turnover of i n  vivo l a b e l l e d nuclear RNA  i n whole brain of adult rats and  found that a l l of the acid-insoluble radioactive l a b e l l o s t from the nucleus i n the f i r s t 18 h a f t e r i n t r a c r a n i a l i n j e c t i o n of  C-cytidine could be accounted f o r by the increased  l a b e l l i n g of cytoplasmic RNA.  That i s , the decrease i n  s p e c i f i c r a d i o a c t i v i t y of newly synthesized nuclear RNA due to i t s rapid conversion to cytoplasmic RNA out of the nucleus.  was  and transport  Within the nucleus of adult brain c e l l s  there would thus appear to be l i t t l e degradation of RNA  to  acid-soluble products of the type reported f o r HeLa c e l l nuclei by H a r r i s . - ' >  00  Nuclear RNA  exhibited a heterogeneous  breakdown pattern with h a l f - l i v e s of from 26 h to 11.5 The s p e c i f i c r a d i o a c t i v i t y of nuclear RNA that of the cytoplasmic RNA  days.  did not f a l l below  f r a c t i o n s i n the manner which one  would expect i f a simple precursor-product r e l a t i o n existed between nuclear and cytoplasmic RNA. large amount of the RNA  This suggests that a  synthesized i n the nucleus i s not  quantitatively transferred to the cytoplasm but remains i n the nucleus.  The extent to which nucleotides released from  heterogeneous nuclear RNA  (HnRNA) during  ~ degradation are  - 25 capable &$ competing with the free nucleotide pool and thus of being r e - u t i l i z e d i n RNA  synthesis i s not known.  has reported that the turnover rate of nuclear RNA rapid i n the adult than i n the infant brain.  Watts^  1  i s more  The combined  evidence suggests that i n the adult, newly synthesized  RNA  i s processed and degraded i n the nucleus as well as transported to the cytoplasm at an enhanced r a t e . I t should be emphasized that the available informat i o n on RNA  turnover i n brain has been derived from a complex  mixture of c e l l - t y p e s which may RNA  d i f f e r considerably i n t h e i r  content and m e t a b o l i s m . 5 9 . 6 0 , 8 6 - 8 9  that RNA  Watson^  0 n a s  reported  turns over more r a p i d l y i n neurons than i n g l i a and  neuronal RNA  turnover i s markedly subject to v a r i a t i o n de-  pending on the functional a c t i v i t y of neurons, the physiol o g i c a l condition of the organism and i t s environmental status.  Information as to the turnover rates of d i f f e r e n t  classes of RNA  (and of p a r t i c u l a r molecular species within  p a r t i c u l a r class) within a single cell^type under various physiological conditions and states of neural a c t i v i t y i s not yet a v a i l a b l e . 1.3 Changes i n Brain RNA Content and Composition Accompanying Sensory Stimulation, P h y s i o l o g i c a l Challenges and Learning Experience Consistent with the brain's s p e c i a l i z a t i o n f o r det e c t i n g and responding  to changes i n i t s ambient stimulus 91-97  6*5  f i e l d there have been numerous reports * J  7J  7 1  of  - 26 -  perturbations i n the steady state content and composition of RNA of adult brain.  Such changes have been demonstrated to  occur as a consequence of ,or concomitant with ,changes i n the functional state of the whole brain, s p e c i f i c brain regions, subregions,  or the a c t i v i t y of s p e c i f i c neurons.  When adult r a t s were kept f i r s t i n the dark f o r 3 days and then exposed to the l i g h t and sounds of a laboratory for  15 minutes, the polysome to monosome r a t i o increased by  8 3 % i n the cerebral cortex but was not affected i n the liver.  Evidence has also been reported i n d i c a t i n g that the  t o t a l RNA of neurons i n the auditory cerebral cortex i s markedly elevated within 1 hour of a 60-minute exposure of rats to moderately intense white noise.  The RNA content of  these neurons returns to control baseline l e v e l s within 24  00  hours.  7  On the other hand, convulsions produced by methionine sulphoximine or electroshock caused a disaggregation of polysomes.  100  ' ^ 10  A more detailed s t u d y  1 0 2  of the time-  course of electroshock showed a decrease i n number of polyribosomes during the f i r s t 15 minutes, and an increase over the following 15 to 30 minutes.  By 60 minutes a f t e r e l e c t r o -  shack no s i g n i f i c a n t difference from control was o b s e r v e d . Other w o r k e r s  10i+  " ^ have reported a rapid turnover 10  of ribonucleoprotein (Nissl substance) and a decrease i n t o t a l c e l l u l a r RNA content under conditions of chronic  103  - 27 -  stimulation and s t r e s s .  On the basis of such observations.  Hyden has proposed that moderate neuronal e x c i t a t i o n produces increases i n RNA as an adaptation to increased funct i o n a l demand on the neurons whereas intense chronic stimul a t i o n produces a decrease i n neuronal RNA content through (1) slowing down RNA synthesis, possibly by d i v e r s t i o n of ATP to other metabolic needs of the c e l l , and/or (2) increasing the rate of RNA degradation. Mild sensory stimulation of the kind which increases neuronal RNA content appears to r e s u l t from a non-specific increase of a l l classes of c e l l u l a r RNA.^  2  That i s , the  increased RNA content i s not due to a p r e f e r e n t i a l or disproportional increase i n the quantity of a p a r t i c u l a r class of RNA molecules,  Hyden  110  has performed base compositions!  analyses of the increased neuronal RNA content e l i c i t e d i n response to increased physiological stimulation of neuronal a c t i v i t y and found that the new RNA has the same base-ratio c h a r a c t e r i s t i c s as the bulk of RNA present i n control c e l l s . Whether the RNA which remains a f t e r intense chronic stimulat i o n exhibits a base composition d i f f e r e n t from that of r e s t i n g c e l l s has not been determined.  Shashoua has reported  that KCl-induced convulsions and generally s t r e s s f u l physiol o g i c a l conditions resulted i n no detectable base composition changes i n the RNA of whole g o l d f i s h b r a i n . H o w e v e r , the occurrence of l o c a l changes i n s p e c i f i c brain regions would not be detected i n such an analysis of whole brain RNA.  -  28  -  Although changes i n RNA content serve as a r e l i a b l e indicator of a l t e r a t i o n s i n the metabolic and functional state of neurons, a deeper understanding of the significance of these changes w i l l require d i r e c t electrophysiological monitoring of the b i o e l e c t r i c a c t i v i t y of neurons as well as a more c a r e f u l examination of the s p e c i f i c contribution of such parameters as stimulus frequency, i n t e n s i t y and duration to the net effects on c e l l u l a r RNA content, composition and metabolism* Since m-, t - , and r-RNA subserve protein synthesis i t has been anticipated that under those conditions i n which neuronal RNA content (or rate of synthesis) i s increased, there might be a comparable, even greater concomitant on protein content and synthesis. data has not accumulated  Sufficient  effect  comparative  to t e s t t h i s hypothesis. '  J  Numerous reports have appeared demonstrating increases 114-117  i n the t o t a l content 1 J 4 118—124 composition  »-"--»-« -»•<-  f  0  as well as changes i n the base  f neuronal RNA during the a c q u i s i t i o n  of a new behavior pattern. ~ * ~ 1 2  1 3 1  Trained mice, compared to  quiet and yoked mice, showed greater incorporation of radioactive uridine into b r a i n ' ' * 1 1  1 3 2  nuclear and ribosomal RNA,  greater incorporation of uridine into polysomes i s o l a t e d from b r a i n , ribosomes.  132  1 1 3  and a higher r a t i o of brain polysomes to free These results have been confirmed by autoradio-  graphic studies with mice ** and rats given s i m i l a r 13  - 29 135,136  training.  These changes were not observed i n l i v e r  and kidney.117,132-133. Reports into RNA  117,125  of increased uridine incorporation  during t r a i n i n g experience cannot always be re-  l i a b l y interpreted as i n d i c a t i n g an actual increase i n the rate of RNA  synthesis since increased l a b e l l i n g could also  r e s u l t from a reduction i n the size of the endogenous unl a b e l l e d (most l i k e l y intranuclear) ribonucleotide pool.  1 3 0  * '' 1 3  precursor  Changes i n ribonucleotide precursor pool sizes  have, i n f a c t , been observed under such c o n d i t i o n s . ^ creased incorporation of l a b e l into RNA i f a decreased rate of RNA unchanged rate of RNA  In-  might also be observed  degradation were combined with an  synthesis.  However, r e s u l t s obtained  following b r i e f pulse times are measures of the i n i t i a l rate of synthesis and are not s i g n i f i c a n t l y influenced by the rate of RNA  degradation.^  neuronal RNA The detected  Measurements of the turnover rate of  under these conditions have not been reported.  increases i n neuronal RNA  i n learning experiments may  content which have been be an unspecific sign of  increased a c t i v i t y , not related to a s p e c i f i c learning or information  consolidation process.  In view of the large i n -  fluence of the organism's behavioral h i s t o r y upon these 112 measures,  139-l40 '-^  controls animals who  i t would seem to be necessary to use  as  have been previously adapted to some of  the simple stimulus components of the t r a i n i n g s i t u a t i o n i n  -  30  -  order to i d e n t i f y changes i n molecular the occurrence  species s p e c i f i c  of i n s t r u m e n t a l l e a r n i n g d u r i n g  to  training  experience. I t has been r e p o r t e d  1 1  ^*  1 1  ®"  1 2 1  t h a t i n c o n t r a s t to  c o n d i t i o n s of i n c r e a s e d p h y s i o l o g i c a l sensory a c t i v i t y i n non-learning  s i t u a t i o n s , the RNA  and/or motor content i n -  c r e a s e s which accompany l e a r n i n g e x h i b i t b a s e - r a t i o changes i n the d i r e c t i o n o f the base composition  characteristics  mRNA ( i . e . , h i g h e r adenine and u r a c i l c o n t e n t ) . i n d i c a t e t h a t i n d i v i d u a l neurons are capable between sensory  of  T h i s would  of d i s c r i m i n a t i n g  i n f o r m a t i o n p r o c e s s i n g c o n t i n g e n t upon the  a c q u i s i t i o n of a new  behavior  e n t a i l the a c q u i s i t i o n o f new  and a c t i v a t i o n which does not information.  Such  specificity  of neurochemical response i n d i c a t e s t h a t the r e g u l a t i o n of neuronal sensory  RNA  metabolism and p o s s i b l y gene e x p r e s s i o n  by  i n f o r m a t i o n i s mediated v i a the r e c e p t i v e p o l e of the  neuron a t which the e l e c t r i c a l l y - c o d e d i n f o r m a t i o n i s t r a n s duced i n t o molecular  messengers.  d i r e c t l y dependent upon or coupled output  RNA  metabolism i s not  to the b i o e l e c t r i c response  or e l e c t r o p h y s i o l o g i c a l a c t i v i t y of  neurons, 3®» 3»15^ 1  l k  but would seem r a t h e r t o be r e g u l a t e d by neurohormones p r e sumably a c t i n g upon r e c e p t o r s of the p o s t - s y n a p t i c membrane. The nature sensory  of the i n t r a c e l l u l a r molecular  i n f o r m a t i o n i s transduced  and  messages i n t o which  the i n t r a c e l l u l a r  sequence of events by which the modulation of RNA  causal  metabolism  - 31 -  i s e f f e c t e d remains t o be e l u c i d a t e d . p o s s i b l e t o i n f e r from the experimental t h a t new molecular  Also>, i t i s not y e t evidence a v a i l a b l e  s p e c i e s o f RNA are s y n t h e s i z e d  through  the r e l e a s e o f genome i n f o r m a t i o n i n order t o account f o r the observed base-composition changes i n neuronal RNA. Although hormonal i n d u c t i o n o f genome i n f o r m a t i o n has been w e l l documented i n other b i o l o g i c a l systems, a l t e r n a t i v e explanations composition  o f the r e p o r t e d a l t e r a t i o n s i n RNA basei n neurons have n o t been adequately  tested.  Such changes might be due t o ( l ) a change i n the q u a n t i t y of one o r more s p e c i e s o f mRNA o r a d i f f e r e n t i a l q u a n t i t a t i v e s h i f t i n the r a t e o f s y n t h e s i s o r degradation  o f two c l a s s e s  o f RNA r e s u l t i n g , f o r example, i n a n e t i n c r e a s e i n mRNA/rRNA (2) enhanced a c t i v i t y o f t e r m i n a l a d d i t i o n enzymes; f o r  ratio,  example, o f the k i n d c a t a l y z i n g the p o s t - t r a n s c r i p t i o n a l adenyl a t i o n o f the 3 ' - t e r m i n a l  end o f mRNA, o r (3)  intercellular  t r a n s f e r o f s p e c i f i c RNA molecules between g l i a and 91 110 neurons.  '  ii'  D i r e c t evidence o f the occurrence  t a t i v e l y new molecular  s p e c i e s o f RNA o r p r o t e i n d u r i n g  l e a r n i n g (which might be obtained tion ^ ' ^ 1  1  1  2  of quali-  by competitive h y b r i d i z a -  o r e l e c t r o p h o r e t i c p r o f i l e s t u d i e s ) i s not y e t  a v a i l a b l e t o supplement the base-composition data and f a c i l i t a t e the i n t e r p r e t a t i o n o f t h e i r s i g n i f i c a n c e t o t h e molecular  mechanisms u n d e r l y i n g l e a r n i n g and memory  formation.  -  32  -  The questions of whether the changes i n neuronal  RNA  base-composition which are correlated with the a c q u i s i t i o n and consolidation of sensory information represent the synthesis of q u a l i t a t i v e l y new species of mRNA and whether 12  eventually new species of proteins  114  •  144  *  also are formed  during the establishment of a new behavioral response have not been d e f i n i t i v e l y answered. However, s u f f i c i e n t data have, nevertheless, accumulated to implicate at least an i n d i r e c t involvement of RNA i n the molecular processes which underlie changes i n neuronal function brought about by changes i n sensory stimulation and learning experience.  The determination of how transient per-  turbations of the steady-state neuronal l e v e l s of RNA  are  effected w i l l require a more detailed understanding of the enzyme systems responsible f o r the synthesis and degradation of RNA  and the mechanisms by which they are regulated, since i t i s  through these mechanisms that changes i n neuronal RNA  are most  l i k e l y coupled to neuronal membrane receptors. The p a r t i c u l a r concern of the present study i s the RNA degradation system of the c e l l , i t s p a r t i c i p a t i o n i n c e l l u l a r RNA metabolism and i t s contribution to the changes i n c e l l u l a r RNA which have been observed to occur i n the course of brain development, aging and during a l t e r a t i o n s i n neuronal function i n the adult brain.  - 33 1.4 The Role of the RNase Enzyme System i n RNA Metabolism I t i s probable that the determination  of the l i f e t i m e  of various RNA molecules by RNases operates as a key factor i n the control of c e l l u l a r biosyntheses.  Considerable  e f f o r t has, therefore, been invested i n studies of the functional aspects of the class of enzymes which have i n common the a b i l i t y to s p l i t phosphodiester bonds i n RNA.  The  p a r t i c i p a t i o n of enzymes cleaving the diester linkages between ribonucleotides has been implicated i n diverse aspects of c e l i u l a r metabolism.  B i o l o g i c a l functions f o r which some  evidence i s available include ( l ) the maturational  processing  of precursor t r a n s c r i p t i o n products, and (2) the degradation of endogenous ribosomal,  messenger, transfer and nuclear RNA.  Other possible functions f o r which experimental evidence i s more tenuous include (3) defense against foreign RNA , and (4) provision of ribonucleotides f o r r e u t i l i z a t i o n by the RNA  synthesizing apparatus. There i s s u f f i c i e n t evidence now to conclude that i n  both procaryotic and eucaryotic c e l l s mRNA ^" ^ 1  i-RNA  151  "  153,1  3 5 t l 5 6 ind t R N A  1 5 7  '  1 5 8  1  0,  ^  are a l l transcribed as  precursor RNA molecules which are subsequently trimmed of t h e i r extra nucleotide sequences by maturation endoribonucleases to form f u n c t i o n a l l y mature RNA molecules. The processing of the large nuclear pool of r a p i d l y turning over  - 34 -  HnRNA appears l i k e l y t o i n v o l v e ( l ) s i t e - s p e c i f i c e n d o r i bonucleases which produce  s e l e c t i v e c l e a v a g e ( s ) o f the  primary t r a n s c r i p t i o n p r o d u c t s , and (2) RNases of more g e n e r a l s p e c i f i c i t y which degrade the n o n - f u n c t i o n a l cleavage products t o m o n o r i b o n u c l e o t i d e s .  Considerable  r e s e a r c h i s c u r r e n t l y being d i r e c t e d toward  elucidating  t h i s c r i t i c a l r o l e o f RNases i n n u c l e a r RNA metabolism and i n the p r o v i s i o n o f f u n c t i o n a l l y mature RNA molecules t o the p r o t e i n - s y n t & e s i z i n g apparatus o f the cytoplasm. The RNase enzyme system a l s o r e g u l a t e s t h e f u n c t i o n a l l e v e l s o f both n u c l e a r and c y t o p l a s m i c RNA content by i t s p a r t i c i p a t i o n i n RNA d e g r a d a t i o n .  The d e t e r m i n a t i o n o f the  l i f e t i m e o f v a r i o u s RNA molecules by RNases i s o f obvious importance  i n r e g u l a t i n g the a v a i l a b i l i t y and l i m i t i n g the  extent o f use o f f u n c t i o n a l l y mature RNA molecules i n protein synthesis. A t h i r d p o s s i b l e f u n c t i o n o f RNases suggested by H e r r i o t - ^ i s the d e g r a d a t i o n o f i n f e c t i o u s v i r a l RNA. 1  That a b a r r i e r e x i s t s t o i n f e c t i o n o f p l a n t and animal by naked v i r a l RNA i s w e l l e s t a b l i s h e d ,  1 6 0  '  1 6 1  cells  and i n t r a -  c e l l u l a r RNase a c t i v i t y i s a l i k e l y candidate f o r t h i s function.  1 6 2  '  1 6 3  ISwfvirJ  iK flantl  4EI Svlrlii  content o f  RNase i n l e a v e s i s not c o r r e l a t e d with s u s c e p t i b i l i t y t o i n f e c t i o n by i n t a c t v i r u s .  164  D i r e c t evidence l i n k i n g RNases  with protection against i n f e c t i o n by RNA  v i r i o n s i n plant,  animal or b a c t e r i a l c e l l s has not yet been reported. F i n a l l y , a possible function of RNA  depolymerizing  enzymes which has received l i t t l e consideration i s the prov i s i o n of ribonucleotide precursors to the RNA synthesizing apparatus of the nucleus.  Some question e x i s t s ^ as to 1 6  the s u f f i c i e n c y of nuclear de novo nucleotide synthesis and nucleotide i n f l u x from the c^jfoplasm as mechanisms f o r the replenishment of ribonucleoside triphosphates within nuclei during RNA  synthesis. I t has been a l t e r n a t i v e l y hypo-  thesized ^' 1 6  1 6 6  that r a t e - l i m i t i n g control of RNA synthesis  may be achieved through the regulation of the free ribonucleotide pool size by the rate at which ribonucleoside-5 phosphates or ribonucleoside-5'-diphosphates are liberated from nuclear polyribonucleotide storage molecules by the action of exoribonucleases within the nucleus.  Such a  mechanism would explain (1) how the processes of RNA thesis and RNA  syn-  degradation are t i g h t l y coupled or co-  ordinated to achieve steady-state turnover rates, (2) RNA  how  synthesis can proceed at rapid rates without rapid  depletion of the small pools of free ribonucleotides i n the n u c l e u s , ^ and (3) a possible function of the r a p i d l y 16  turning over h o m o p o l y r i b o n u c l e o t i d e s  167  "  170  and other  nuclear RNA which does not reach the cytoplasm and whose  - 36 function i n the nucleus i s to date  undetermined.  171  " ^ 17  Ribonucleoside-5'-phosphate-forming ribonuclease a c t i v i t i e s capable of f u l f i l l i n g such a putative function have been i d e n t i f i e d i n guinea pig l i v e r , liver  1 7 8  1 7 6  *  1 7 7  rat  and E h r l i c h ascite carcinoma ^ c e l l n u c l e i . 17  The ribonuclease enzyme system thus modulates the l e v e l s of functional RNA  molecules i n the c e l l by p a r t i c i -  pating i n the biosynthesis and degradation of f u n c t i o n a l l y mature RNA.  Much remains to be learned as to the s p e c i f i c  contribution of t h i s group of enzymes and i t s i n d i v i d u a l components to the s p e c i f i c changes i n c e l l u l a r metabolism accompanying each stage of the c e l l cycle ( c e l l d i v i s i o n , c e l l d i f f e r e n t i a t i o n , c e l l growth, c e l l maintenance and c e l l degeneration).  1,5 Regulation of RNase A c t i v i t y and RNA  Degradation  It must be postulated out of l o g i c a l necessity that a l l tissues and c e l l s possess an enzyme system f o r degrading RNA molecules.  I f t h i s enzymatic apparatus were allowed to  function uncontrolled i t would completely hydrolyze the c e l l u l a r RNA.  Since t h i s does not occur, i t follows that  Regulatory mechanisms must exist which control the functional l e v e l s of RNase a c t i v i t y and prevent the indiscriminate breakdown of RNA  within the c e l l .  -  37  -  The molecular mechanisms by which i n t r a c e l l u l a r RNase a c t i v i t y and RNA degradation are regulated are l i t t l e understood.  The question as to which or whether a p a r t i c u l a r  type of RNase i n a c e l l s p e c i a l i z e s i n attacking only one class of RNA has not been d e f i n i t i v e l y answered.  While a l l  classes of RNA are l i k e l y to be competent substrates f o r any one RNase i n variously manipulated i n v i t r o conditions, the i n t r a c e l l u l a r s i t u a t i o n of the RNA and of the enzyme may impose considerable r e s t r i c t i o n i n vivo.  Thus, apart from  the degree of s p e c i f i c i t y inherent i n the enzyme-substrate i n t e r a c t i o n which may e n t a i l recognition of primary, secondary and t e r t i a r y s t r u c t u r a l features of the substrate, additional constraints on substrate-enzyme i n t e r a t i o n may depend on the functional l e v e l of various RNases as well as t h e i r a c c e s s i b i l i t y to substrate.  Several means by which  such regulation or r e s t r i c t i o n of enzyme-substrate i n t e r action may be achieved i n vivo include» ( l ) s p a t i a l segregat i o n of substrate and enzyme by d i f f e r e n t i a l i n t r a c e l l u l a r compartmentalizatiom  ( 2 ) complexing of RNA with protein to  form ribonucleoprotein structures r e s i s t a n t to the action of RNaSes? ( 3 ) complexing of enzymes with i n h i b i t o r  molecules;  ( 4 ) a l l o s t e r i c modulation of the substrate-binding or catal y t i c ^ o f R ^ K s e s by small e f f e c t o r molecules, V  and ( 5 ) a l t e r a -  tions i n the rate of de novo synthesis or turnover of RNases.  - 38 -  S p a t i a l s e g r e g a t i o n o f the enzyme i s c l e a r l y i n v o l v e d i n the p r e v e n t i o n o f i n d i s c r i m i n a t e d e g r a d a t i o n  o f RNA by i fin  the l a r g e amounts o f RNase present i n lysosomes.  Lyso-  somal RNase i s u n l i k e l y t o be i n v o l v e d i n the turnover o f a s i n g l e s p e c i f i c c l a s s o f RNA.  The a c t i v i t y o f lysosomal  RNase depends on the f u s i o n o f v a c u o l i z e d RNA p a r t i c l e s  with  the lysosomes o r the r l e a s e o f lysosomal RNases i n t o t h e c y t o s o l f o l l o w i n g a change i n lysosomal membrane permeability.  Such lysosomal membrane changes have been r e p o r t e d T  to  fin i fti  occur under c o n d i t i o n s o f s t r e s s  •  and are f o l l o w e d  by a c o n s i d e r a b l e breakdown o f messenger and r&bosomal RNA. Upon b e i n g s y n t h e s i z e d most RNA becomes a s s o c i a t e d with s p e c i f i c p r o t e i n s . RNA  The p r o t e i n - a s s o c i a t e d s t a t e o f the  p r o v i d e s an obvious means o f r e g u l a t i n g i t s a c c e s s i -  b i l i t y and s u s c e p t i b i l i t y t o degradative  enzymes.  That RNA-  a s s o c i a t e d p r o t e i n s r e s t r i c t the a c t i v i t y o f RNases i n v i v o i s i n d i c a t e d by the f a c t t h a t i n i n t a c t polysomes, rRNA i s u n a f f e c t e d by l e v e l s o f RNase which r e a d i l y a t t a c k the ribosomal subunits.  segments o f mRNA and the rRNA o f f r e e ftfi  1 Op  *  RNase without  inter-  ribosomal  R i b o n u c l e o p r o t e i n p a r t i c l e s may even b i n d 183 184  d e s t r u c t i o n o f t h e i r RNA.  c r e a t i c RNase A - c a t a l y z e d d e g r a d a t i o n  Bovine pan-  o f ribosomes has been  demonstrated t o depend on t h e i r f u n c t i o n a l s t a t e , and J354 Yanofsky has  suggested  t h a t mRNA while a s s o c i a t e d with  ribo-  somes i s a f f o r d e d some p r o t e c t i o n from the a c t i o n o f RNases.  -  39  -  A s s o c i a t e d p r o t e i n s thus c o n f e r r e s i s t a n c e upon the RNA  of r i b o n u c l e o p r o t e i n complexes by imposing c o n s t r a i n t s  on the s t e r i c a c c e s s i b i l i t y to RNases of s p e c i f i c i n the RNA of RNA  molecules.  sequences  M a t u r a t i o n a l p r o c e s s i n g and  must, t h e r e f o r e , proceed  i n a r e g u l a t e d sequence of  steps with exposed cleavage s i t e s being a t t a c k e d p o s s i b l y by s i t e - s p e c i f i c e n d o r i b o n u c l e a s e s . vages would be expected  turnover  first  Initial  clea-  to induce c o n f o r m a t i o n a l changes i n  the r i b o n u c l e o p r o t e i n s t r u c t u r e p o s s i b l y accompanied by the d i s s o c i a t i o n or l o o s e n i n g of bound p r o t e i n and hence the exposure of new  cleavage s i t e s which c o u l d then be a t t a c k e d  by RNases of more g e n e r a l s p e c i f i c i t y . of a s i n g l e RNA  molecule might e n t a i l the p a r t i c i p a t i o n of  s e v e r a l d i f f e r e n t RNases a t d i f f e r e n t tion.  Thus, the d e g r a d a t i o n  stages of i t s degrada-  A l s o , the concerted a c t i o n o f RNases and p r o t e a s e s i n  the d e g r a d a t i o n of r i b o n u c l e o p r o t e i n i s suggested by the synchronous turnover of the RNA ribosomes.  7 0  Any  and p r o t e i n components of  impairment i n the f i d e l i t y  c r i p t i o n would r e s u l t i n d e f e c t i v e RNA  of t r a n s -  molecules which c o u l d  not p a r t i c i p a t e i n ribosome and informosome assembly. d e f e c t i v e RNA  This  unprotected by f u n c t i o n would thus be more  s u s c e p t i b l e to a t t a c k by RNases and,  t h e r e f o r e , would be  degraded r a p i d l y , whereas the f u n c t i o n a l p o o l of RNA undergo normal t u r n o v e r .  would  Such a mechanism of d i f f e r e n t i a l  d e g r a d a t i o n of f u n c t i o n a l versus n o n - f u n c t i o n a l RNA  molecules  - 40  -  would minimize the m a n i f e s t a t i o n of t r a n s c r i p t i o n a l e r r o r s at the  l e v e l of p r o t e i n and  of RNA  turnover c o u l d  c o n c e n t r a t i o n s and  c e l l function.  Thus, the  control  depend g r e a t l y on changes i n l o c a l i o n  other f a c t o r s which d i s s o c i a t e  ribonucled-  p r o t e i n complexes as w e l l as upon more d i r e c t r e g u l a t i o n the f u n c t i o n a l l e v e l s and The b i t o r s has  1 8 5  a c t i v i t i e s of s p e c i f i c RNases.  complexing of enzymes with s p e c i f i c p r o t e i n  a c t i v i t y of such c a t a b o l i c enzymes as p r o -  deoxyribonuclease  f u n c t i o n a l l e v e l of the  1 8 6  could  be r e g u l a t e d -l  balance.  The  reported  pH  7.8  RNase.  inhibitor-bound  by any  The the  forms of  the  factors affecting this  absence of d e t e c t a b l e RNase i n h i p a r t i a l l y account f o r the  i n the n u c l e u s , whereas RNA  of the nucleus i n t o the  1 8 7  DO  b i t o r p r o t e i n i n n u c l e i may turnover of RNA  and  l a t t e r enzyme thus depends on  p a r t i t i o n i n g between a c t i v e and enzyme and  inhi-  been demonstrated i n some c e l l s as a mechanism f o r  c o n t r o l l i n g the teases,  of  rapid  transported  c y t o s o l which c o n t a i n s a l a r g e  out excess  of RNase i n h i b i t o r , escapes r a p i d d e g r a d a t i o n . 189 Some evidence does e x i s t functional  7  f o r the  and  The  f o r e f f e c t i n g t h i s c o n t r o l are unknown. l i f e t i m e and  regula-  t r a n s l a t i o n of the mRNA  molecules coding f o r these enzymes.  unknown.  of  l e v e l s of i n t r a c e l l u l a r RNases through the  t i o n of the r a t e of s y n t h e s i s  l a t i n g the  control  factors The  responsible  f a c t o r s regu-  turnover of s p e c i f i c RNases are  also  - 41 -  Much remains to he understood as to the mechanisms by which RNA degradation i s regulated and coordinated with RNA synthesis.  1.6 I n t r a c e l l u l a r RNases of Non-Neural Mammalian Tissues I n t r a c e l l u l a r RNases of mammalian tissues have not been p u r i f i e d to homogeneity and knowledge of t h e i r structure and function does not approach i n completeness that f o r bovine pancreatic RNase A or RNase T^.  Extensive  study of  t h i s enzyme system i n animal tissues has, nevertheless, revealed i t to be complex, consisting of many distinguishable components widely divergent i n t h e i r s t r u c t u r a l and functional ppoperties.  The i n t r a c e l l u l a r RNase system of r a t l i v e r has  been studied more extensively than that of any other t i s s u e , and the following discussion w i l l hence deal primarily with those components of the RNase system which have been ident i f i e d and more or less thoroughly characterized i n t h i s tissue. 1.61 Acid RNase An RNase with an acid pH optimum has been characterized and p a r t i a l l y p u r i f i e d by a number of i n v e s t i gators. 190-193  _«his enzyme i s highly unstable and i s r a p i d l y  inactivated by heat and d i l u t e acid.  I t has a molecular  weight of about 24,000 to 28,000 D a l t o n s , ^ 1  a pH optimum  - 42 -  of 5*8, and no metal i o n requirement.  A c i d RNase has been  r e p o r t e d to degrade p a r t i a l l y h y d r o l y z e d RNA more r a p i d l y 192 than h i g h molecular weight RNA,  t o hydrolyze a l l p u r i n e  7  and p y r i m i d i n e phosphodiester bonds (although i t has a p y r i m i d i n e bond p r e f e r e n c e ) t o 3 ' - r i b o n u c l e o t i d e s with the i n t e r m e d i a t e f o r m a t i o n of 2•, 3 ' - c y c l i c r i b o n u c l e o s i d e 191-195 phosphates  7 J  and hence t o leave no u n d i a l y z a b l e RNA  196 197 core.  7  '  ' The s i n g l e - s t r a n d e d form o f RNA i s s p e c i f i -  7 1  c a l l y or p r e f e r e n t i a l l y a t t a c k e d .  Although a h i g h p u r i f i -  c a t i o n o f t h i s enzyme from r a t mammary carcinomas has 189 r e c e n t l y been r e p o r t e d ,  ' d e f i n i t i v e substrate s p e c i f i c i t y  s t u d i e s with h i g h l y p u r i f i e d enzyme p r e p a r a t i o n s have not y e t been done. I n i t i a l s t u d i e s of the i n t r a c e l l u l a r d i s t r i b u t i o n o f t h i s enzyme found i t t o be a s s o c i a t e d p r i m a r i l y with the 198 199 200 crude m i t o c h o n d r i a l f r a c t i o n .  •  7  7  De Duve e t a l .  subsequently demonstrated t h a t t h i s enzyme along with other a c i d h y d r o l a s e s i s a s s o c i a t e d not with mitochondria p e r se ibuti With a separate c l a s s o f o r g a n e l l e s which they named 196 "lysosomes." concluded  R e i d and Nodes  7  obtained s i m i l a r r e s u l t s and  t h a t a c i d RNase was present i n c e l l  particulates  i n t e r m e d i a t e i n s i z e between mitochondria and t h a t c l a s s o f 201 lysosomes c o n t a i n i n g a c i d phosphatase.  Rahman  also  s t u d i e d the i n t r a c e l l u l a r d i s t r i b u t i o n o f a c i d RNase i n comparison with t h a t of s e v e r a l other a c i d h y d r o l a s e s and  - 43 found i t t o be present i n a c l a s s o f lysosomes s i m i l a r t o that  c o n t a i n i n g c a t h e p s i n D but d i s t i n g u i s h a b l e  from those  lysosomes c o n t a i n i n g a c i d phosphatase and c a t h e p s i n C.  De  202 20? Lamirande and A l l a r d not  •  J  a l s o found t h a t a c i d RNase i s  contained i n the same c l a s s o f c e l l p a r t i c u l a t e s  phosphatase, but concluded on the b a s i s  as a c i d  o f the s i m i l a r i t y o f  i t s d i s t r i b u t i o n with glutamate dehydrogenase t h a t be present predominantly i n m i t o c h o n d r i a .  i t must  Considerable  amount of the t o t a l c e l l u l a r a c i d RNase a c t i v i t y has a l s o been recovered i n the h i g h speed supernatant f r a c t i o n o f 194 202 c e l l horaogenates.  7  *  I t i s not c l e a r whether t h i s  f r a c t i o n o f the enzyme a c t i v i t y a c t u a l l y e x i s t s i n s i t u i n the  soluble  phase o f the cytoplasm or whether i t r e p r e s e n t s  enzyme r e l e a s e d  from lysosomes d u r i n g the t i s s u e homogeniza-  t i o n and s u b c e l l u l a r f r a c t i o n a t i n g procedures. 194 has  been obtained  suggesting t h a t  7  Some evidence  the a c i d RNase a c t i v i t y  of the c y t o s o l may be due t o an enzyme d i f f e r e n t from  that  found i n the crude m i t o c h o n d r i a l f r a c t i o n . 1.62  pH 9.5  RNase  An a l k a l i n e RNase with a pH optimum o f 9$5  was f i r s t  204 205 d e t e c t e d by Rahman  '  J  i n rat liver.  b i t e d by most monovalent and d i v a l e n t  I t i s strongly cations  and t h i s  i n h i b i t o r y e f f e c t i s not due t o i o n i c s t r e n g t h . a c o n c e n t r a t i o n o f 0.5 divalent  cations,  t o 1.0 x 10  J  M Mg  inhi-  However, a t  , unlike  other  produces a 30 t o 40$ a c t i v a t i o n o f t h i s  enzyme.  I n h i b i t i o n m EDTA can also be overcome by Mg++  at a suitable concentration.  This enzyme i s heat and acid  l a b i l e and exhibits substrate i n h i b i t i o n with an RNA concent r a t i o n of more than 2.0 mg/ml. 204 have been reported  The above c h a r a c t e r i s t i c s  f o r the unpurified enzyme and no p u r i -  f i c a t i o n procedure or s u b s t r a t e - s p e c i f i c i t y data have yet been published.  In a study of i t s i n t r a c e l l u l a r d i s t r i b u t i o n  reported the pH 9 . 5 RNase to be associated prima-  Rahman  r i l y with mitochondria and microsomes.  L i t t l e of the t o t a l  c e l l u l a r content of t h i s enzyme was recovered i n the l y s o somal and supernatant  f r a c t i o n s , although lysosomes possessed  the highest s p e c i f i c pH 9 » 5 RNase a c t i v i t y . 1 . 6 3 pH 7 . 8 RNase An alkaline RNase with a pH optimum of 7 . 8 has been extensivley studied and p a r t i a l l y p u r i f i e d . 1 9 1 - 1 9 3 . 1 9 9 , 2 0 7 This enzyme exhibits a broad a c t i v i t y maxima between pH 6 . 7 and 8 . 5 ,  i t has no divalent cation requirement, and i s  stable at 7 0 ° f o r 5 minutes.  I t hydrolyzes RNA more rapidly  than poly U and poly C and shows no a c t i v i t y toward poly A and poly G.  I t appears to s p l i t p r i n c i p a l l y pyrimidine  phosphodiester  bonds to y i e l d  ribonucleoside-3'-phosphates  v i a 2 * , 3 ' - c y c l i c pyrimidine nucleotide intermediates, and leaves a r e s i s t a n t undialyzable oligoribonucleotide core.  y  This enzyme i s thus s i m i l a r i n i t s functional properties to bovine pancreatic RNase A.  pH 7 * 8 RNase has been reported  '  -  45  -  to have a molecular weight of between 1 1 , 5 0 0 to 12,000 208  \qL  Daltons.  Beard and Razzell  have achieved a 3,000-  f o l d p u r i f i c a t i o n of t h i s enzyme from hog l i v e r and Gordon °^ has p u r i f i e d r a t l i v e r pH 7.8 RNase 6,000-fold. 2  Results obtained by various workers concerning the i n t r a c e l l u l a r d i s t r i b u t i o n of t h i s enzyme are not e n t i r e l y 207 i n agreement.  Roth  ' separated the r a t l i v e r homogenate  into only three f r a c t i o n s — n u c l e i , supernatant—and  crude mitochondria, and  reported that the t o t a l pH 7.8 RNase  a c t i v i t y was recovered i n the mitochondrial and supernatant fractions with a small amount of enzyme i n the n u c l e i .  Reid  196 and Nodes  7  achieved a further fractionation  of the crude  mitochondrial f r a c t i o n into mitochondria and lysosomes and reported that the highest s p e c i f i c a c t i v i t y of pH 7.8 RNase occurred not i n mitochondria but i n a class of lysosomes " l e s s r e a d i l y sedimented than those p a r t i c l e s containing acid phosphatase."  However, these investigators did not  present t h e i r data i n terms of the per cent d i s t r i b u t i o n of the t o t a l a c t i v i t y .  Rahman  subsequently confirmed that  the lysosomal f r a c t i o n possessed the highest s p e c i f i c pH 7.8 RNase a c t i v i t y (3-fold that of any other f r a c t i o n ) and found that the per cent recovery of t o t a l a c t i v i t y was lowest i n the lysosomal and highest f o r the mitochondrial f r a c t i o n . F i f t y per cent of the t o t a l recovered pH 7.8 RNase a c t i v i t y was also approximately equally distributed  between the  - 46 -  microsomal and supernatant f r a c t i o n s . 199 Allard  7 7  De Lamirande and  under t h e i r subcellular f r a c t i o n a t i o n and assay-  conditions found pH 7.8 RNase to be evenly d i s t r i b u t e d among mitochondria, lysosomes and microsomes.  Discrepancies i n  the i n t r a c e l l u l a r d i s t r i b u t i o n data probably r e f l e c t d i f f e rences i n assay procedures employed i n d i f f e r e n t laboratories. 1.64 RNase I n h i b i t o r 210 P i r o t t e and Desreux f i r s t described an RNase i n h i b i t o r protein i n the cytosol of guinea p i g l i v e r . The properties of the i n h i b i t o r from r a t l i v e r have been studied,187,211,212,213,155.158 purified.  214  a  n  d  i  t  h  a  g  b  e  e  n  p  a  r  t  i  a  l  l  y  215 I t has also been studied i n mouse t i s s u e s ,  mouse ascites tumours,  J  ? 16  r a t adipose tissue  ?17  ' and blood.  ? i ft  The r a t l i v e r RNase i n h i b i t o r i s a l a b i l e protein r e a d i l y inactivated by acid, heat, heavy metal ions such as Hg++ and Pb++ and by sulfhydryl-blocking reagents such as p-chloromercuribenzene sulfonic a c i d .  '*  The free i n h i b i t o r i s present i n large excess i n the rat l i v e r supernatant f r a c t i o n as indicated by the i n h i b i t i o n by t h i s f r a c t i o n of sizeable quantities of added bovine pancreatic RNase A or free pH 7.8 RNase.  There i s also i n  most mammalian tissues a considerable quantity of i n h i b i t o r present which i s complexed to the endogenous pH 7«8  - 47  RNase." ""''• " 1  tJ  -  On treatment of the  L  supernatant f r a c t i o n with  s u l f h y d r y l - b l o c k i n g reagents, a c i d , or heat the i n a c t i v a t e d and released  the i n h i b i t o r - b o u n d  inhibitor is  7*8  l a t e n t pH  RNase i s  211 i n a c t i v e form.  Some c h a r a c t e r i s t i c s o f the i n h i b i t o r and  i n t e r a c t i o n between RNase  m o d i f i e d d e r i v a t i v e s of bovine  pancreatic  219 RNase A have been s t u d i e d . r e a c t i o n between the two  *  proteins  a c t i v e s i t e of the RNase, nor the  enzyme.  determined t h a t  i s not  on the  dependent on  the  f r e e amino groups of  configuration  There i s c o n s i d e r a b l e  of the RNase and  suggested t h a t  since pancreatic  RNase A has  groups, the f o r m a t i o n of a d i s u l p h i d e inhibitor i s unlikely. i n h i b i t o r may  on  on hydrogen  evidence t h a t f r e e s u l f h y d r y l 211  groups are e s s e n t i a l f o r i n h i b i t o r a c t i v i t y .  the  the  I n t e r a c t i o n i s , however, s t r o n g l y dependent  a r e l a t i v e l y native bonding.  I t was  Rather, the  Roth no  has  free sulfhydryl  bond between RNase  and  s u l f h y d r y l group(s) of  p a r t i c i p a t e i n the f o r m a t i o n of hydrogen  bond(s) with the enzyme, or may  be r e q u i r e d  to m a i n t a i n  the  a c t i v e conformation of the i n h i b i t o r . RNase i n h i b i t o r i s a c t i v e p r i n c i p a l l y i n the pH between 7 and no  9 and  has  been r e p o r t e d  i n h i b i t i o n of a c i d R N a s e  1 9 5  *  2 1 3  to produce l i t t l e  or pH  9.5  s p e c i f i c i t y of the i n h i b i t o r a c t i o n i s not i n terms of the pH a c t i o n s i n c e the  dependence of the  RNase. ^ 20  range or The  wholly accountable  inhibitor-enzyme i n t e r -  i n h i b i t o r produces s i g n i f i c a n t i n h i b i t i o n of  - 48 -  bovine pancreatic RNase A assayed at pH 5 . 8 ,  1  8  7  ,  2  1  whereas  3  RNase Tj which i s similar i n several respects to pancreatic RNase A was not inhibited when assayed at pH  7 . 8 .  2  1  3  RNase i n h i b i t o r s isolated from d i f f e r e n t mammalian sources are not s p e c i f i c and do cross-react i n varying 212  degrees with heterologous pH 7 . 8 RNases.  There are considerable t i s s u e - and s p e c i e s - s p e c i f i c differences i n the r e l a t i v e amounts of free and RNasebound i n h i b i t o r .  1 8 7  RNase i n h i b i t o r has recently been p u r i f i e d to near 220  homogeneity  221  •  and i t has been found to be an a c i d i c  protein with a molecular weight of  50,000  to  60,000  Daltons. 1 , 6 5 Ribosomal RNase 2 2 2  Another a l k a l i n e RNase has been detected i n ribosome and polysome preparations from r a t l i v e r .  2 2 3  "  2 2 5  *  This  ribosomal RNase has a pH optimum of 8 . 5 . i s r e l a t i v e l y stable i n acid, but r a p i d l y inactivated above 5 5 ° . I t i s stimulated by an optimal divalent cation (Ca.++ or Mg++) concentration of 1 0 mM and a monovalent cation (K+ or Na+) of 5 0 mM. 37,000  concentration  I t s molecular weight has been estimated to be  Daltons.  Unlike the pH  7.8  RNase of the c e l l  sap,  and mitochondrial,fractions, ribosomal RNase does not degrade poly U.  Like the pH 7 . 8 RNase of the c e l l sap i t  exists i n a latent form bound to RNase i n h i b i t o r .  Although  - 49 -  d i r e c t i n h i b i t i o n of the p u r i f i e d enzyme by the cytosol or p u r i f i e d i n h i b i t o r has not yet been demonstrated, several workers have reported thatppolysomes are s t a b i l i z e d by RNase inhibitor  preparations.  The f a c t that, despite considerable  e f f o r t , i t has not  been possible to i s o l a t e polysomes free of RNase a c t i v i t y suggests that the bound RNase may be an i n t e g r a l part of polysome structure and may be involved i n t h e i r normal O O f\  function.  Roth  has made the i n t e r e s t i n g observation  that  the a c t i v i t y of the a l k a l i n e RNase present i n r a t l i v e r microsomes must be f u n c t i o n a l l y i n t a c t f o r the incorporation of 14 C-l-leucine into protein.  This suggests that t h i s enzyme  may play a c r i t i c a l role i n the regulation of protein synthesis. 1.66 Nuclear RNases Many investigators have attempted to detect the enzymes responsible f o r the rapid turnover of RNA i n the nucleus since the a c t i v i t y of these enzymes may regulate the kind and amount of RNA that does f i n d i t s way into the cytoplasm. op o  Heppel et a l .  op  '*  Q  have detected an a l k a l i n e RNase  with a pH optimum between 8 . 5 and 9 . 5 i n n u c l e i of guinea p i g liver.  This enzyme i s inactivated below pH 6 and above 5 0 ° .  I t hydrolyzes poly A and poly U to  ribonucleoside-5'-phosphates  -  and  50  -  o l i g o r i b o n u c l e o t i d e s of two  minated by 5 ' - p h o s p h a t e s . t h i s enzyme has  to s i x u n i t s i n l e n g t h t e r -  An 8 0 - f o l d purj purification  1 7 6  of  by R a z z e l l . '229  been achieved  230 Cunningham and S t e i n e r  have r e p o r t e d f u r t h e r  J  p r o p e r t i e s of what appears to be the same enzyme. A p o l y r i b o n u c l e o t i d e phosphorylase has r e p o r t e d to be present HeLa c e l l n u c l e i .  2 3 1  a l s o been  i n guinea p i g l i v e r n u c l e i  and  1 7 7  T h i s enzyme i s supposedly a s s o c i a t e d  with chromosomes and may  p a r t i c i p a t e i n the breakdown of  newly s y n t h e s i z e d heterogeneous n u c l e a r RNA  (HnRNA) to  ribonucleoside-5'-diphosphates. An RNase a c t i v i t y has a l s o been d e t e c t e d i n the n u c l e o l a r f r a c t i o n obtained from n o r m a l , 3 * 3 3 2  plastic  233 234 * and r e g e n e r a t i n g J J  from H e L a  *  2 3 7  2  neoflsiiifei©  235 r a t l i v e r as w e l l as  J  2 3 6  2  J J  and L c e l l s . ® 23  T h i s enzyme i s an  endori-  bonuclease and has been i m p l i c a t e d i n the p r o c e s s i n g 4 5 S p r e c u r s o r ribosomal RNA  of  i n t o mature 18 S and 28 S tRNA.  However, t h i s p u t a t i v e f u n c t i o n remains to be  definitively  demonstrated. Other endoribonucleases logous  to those  analogous and  p o s s i b l y homo-  f u n c t i o n i n g i n the m a t u r a t i o n a l  of precursor t R N A  2 3 9 , 2 2 + 0  and p r e c u r s o r r R N A  2 k l  "  processing 2 k 3  in  b a c t e r i a have not y e t been demonstrated i n mammalian c e l l s . 244 S t e i n and Hausen  245 '  enzyme (RNase H or hybridase)  J  have r e p o r t e d an  interesting  from c a l f thymus which  - 51  s p e c i f i c a l l y degrades the RNA The  -  moiety of DNA-RNA h y b r i d s .  r e c e n t f i n d i n g t h a t RNase H a c t i v i t y appears to be a 246  247  u n i v e r s a l p r o p e r t y of r e v e r s e t r a n s c r i p t a s e s RNA  '  ' from  tumor v i r u s e s has l e d to the s u g g e s t i o n t h a t the b i o -  l o g i c a l r o l e of t h i s RNase a c t i v i t y i s to remove the  oligori-  b o n u c l e o t i d e which normally f u n c t i o n s as a primer i n DNA 248 249 2 50 2 51 replication ' and which becomes c o v a l e n t l y l i n k e d »<•-'•»• 7  J  to the newly s y n t h e s i z e d DNA  chain.  t r a n s c r i p t i o n by r a t l i v e r RNA l i v e r DNA  and  chromatin  S t i m u l a t i o n of i n v i t r o  polymerase B of n a t i v e r a t  has a l s o been r e p o r t e d ! 2  i n the presence of r a t l i v e r RNase H.  2  to  occur  T h i s s t i m u l a t i o n was  a t t r i b u t e d to the supposed p a r t i c i p a t i o n of t h i s RNase i n the r e l e a s e of newly s y n t h e s i z e d RNA  from the  template.  2 5 2  I n g e n e r a l , s t u d i e s of n u c l e a r RNases have not achieved a high degree of r e s o l u t i o n . Those enzymes which have been d e t e c t e d have not been e x t e n s i v e l y p u r i f i e d characterized.  F u r t h e r work i s r e q u i r e d to d e f i n e the  of these enzymes i n the turnover o f RNA the n u c l e a r maturation  1.7  o f cytoplasmic  or role  i n the nucleus &n_d i n RNA.  Changes i n RNase A c t i v i t i e s under V a r i o u s P h y s i o l o g i c a l and Pathological Conditions A number of workers have monitored v a r i a t i o n s i n the  l e v e l s and  i n t r a c e l l u l a r d i s t r i b u t i o n of v a r i o u s components  of the RNase enzyme system i n s e v e r a l t i s s u e s under v a r i o u s  -  52 -  p h y s i o l o g i c a l and p a t h o l o g i c a l c o n d i t i o n s w i t h the expectat i o n t h a t such i n f o r m a t i o n  might y i e l d a b e t t e r understanding  of the f u n c t i o n a l r o l e o f these enzymes i n the r e g u l a t i o n o f c e l l u l a r RNA content and t u r n o v e r . C e l l u l a r RNase a c t i v i t y has been found t o be e l e v a t e d under s e v e r a l circumstances i n which the RNA/DNA r a t i o i s reduced  '  2 5 3  and p r o t e i n s y n t h e s i s  2 5 5  1 8 9 , 2  53-267  ^  Thus, decreases i n RNase i n h i b i t o r or i n c r e a s e s 7.8  g  r e (  }  U c e (  i  t  i n f r e e pH  RNase a c t i v i t y l e v e l s have been observed i n t i s s u e s  e x h i b i t i n g a low r a t e o f p r o t e i n s y n t h e s i s their catabolic a c t i v i t i e s . increased  "  I n some  2 6 7  cases  These o b s e r v a t i o n s  8  9  ,  2  5  7  ,  2  5  8  ,  suggest t h a t the de-  p r o t e i n s y n t h e s i s under these c o n d i t i o n s  elevated  1  2  a c i d RNase a c t i v i t y has a l s o been demonstrated under  similar conditions. creased  2 5 7  o r an i n c r e a s e i n  i s due t o  f u n c t i o n a l l e v e l s o f v a r i o u s c e l l u l a r RNase  acti-  v i t i e s and hence enhanced d e g r a d a t i o n o f c e l l u l a r RNA. C o n v e r s e l y , i n c r e a s e d RNA/DNA r a t i o s and i n c r e a s e d 268—? 7 5 p r o t e i n s y n t h e s i s have been demonstrated  "  (  J  t o be  p o s i t i v e l y c o r r e l a t e d with decreased f u n c t i o n a l l e v e l s o f s e v e r a l c e l l u l a r RNase a c t i v i t i e s .  Thus, i n c r e a s e s  i n the  r a t i o o f RNase i n h i b i t o r / f r e e pH 7 . 8 RNase a c t i v i t y have been observed i n t i s s u e s c h a r a c t e r i z e d by a high r a t e o f RNA oot  OAft  synthesis  and/or c e l l d i v i s i o n  rat l i v e r  2  nephrotic  kidney  7  6  '  2  7  •  f  D  such as i n r e g e n e r a t i n g  and l i v e r o f hypophysectomized r a t s ,  7  2 70 C ( y  2 fiO  *  and d u r i n g  compensatory r e n a l  2 5 5  *  2 7 8  6  5  - 53 hypertrophy f o l l o w i n g u n i l a t e r a l nephrectomy.'' " ,J  of nephrotic  L  I n the case  decreases i n pH 9.5 RNase and a c i d  kidney  RNase were a l s o found t o be p o s i t i v e l y c o r r e l a t e d w i t h the e l e v a t e d r a t e of p r o t e i n s y n t h e s i s .  These o b s e r v a t i o n s  suggested t h a t the i n c r e a s e d p r o t e i n s y n t h e s i s  have  i n these con-  d i t i o n s may r e s u l t from the p r e s e r v a t i o n o f mRNA and p o s s i b l y a l s o tRNA and rRNA. p Qp  RNA turnover  studies  p Q0  '  J  have i n d i c a t e d t h a t i n  these s i t u a t i o n s the changes i n c e l l u l a r RNA content are due predominantly t o changes i n the r a t e o f RNA c a t a b o l i s m than i n the r a t e o f RNA s y n t h e s i s . 2 72 f i n d i n g s , Shortman  '  rather  On the b a s i s o f these  has hypothesized t h a t the changes i n  c e l l u l a r RNA content and c e l l u l a r metabolism accompanying changes i n p h y s i o l o g i c a l c o n d i t i o n s are e f f e c t e d through RNases by i n f l u e n c i n g the r a t e o f RNA d e g r a d a t i o n .  T h i s work  thus p o i n t s t o a need f o r a thorough i n v e s t i g a t i o n o f the c o n t r i b u t i o n of the RNase enzyme system t o the changes i n c e l l u l a r RNA o f b r a i n which have been r e p o r t e d  t o occur i n  response to changes i n p h y s i o l o g i c a l s t i m u l a t i o n and d u r i n g l e a r n i n g and memory  formation.  1.8 The RNase Enzyme System o f B r a i n Whereas some i n f o r m a t i o n  e x i s t s concerning  i n v o l v e d i n the s y n t h e s i s o f RNA i n b r a i n , little  the enzymes  comparatively  i s known of the molecular processes by which c e l l u l a r  - 54 -  RNA content i s p o s t - t r a n s c r i p t i o n a l l y controlled.  Infor-  mation as to the enzymes p a r t i c i p a t i n g i n the maturational processing of precursor t r a n s c r i p t i o n products into f u n c t i o n a l l y mature transfer, ribosomal, and messenger RNA as well as i n the degradation of these molecules i s not available i n the same degree of d e t a i l f o r brainnas f o r many other mammalian organs. Roth  1 8 7  i n a comparative study of various r a t tissues  found that RNase i n h i b i t o r a c t i v i t y was highest i n brain and hence presumably has a p a r t i c u l a r l y important role i n regul a t i n g the metabolism of RNA i n t h i s t i s s u e .  Ellem and  215  Colter, however, i n a similar study of mouse tissues found that mouse brain contained very low l e v e l s of both pH 7.8 284 J  RNase and RNase i n h i b i t o r a c t i v i t y .  Guroff  i n a develop-  mental study of r a t brain RNase a c t i v i t y assayed at pH 5 found that the l e v e l of RNase a c t i v i t y expressed at t h i s pH was lower i n older animals than i n young animals. and T a k a h a s h i  285  Suzuki  studied the regional d i s t r i b u t i o n of RNase  i n h i b i t o r i n rabbit brain and found that those areas (cereb r a l cortex, cerebellum, hippocampus) r i c h i n neurons corresponded to the areas having highest RNase i n h i b i t o r a c t i v i t y . The maximal v a r i a t i o n i n the l e v e l of RNase i n h i b i t o r a c t i v i t y between the d i f f e r e n t brain regions studied was not more than 30$.  These investigators have also reported  a developmental p r o f i l e f o r RNase i n h i b i t o r a c t i v i t y i n r a t  - 55 -  cerebral cortex.  This component of the RNase system exhibits  a sharp peak between the 5th and 10th day after b i r t h , f a l l s to near neonatal l e v e l s by the 13th day, and subsequently 221  remains r e l a t i v e l y constant.  Takahashi,  Mase and Suzuki  have also recently reported a high p u r i f i c a t i o n of RNase i n h i b i t o r from p i g cerebral cortex.  Datta and co-workers  have detected two RNase a c t i v i t i e s with pH optima of 5»4 and 7.9 i n ribosomes isolated from goat cerebral cortex.  Detec-  t i o n of s i g n i f i c a n t a c t i v i t y required incubation periods of 14 to 18 hours.  Both a c t i v i t i e s were t i g h t l y bound to the  ribosomes and could not be s o l u b i l i z e d .  These workers have  suggested that these ribosomal RNase a c t i v i t i e s may be responsible f o r the chromatolytic changes which ribosomes undergo during neuronal s t r e s s . of 7.6 has been detected humans.  An RNase a c t i v i t y with a pH optimum ' i n the cerebrospinal f l u i d of  Although i t s source and function were not deter-  mined, i t was observed that the spinal f l u i d of patients with damaged CNS tissue (cerebral i n f a r c t i o n , neoplasm, and demyelination due to multiple s c l e r o s i s ) exhibited elevated l e v e l s of t h i s RNase a c t i v i t y . In general, the available information on the RNase enzyme system of brain i s fragmentary and unconfirmed.  -  1.9 The P r e s e n t  56  -  Investigation  Because the RNase enzyme system p l a y s  such an  important r o l e i n r e g u l a t i n g i n t r a c e l l u l a r RNA content and hence i n r e g u l a t i n g the a v a i l a b i l i t y of f u n c t i o n a l l y mature RNA molecules t o the p r o t e i n - s y n t h e s i z i n g  apparatus  o f the c e l l ,  charac-  i t i s p o s s i b l e t h a t any d i s t i n c t i v e  t e r i s t i c s o f t h i s enzyme system  i n b r a i n may have a d i r e c t  r e l a t i o n s h i p t o the s p e c i a l i z e d f u n c t i o n o f t h i s t i s s u e . A M Q C S inasmuch as changes i n b r a i n c e l l u l a r RNA content have been demonstrated  t o occur i n response t o l e a r n i n g and sensory  s t i m u l a t i o n , the r o l e of RNases may achieve s p e c i a l s i g n i f i c a n c e i n b r a i n i n comparison  with other t i s s u e s such as  l i v e r which have been more e x t e n s i v e l y  studied.  Thus, f o r  example, the enhanced l e v e l s o f neuronal RNA r e s u l t i n g from sensory experience may be predominantly due t o decreased d e g r a d a t i o n which, i n t u r n , may be due t o decreased  RNA  functional  l e v e l s of i n t r a c e l l u l a r RNase a c t i v i t y . The  i n v e s t i g a t i o n reported  upon i n t h i s t h e s i s was  hence aimed a t o b t a i n i n g more d e t a i l e d and comprehensive information system  about the c h a r a c t e r i s t i c s o f the RNase enzyme  i n r a t b r a i n and a t a c h i e v i n g  some understanding o f  the c o n t r i b u t i o n of the i n d i v i d u a l components of t h i s enzyme system to the r e g u l a t i o n of RNA metabolism  i n this tissue.  I I . MATERIALS AND  METHODS  2.0 M a t e r i a l s 2.01  Experimental  animals  Rats of the Wistar s t r a i n were obtained from the V i v a r i u m of the Department of Zoology, U n i v e r s i t y of British  Columbia. 2.02  Chemicals  T r i t o n X100 pure  was  a product of Rohm and Haas.  Ultra-  sucrose and ammonium sulphate were obtained from  Swartz/Mann.  P a r a - c h l o r o - m e r c u r i b e n z o i c a c i d was  of N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n .  T h i s reagent  d i s s o l v e d i n .02 M T r i s - H C l b u f f e r , pH 8.9. ( C l e l a n d ' s reagent) was  a product  Dithiothreitol  a product of Calbiochem.  Solutions  of d i t h i o t h r e i t o l were f r e s h l y prepared i n d i s t i l l e d immediately p r i o r to use. Organic Chemicals and was man.  G e l a t i n was  was  water  a product of Eastman  p u r i f i e d by the method of S h o r t -  DEAE c e l l u l o s e with an exchange c a p a c i t y of  m i l l i e q u i v a l e n t monovalent anion per gram was BioRad L a b o r a t o r i e s and was  0.84  obtained from  p u r i f i e d by the method of  288 P e t e r s o n and Sober. were "reagent grade"  A l l other common l a b o r a t o r y chemicals and were used without f u r t h e r  cation.  - 57 -  purifi-  - 58 -  Fleischmann's y e a s t s-RNA prepared by the method of 289 Holley  7  was  the product of Calbiochem.  S o l u t i o n s of  t h i s s u b s t r a t e prepared i n d i s t i l l e d water c o u l d be at  4° without e x h i b i t i n g s i g n i f i c a n t i n c r e a s e i n a c i d - s o l u b l e  A260. was  stored  H i g h l y polymerized double-stranded calf-thymus  a product of Calbiochem,  NaCl.  and was  B i i f - ( p - n i t r o p h e n y l ) phosphate  DNA  d i s s o l v e d i n .01 M (Na) was  a product of  Sigma. C r y s t a l l i n e bovine p a n c r e a t i c RNase A ( c a . 50 K u n i t z units/mg) was tion.  a product of N u t r i t i o n a l B i o c h e m i c a l s Corpora-  Bovine serum albumin was  a product of  Calbiochem.  2.1 Methods 2.11  P r e p a r a t i o n of t i s s u e homogenates and e x t r a c t s  W i s t a r s t r a i n white r a t s of body weight 200-400 g were d e c a p i t a t e d and the whole b r a i n q u i c k l y removed from the cranium and p l a c e d i n a g l a s s p e t r i d i s h kept on i c e . The t i s s u e was T r i t o n X100 weight at  weighed, suspended  or 0.32  tissues  i n i c e - c o l d 0.1$  (v/v)  M sucrose i n the r a t i o of one gram wet  9 ml of homogenizing  medium, and homogenized  0° with n i n e to t e n s t r o k e s i n a g l a s s homogenizer  f i t t e d ,with a t e f l o n p e s t l e 0.13-0.18  mm).  (Arthur H. Thomas Co.; c l e a r a n c e ,  -  Tissue  59  -  used f o r p u r i f i c a t i o n work was  processed  as  above with the e x c e p t i o n of b e i n g homogenized at 0° i n a for 30  s t a i n l e s s s t e e l OmniMixer (Ivan S o r v a l l Inc.) seconds at speed c o n t r o l 5 t  3 0 seconds pause,and 3 0 seconds  at speed c o n t r o l 6 . The 3 , k  10$  (w/v)  t i s s u e homogenates were c e n t r i f u g e d  x g f o r 6 0 minutes at 0-4°  800  decanted and 2.12  For  r e f e r r e d to as the  and  the  at  supernatant  "extract."  F r a c t i o n a t i o n of i s o t o n i c sucrose homogenates by d i f f e r e n t i a l centrifugation subcellular d i s t r i b u t i o n studies  i s o t o n i c sucrose  homogenates were f r a c t i o n a t e d by d i f f e r e n t i a l at 0° i n a type S S - 3  k  r o t a r o f a S o r v a l l RC2  centrifugation centrifuge  and  a Spineo r o t a r # 5 0 of a Beckman Model L u l t r a c e n t r i f u g e . Isotonic 2.11 800  sucrose homogenates prepared as d e s c r i b e d  were c e n t r i f u g e d  at 800  x g f o r 10 minutes, the  sucrose equal to l / 5 the volume of the c a r r i e d out  pestle being gently a t 800  i n i t i a l homogenate  i n a g l a s s homogenizer,  r o t a t e d by hand), and  centrifuged  x g f o r 10 minutes to y i e l d the f i n a l 800  pellet.  Supernatants from the p r e c e d i n g two  were pooled and y i e l d the  crude  resuspended i n a volume of i c e - c o l d 0 . 3 2 M  x g p e l l e t was  ( r e s u s p e n s i o n was  i n section  centrifuged  the again  x g nuclear  centrifugations  a t 8,000 x g f o r 20 minutes to  8,000 x g m i t o c h o n d r i a l  pellet.  The  8,000 x g  - 60 -  supernatant was then c e n t r i f u g e d  a t 105,000 x g f o r 60  minutes (Spinco r o t a r #50) t o y i e l d the 105,000 x g microsomal p e l l e t and the 105,000 x g supernatant o r cytosol f r a c t i o n .  The f i n a l p a r t i c u l a t e f r a c t i o n s were  each suspended i n and brought t o a f i n a l volume with i c e c o l d 0.32 M sucrose equal t o one-half the s t a r t i n g volume of the homogenate, and rehomogenized a t 0° w i t h strokes  five  i n a g l a s s homogenizer. 2.13 P u r i f i c a t i o n procedure 2.131  Ammonium sulphate f r a c t i o n a t i o n  Powdered u l t r a p u r e onto t h e s u r f a c e  ammonium sulphate was s p r i n k l e d  o f a m a g n e t i c a l l y s t i r r e d crude enzyme  e x t r a c t maintained a t 0° i n an ice-water bath.  The r a t e o f  a d d i t i o n o f ammonium sulphate was c o n t r o l l e d so as not t o exceed t h e r a t e a t which the s a l t d i s s o l v e d . ammonium sulphate added to g i v e a d e s i r e d  The amount o f  s a t u r a t i o n was 290  c a l c u l a t e d a c c o r d i n g to the formula o f Noda and Kuby.  7  A f t e r 30 minutes o f a d d i t i o n a l s t i r r i n g , the p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n a t 3 ,800 x g f o r 15 minutes, k  suspended i n i c e - c o l d .02 M T r i s - H C l , p H ? . k : !  °  i n the c o l d room a g a i n s t  k  and d i a l y z e d a t  the same b u f f e r .  Nessler's  reagent was used t o t e s t f o r complete removal o f (NHu^SOk from the d i a l y s a t e s a c c o r d i n g t o Umbreit, B r u n i s and Stauffer.  2 3 6  *  -  61  -  2 . 1 3 2 DEAE c e l l u l o s e column chromatography of ammonium sulphate f r a c t i o n s Diethylaminoethylcellulose  was treated according to p OQ  the procedure of Peterson and Sober.  The washed DEAE  c e l l u l o s e , suspended i n . 0 2 M Tris-HCl, pH 7»4 e q u i l i brating buffer, was poured into columns at 4° i n the cold room and allowed to s e t t l e under gravity.  The packed  columns were washed with 5 0 0 ml e q u i l i b r a t i n g buffer p r i o r to being loaded with the dialyzed ammonium sulphate f r a c tions.  After the protein solution had completely entered  the column matrix i t was washed i n with ten or more bed volumes of e q u i l i b r a t i n g buffer and then eluted with a l i n e a r gradient  of NaCl i n e q u i l i b r a t i n g buffer.  Three to  7 ml eluate fractions were collected at a flow rate of 2 5 to 3 5 ml per hour.  The absorbance (at 2 6 0 and 280 nano-  meters) of the eluate f r a c t i o n s was sampled with a Beckman Model DU  spectrophotometer.  2.14 Enzyme assays 2.141 Determination of RNase a c t i v i t y RNase a c t i v i t y was determined increase i n  by measuring the  A260 of the acid-soluble ribonucleotides  and  oligoribonucleotides which are released as the degradation products of RNA.  The standard reaction mixture contained  f i n a l concentrations of  .05  M buffer,  .025$  (w/v)  Fleischmann's yeast s-RNA ( i n d i s t i l l e d H2O),  (.25  mg/ml)  and various  - 62 -  amounts of tissue sample, in a total volume of 2.0 ml contained in heavy-walled pyrex Sorvall centrifuge tubes (catalog No. 119)•  Where necessary, the enzyme solution  was diluted to ensure that the release of acid-soluble A260 was linear with incubation time.  The reaction  mixture was incubated for 3 ° or 60 minutes at 37° in a thermostatically controlled shaking water bath. reaction was terminated by placing the  :  _  The  incubation  tubes in a crushed ice bath and adding 2.0 ml ice-cold 1.4 N perchloric acid followed by thorough mixing.  The  assay tubes were allowed to stand 20 minutes in ice and then were centrifuged at  12,100  x g for  10  minutes at  0 ° to 4 ° in a type SS-34 rotar of a Sorvall RC2 centrifuge.  The supernatant was carefully decanted and its  absorbance at 260 nm measured immediately in s i l i c a cuvettes of 1 cm path length with a Beckman Model DU spectrophotometer.  Appropriate controls without added  enzyme and without added substrate were incubated and assayed concurrently in order to correct for any nonenzymatic depolymerization of RNA, the acid-soluble A260 material endogenous to the tissue sample, and the release of acid-soluble A260 from RNA endogenous to the tissue sample.  Experimental assay samples were corrected by  subtracting the total acid-soluble A260 released in controls,and the net RNase activity was expressed as the increase in  A260 (&A260)  per 30 minute incubation at 37° •  -  63  -  A unit of RNase a c t i v i t y was defined as the amount of enzyme which liberated acid-soluble material causing a net absorbancy increment of 1.0 O.D. 260 per ml per 3 0 minute incubation at  37°•  S p e c i f i c a c t i v i t y was ex-  pressed as units per mg protein. 2.142 Assay f o r deoxyribonuclease a c t i v i t y 291 The assay procedure f o r measuring DNase a c t i v i t y  7  was the same as that used f o r measuring RNase a c t i v i t y except highly polymerized  double-stranded c a l f thymus DNA  at a f i n a l concentration of  .025$  (w/v) was used as sub-  Incubation was f o r 6 0 minutes at 3 7 ° . 2.143 Assay f o r phosphodiesterase a c t i v i t y  strate.  Phosphodiesterase a c t i v i t y was measured by the l i b e r a t i o n of p-nitrophenol from the synthetic substrate 2 92 bis-(p-nitrophenyl) phosphate (Na).  7  The amount of p-  nitrophenol released was detected by i t s absorbance at 400 nm.  293  The reaction mixture contained 3 3 mM buffer,  0 . 4 mM bis-(p-nitrophenyl) phosphate (Na), and enzyme sample i n a t o t a l volume of 3 * 0 ml. minutes at  37°•  Incubation was f o r 60  The reaction was terminated  by t r a n s f e r r i n g  the incubation tubes to an i c e bath and adding 3 * 0 ml of 0.04 N NaOH to each tube.  The A400 was measured immediately.  - 64 -  2.144 Assay f o r pH 7 . 8 RNase inhibitor activity RNase i n h i b i t o r a c t i v i t y was determined by measuring the reduction i n the a c t i v i t y of a standard amount of bovine pancreatic RNase A i n the presence of various amounts of inhibitor sample.  187  C r y s t a l l i n e bovine pancreatic RNase A  (ca 5 0 Kunitz units per mg) was dissolved i n 0 . 1 $ (w/v) p u r i f i e d gelatin solution to a concentration of 0 , 0 1 ;ug/ml. The standard RNase i n h i b i t o r assay contained  . 0 5 ml of the  above stock solution ( 0 . 5 ng bovine pancreatic RNase A), 5 0 mM Tris-HCl pH 7 . 8 , 1 mM EDTA diNa, various d i l u t i o n s of tissue sample to give a l e v e l of i n h i b i t i o n between 3 0 $ and 70$,  and  .025$  (w/v) sRNA i n a t o t a l volume of  2.0  ml. The  substrate was added l a s t after mixing the previous components.  Controls f o r the a c t i v i t y of 0 . 5 ng c r y s t a l l i n e  bovine pancreatic RNase A alone, and f o r the RNase a c t i v i t y of the tissue samples alone, as well as appropriate blanks were incubated and assayed concurrently.  reagent  Corrections  f o r any RNase a c t i v i t y i n the i n h i b i t o r samples were made i n calculating  the per cent i n h i b i t i o n of control pancreatic  RNase A a c t i v i t y .  Incubation was f o r 3 ° minutes at 3 7 ° .  A l l other aspects of the assay procedure were the same as that previously described f o r the assay of RNase a c t i v i t y . The l e v e l of RNase i n h i b i t o r a c t i v i t y i n the tissue samples was interpolated from experimentally constructed  standard  -  65  -  curves r e l a t i n g per cent o f c o n t r o l bovine p a n c r e a t i c RNase A a c t i v i t y t o p r o t e i n c o n c e n t r a t i o n o f the i n h i b i t o r sample. A u n i t o f RNase i n h i b i t o r a c t i v i t y was d e f i n e d as t h a t amount of i n h i b i t o r which gave $0% i n h i b i t i o n of the cont r o l a c t i v i t y o f 0 . 5 ng bovine p a n c r e a t i c RNase A under these c o n d i t i o n s and i s hence n u m e r i c a l l y e q u i v a l e n t t o the weight o f bovine p a n c r e a t i c RNase A 2.15  Protein  inhibited.  determination  P r o t e i n was measured a c c o r d i n g t o the method of Lowry 29  et a l .  k  u s i n g bovine serum albumin as standard.  I I I . EXPERIMENTAL RESULTS  3 . 0 C h a r a c t e r i s t i c s o f the RNase A c t i v i t i e s o f A d u l t Rat Whole B r a i n Homogenates and E x t r a c t s I n i t i a l experiments were aimed a t d e t e c t i n g RNase a c t i v i t i e s i n a d u l t r a t whole b r a i n homogenates and e x t r a c t s , and determining the e f f e c t on these  activities  of such v a r i a b l e s as the medium used f o r homogenization o f the t i s s u e , and the pH, b u f f e r , and i o n i c s t r e n g t h of the i n c u b a t i o n medium.  These experiments y i e l d e d i n f o r m a t i o n  as to some g e n e r a l c h a r a c t e r i s t i c s o f the RNase enzyme system i n b r a i n and p e r m i t t e d s e l e c t i o n of o p t i m a l c o n d i t i o n s of assay f o r the RNase a c t i v i t i e s which were d e t e c t e d . 3 . 0 1 E f f e c t s o f pH and b u f f e r system on the RNase a c t i v i t i e s assayed i n i s o t o n i c sucrose homogenates F i g u r e 1 shows three r e p r e s e n t a t i v e curves o f the RNA-depolymerizing a c t i v i t y assayed i n f r e s h l y prepared r a t whole b r a i n .  a t d i f f e r e n t pH values  i s o t o n i c sucrose homogenates o f a d u l t  RNase a c t i v i t y i s d e t e c t a b l e  throughout  the e n t i r e range o f hydrogen i o n c o n c e n t r a t i o n s t e s t e d w i t h Tris-HCl buffer.  P o o r l y d e f i n e d a c t i v i t y maximae  betwen pH 6 t o 7 and between pH 8 t o 9 » a c t i v i t y expressed  R e l a t i v e to the  i n .05 M T r i s - H C l , equimolar  t i o n s of imidazole-HCl b u f f e r y i e l d 6 5 $ higher -  66  -  occur  concentraactivity  - 67 -  .5 O  CD  ° "5 .4  .y a  M—  a v> CO  .3  c  \ ~  .2  V  ~A  .1 JL_i  7  L.  J__J  1_  8  10  PH  FIGURE 1. The e f f e c t o f pH on the a c t i v i t y o f RNase i n i s o t o n i c sucrose homogenates o f a d u l t r a t whole b r a i n .  A l i q u o t s o f 0.1 ml were assayed from a homogenate c o n t a i n i n g 11.3 gprotein/ml. I n c u b a t i o n was f o r 60 minutes. The b u f f e r systems used were T r i s - H C l —A— , NHty-acetate —<5ri m i d a z o l e - H C l --+-- , and the f i n a l b u f f e r c o n c e n t r a t i o n was 50 mM i n a l l experiments u n l e s s s t a t e d o t h e r w i s e . m  -  6.6.  a t pH  68  -  about 3 0 % lower with e q u i -  A c t i v i t y l e v e l s are  molar c o n c e n t r a t i o n s of NHif-acetate b u f f e r maxima i s s h i f t e d to more a c i d pH  and  the a c t i v i t y  values.  Since homogenization i n i s o t o n i c sucrose i s most l i k e l y to p r e s e r v e the o r g a n e l l e s i t may  be  s t r u c t u r a l i n t e g r i t y of i n t r a c e l l u l a r  i n f e r r e d that  the  l e v e l of RNase a c t i -  v i t y found under these c o n d i t i o n s r e f l e c t s the a c c e s s i b i l i t y of the  enzymes to the  p a r t i c l e s are  added s u b s t r a t e when the  s t i l l relatively intact.  The  subcellular  amount of RNase  a c t i v i t y expressed i n i s o t o n i c sucrose homogenates i s about 50,  ? 6 , and  of the X100  6 8 per  cent a t pH  t o t a l extractable  homogenates.  6 . 7 » 7 * 8 and  9.5  respectively  RNase a c t i v i t y assayed i n 0.1%  Hence, i t may  be t e n t a t i v e l y concluded  under c o n d i t i o n s which most c l o s e l y approximate the state  of these enzymes, more of the  extractable  3.02  situ  t o t a l detergentacid  to added s u b s t r a t e .  r a t whole b r a i n i n  of the n o n - i o n i c detergent T r i t o n X100,  0.1%  which i s known  t o d i s r u p t , a n d s o l u b i l i z e l i p o p r o t e i n membranes, r e s u l t e d l e v e l s of RNase a c t i v i t y which were 5 0 , 24, h i g h e r at pH  that  S o l u b i l i z a t i o n of l a t e n t RNase a c t i v i t i e s by homogenization i n 0,1% T r i t o n X100  Homogenization of a d u l t (v/v)  in  a l k a l i n e RNase a c t i v i t y (as compared to  RNase) of b r a i n i s a c c e s s i b l e  Triton  6.7,  7 . 8 , and  9*5 respectively  expressed i n i s o t o n i c sucrose homogenates.  and  3 2 per  in  cent  than t h a t The  0,1%  Triton  - 69  homogenate and the 0.1$  -  T r i t o n e x t r a c t prepared from i t  a c c o r d i n g t o the procedure d e s c r i b e d Methods e x h i b i t e d n e a r l y a c t i v i t y curves.  i n s e c t i o n 2.11  of  congruent pH v e r s u s RNase  Ninety-five  per cent of the t o t a l RNase  a c t i v i t y measured i n T r i t o n homogenates was  consistently  r e c o v e r e d i n the e x t r a c t s .  This observation  indicates  t h a t homogenization  T r i t o n XI00 was  effective i n  producing complete  i n 0.1$  c e l l breakage  and i n r e l e a s i n g i n  s o l u b l e form any a c t i v e RNase bound t o or sequestered w i t h i n sedimentable i n t r a c e l l u l a r p a r t i c l e s . The a l k a l i n e t o a c i d RNase a c t i v i t y r a t i o i s cons i d e r a b l y lower f o r the 0.1$  T r i t o n X100  extract  (see  F i g . 2 on page 72) compared t o t h a t f o r the i s o t o n i c sucrose homogenate, thus i n d i c a t i n g t h a t homogenization  of  the t i s s u e i n detergent as compared with i s o t o n i c sucrose r e s u l t e d i n the e x t r a c t i o n of r e l a t i v e l y more a c i d RNase a c t i v i t y than a l k a l i n e RNase a c t i v i t y . between the l e v e l s o f measurable two homogenization  conditions  The l a r g e  difference  RNase a c t i v i t y under these  suggests t h a t a l a r g e f r a c t i o n  of the t o t a l RNA-degrading c a p a c i t y  of b r a i n c e l l s may  be  p r e s e n t i n s i t u i n an i n a c t i v e or l a t e n t s t a t e adsorbed to or compartmentalized  within i n t r a c e l l u l a r  organelles.  However, these r e s u l t s do not permit any statement as t o the r e l a t i v e c o n t r i b u t i o n of these two p o s s i b l e i n s i t u  -  70 -  enzyme s t a t e s to the observed l a t e n c y i n enzyme a c t i v i t y . A l s o , no study was made o f whether the r a t i o o f RNase a c t i v i t y i n sucrose homogenates v a r i e s with developmental age. T r i t o n most l i k e l y a c t s t o r e l e a s e and a c t i v a t e those enzymes present  i n a l a t e n t , non-functional  s t a t e by s o l u b i -  l i z i n g i n t r a c e l l u l a r l i p o p r o t e i n membrane s t r u c t u r e s .  The  p o s s i b i l i t y t h a t Triton-enhancement o f expressed l e v e l s o f RNase a c t i v i t y may be p a r t i a l l y due t o a more d i r e c t a c t i o n on the enzyme molecules p e r se or t o i n a c t i v a t i o n o f i n h i b i t o r s of RNase a c t i v i t i e s has not been excluded.  A study  o f the e f f e c t o f T r i t o n X100 on the p a r t i a l l y p u r i f i e d enzymes i n d i c a t e d 50$ s t i m u l a t i o n o f a c i d RNase a c t i v i t y and no s i g n i f i c a n t e f f e c t on a l k a l i n e RNases assayed a t t h e i r pH optima (see Table V I I , page i l l  .  respective  Since the e f f e c t o f  added T r i t o n on the assay o f RNase a c t i v i t y i n more crude but s o l u b l e enzyme p r e p a r a t i o n s  (such as hypotonic b u f f e r  extracts  prepared without detergent) was not determined, i t must be considered  t h a t T r i t o n may a c t a t l e a s t i n p a r t by d i s s o c i a t i n g  or p r e v e n t i n g  the formation  o f n o n - s p e c i f i c molecular aggre-  gates i n v o l v i n g RNase enzymes, and by m i n i m i z i n g the i n t e r a c t i o n o f added s u b s t r a t e w i t h n o n - s p e c i f i c I n summary, the p r e c e d i n g the importance o f ensuring  proteins.  r e s u l t s c l e a r l y demonstrate  complete d i s r u p t i o n o f i n t r a -  c e l l u l a r membrane compartment i f the f u l l RNase c a p a c i t y  - 71 -  of b r a i n t i s s u e i s t o be measured.  I n v e s t i g a t o r s o f the  components o f the RNase enzyme system i n r a t l i v e r have reported  s i m i l a r observations  i n t h a t maximal RNase  a c t i v i t y c o u l d o n l y be detected  upon d i s r u p t i o n o f i n t r a -  c e l l u l a r o r g a n e l l e membranes by hypotonic shock5 tion,  296 7  repeated freeze-thaw c y c l e s ,  detergent treatment.  sonica-  196 214 * or non-ionic  7  204 295 20*4 • Rahman, f o r instance, y  j  found  t h a t a c i d RNase i n r a t l i v e r i s o t o n i c sucrose homogenates e x h i b i t e d only 5 t o 10% o f the t o t a l a c i d RNase a c t i v i t y e x t r a c t a b l e with 0.1% T r i t o n X100.  He a l s o r e p o r t e d  that  RNase a c t i v i t i e s assayed a t both pH 7»8 and pH 9*5 were e q u a l l y a c t i v a t e d about 60% by 0.1% T r i t o n X100. 1 3.03 E f f e c t s o f pH, b u f f e r system, i o n i c s t r e n g t h , and NaCl on RNase a c t i v i t i e s assayed i n 0.1% T r i t o n X100 e x t r a c t s Buffer-dependent d i f f e r e n c e s i n the a c t i v i t y and pH optimum o f RNA-depolymerizing enzymes assayed i n a f r e s h l y prepared 0.1% T r i t o n X100 e x t r a c t o f a d u l t r a t whole b r a i n are shown i n F i g u r e 2.  The pH optimum o f the a c i d RNase  a c t i v i t y v a r i e s w i t h the b u f f e r system used,  A distinct  pyramidal a c t i v i t y maxima i s observed a t pH 6 . 7 w i t h both imidazole-HCl  and T r i s - H C l b u f f e r s , and a t pH 6 , 4 and 5.9  w i t h NH4-acetate and Na-phosphate b u f f e r s r e s p e c t i v e l y .  - 72 -  FIGURE 2 . The e f f e c t of pH on the a c t i v i t y of RNase i n 0.1$ (w/v) T r i t o n X100 e x t r a c t s ^ a d u l t r a t whole b r a i n .  A J i q b o t s f o f 0 . 1 mi of a frfeshly prepared e x t r a c t c o n t a i n i n g 5»2 mg.protein/ml were assayed i n v a r i o u s b u f f e r systems as a f u n c t i o n o f the hydrogen i o n concent r a t i o n of the i n c u b a t i o n .mixture. I n c u b a t i o n was f o r 60 minutes. The b u f f e r systems used were Tris-H61 — • — , NH4a c e t a t e — A — , imidazole-HCl , and Na-phosphate --o-- .  -  73  -  Of the v a r i o u s b u f f e r s t e s t e d , maximal a c i d RNase a c t i v i t y was  obtained with i m i d a z o l e - H C l .  T r i s - H C l and  NH4-  a c e t a t e b u f f e r s y i e l d e d a c t i v i t i e s which were about 8 7 % and 6 9 % r e s p e c t i v e l y of t h a t obtained with i m i d a z o l e - H C l ,  The  at t h e i r r e s p e c t i v e pH optimae i s n e a r l y  l e v e l of a c t i v i t y  i d e n t i c a l i n equimolar phosphate b u f f e r s .  c o n c e n t r a t i o n s of T r i s - H C l and  Na-  However, i n Na-phosphate b u f f e r t h e r e  occurs with d e c r e a s i n g hydrogen i o n c o n c e n t r a t i o n a r a p i d f a l l to very low l e v e l s of a c t i v i t y by pH 6 . 7 » The  c o n c e n t r a t i o n o f the b u f f e r was  found  to have a  c o n s i d e r a b l e e f f e c t on both a c i d RNase a c t i v i t y and RNase a c t i v i t y assayed  a t pH  7 . 9 (Table I ) .  The  alkaline  activity  a c i d RNase d e c l i n e s and a s h i f t to more a c i d pH optima with i n c r e a s i n g i o n i c s t r e n g t h ( F i g . 3 ) » s t r e n g t h of an equimolar  The  higher  of  occurs  ionic  c o n c e n t r a t i o n of Na-phosphate b u f f e r ,  as compared with the monovalent b u f f e r systems t e s t e d , thus account f o r the more a c i d pH optimum obtained with  may this  buffer. I n view of the dependence o f a c i d RNase a c t i v i t y  and;.  pH optimum upon i o n i c s t r e n g t h , i t seems l i k e l y t h a t apparent d i s c r e p a n c i e s i n the maximal a c t i v i t y and pH optimum r e p o r t e d by s e v e r a l workers f o r t h i s enzyme i n other mammalian t i s s u e s i s due  t o d i f f e r e n c e s i n the i o n i c s t r e n g t h under which 204  a c t i v i t y was a pH  assayed.  optimum of 5*5  assayed  i n 200  mM  Thus, f o r example, Rahman  reported  f o r a c i d RNase from r a t l i v e r homogenate  acetate b u f f e r .  - 7  TABLE I .  k  -  EFFECT OF BUFFER CONCENTRATION ON RNASE ACTIVITY OF 0.1$ TRITON X100 EXTRACTS OF ADULT RAT WHOLE BRAIN Buffer system  F i n a l b u f f e r Increase i n A 2 6 0 per c o n c e n t r a t i o n (mM) 6 0 minute i n c u b a t i o n  6.4  Na-phosphate  20  .549  6.4  N.a-phosphate  50  .369  7.9  Na-phosphate  20  .167  7.9  Na-phosphate  50  .056  Tris-HCl  50  .266  .7.8  A s i m i l a r interdependence of i o n i c s t r e n g t h and  pH 297  optimum has been observed with bovine p a n c r e a t i c RNase A 298 and  bovine b r a i n a c i d DNase.  7  Low  i o n i c s t r e n g t h and  pH f a v o r the d e n a t u r a t i o n of p o l y n u c l e o t i d e  low  substrates.  I n c r e a s i n g the s a l t c o n c e n t r a t i o n or i o n i c s t r e n g t h , on other hand, by s h i e l d i n g the mutually  '  the  r e p e l l e d charged  phosphate groups of the p o l y n u c l e o t i d e c h a i n , s t a b i l i z e s  the  hydrogen-bonded secondary s t r u c t u r e and f a v o r s a more t i g h t l y c o i l e d double-stranded  configuration.  Thus, the  inhibitory  e f f e c t on enzyme a c t i v i t y of i n c r e a s i n g i o n i c s t r e n g t h be p a r t i a l l y ,  though not completely,  c u r r e n t l y d e c r e a s i n g the pH, stranded  thereby  counteracted  by  may  con-  maintaining a s i n g l e -  form of the p o l y n u c l e o t i d e s u b s t r a t e .  Assuming  t h i s to be the primary mechanism of a c t i o n of s a l t , i t can . be i n f e r r e d t h a t a c i d RNase, l i k e bovine p a n c r e a t i c RNase A, p r e f e r e n t i a l l y a t t a c k s s i n g l e - s t r a n d e d sequences of  RNA.  - 75 -  Molarity  of  NaCl  FIGURE 3. The e f f e c t o f NaCl on a c i d RNase  activity,  A l i q u o t s o f 0 . 1 ml o f 0 . 1 % T r i t o n X 1 0 0 e x t r a c t s o f a d u l t r a t whole b r a i n were assayed (30 minute i n c u b a t i o n ) at the f o l l o w i n g pHi NH-4-acetate, p H 4 — o — ; NH4-acetate, p H 5 —w— ; NH4-acetate, pH.6—v— ; T r i s - H C l , p H 7 "+*- . The a c t i v i t y assayed i n T r i s - H C l a t pH 6 . 4 i n the absence o f added NaCl i s a l s o shown , RNase a c t i v i t y i s expressed as the i n c r e a s e ( c o r r e c t e d f o r s u b s t r a t a and.enzyme b l a n k s ) i n A 2 6 0 per 3° minute incubation.  -  I n a s i n g l e experiment,  76  -  i t was indeed found t h a t the a c t i v i t y  of a p a r t i a l l y p u r i f i e d a c i d RNase p r e p a r a t i o n was 3 0 % h i g h e r w i t h heat-denatured r a t b r a i n n u c l e a r RNA than with n a t i v e n u c l e a r RNA.  However, u n t i l t h i s p u t a t i v e "explanation i s  more c o n c l u s i v e l y t e s t e d , the d i r e c t e f f e c t o f i o n i c s t r e n g t h on enzyme conformation, s t a b i l i t y and s o l u b i l i t y must a l s o be taken i n t o c o n s i d e r a t i o n i n attempting t o account f o r the observed i o n i e - s t r e n g t h - d e p e n d e n t changes i n the a c t i v i t y and pH optimum o f a c i d RNase. In the a l k a l i n e r e g i o n , the pH-RNase a c t i v i t y  profile  o b t a i n e d with T r i s - H C l b u f f e r ( F i g . 2) has a composite p a t t e r n w i t h the appearance 7.5  and  9«5»  o f s e v e r a l superimposed  curves between pH  T h i s p r o f i l e i s s u g g e s t i v e o f the presence of a t  l e a s t two a l k a l i n e RNase a c t i v i t i e s with o v e r l a p p i n g pH c u r v e s . Phosphate and i m i d a z o l e b u f f e r s y i e l d very low a c t i v i t i e s i n the a l k a l i n e pH range w i t h i n which they r e t a i n  effective 213  buffering capacity.  T h i s i s p o s s i b l y due t o the known  J  c h e l a t i n g a c t i v i t y o f these b u f f e r s which may s t a b i l i z e the pH 7,8  R N a s e - i n h i b i t o r complex o r prevent the i n a c t i v a t i o n o f  RNase i n h i b i t o r by metal i o n s .  Such an i n t e r p r e t a t i o n i s  suggested by t h e o b s e r v a t i o n s t h a t (1) EDTA does not apprec i a b l y suppress RNase a c t i v i t y assayed a t pH 7 . 8 i n Na-phosphate b u f f e r , and (2) the l e v e l o f RNase a c t i v i t y assayed a t pH 7 . 8 i n the presence o f 0.2 mM pCMB i s the same i n both T r i s - H C l  -  77  and Na-phosphate "buffers (Table and  imidazole  -  II).  A l t e r n a t i v e l y phosphate  ions may i n t e r a c t w i t h the a l k a l i n e RNase,  whose a c t i v i t y i s normally expressed i n t h i s pH range when assayed with T r i s - H C l b u f f e r , t o produce a l e s s a c t i v e mation of the enzyme molecule.  confor-  I t may be worth n o t i n g f o r  the purpose o f comparison t h a t bovine p a n c r e a t i c  RNase A, a  299 b a s i c p r o t e i n with p l = 7 . 8 ,  i s known  7  t o have a s t r o n g  a f f i n i t y f o r m u l t i v a l e n t anions such as orthophosphate. TABLE I I . EFFECT OF pCMB AND EDTA ON RNASE ACTIVITY ASSAYED IN 0.1$ TRITON X100 EXTRACTS OF ADULT RAT WHOLE BRAIN  pH  Reagent p r e s e n t a t I n c r e a s e i n A260 p e r B u f f e r system* ffnailconceniftfration 6 0 minute i n c u b a t i o n  7 . 8 Tris-HCl  .266  7 . 9 Na-phosphate  .049  7 . 9 Na-phosphate  EDTA ( l t O mM)  .029  7 . 9 Na-phosphate  EDTA ( 2 . 0 mM)  .044  7 . 8 Tris-HCl  pCMB ( 0 . 2 mM)  .687  7 . 9 Na-phosphate  pCMB ( 0 . 2 mM)  .692  • F i n a l buffer concentration Several and  i n a l l cases was 5 0 mM.  other b u f f e r systems (Tes-H61, glycine-NaOH,  Na2C03-NaHC03) with e f f e c t i v e b u f f e r i n g c a p a c i t i e s i n the  a l k a l i n e pH range were found t o y i e l d very low RNase a c t i v i t i e s compared with T r i s - H C l .  - 78 3.04 Evidence i n d i c a t i n g the presence of a protein i n h i b i t o r of pH 7»8 RNase a c t i v i t y i n brain 3.041 Time-dependent a c t i v a t i o n of pH 7.8 RNase a c t i v i t y i n stored enzyme preparations A time-dependent  a c t i v a t i o n of RNase a c t i v i t y assayed  at pH 7»8 was observed upon storage of f r e s h l y prepared enzyme preparations at 0°. RNase a c t i v i t y assayed at pH 7«8 reached a maximum a f t e r about 6 days storage of 0.1% T r i t o n X100 extracts at 0°, and subsequently remained r e l a t i v e l y constant up to 14 days.  A c t i v i t y a f t e r 6 days storage at 0° was 86%  greater than the a c t i v i t y assayed i n freshly prepared extracts.  Figure 14 on page 138  shows the pH versus RNase  a c t i v i t y p r o f i l e s of a cytosol f r a c t i o n assayed immediately upon preparation and a f t e r two weeks storage at 0°. Maximal a c t i v a t i o n of RNase a c t i v i t y i s observed at about pH 8,3 and the a c t i v i t y at t h i s pH i s 476% above that of f r e s h l y prepared cytosol.  A 10% increase i n RNase a c t i v i t y assayed at  pH6.7 also occurred i n the l4 day old cytosol. 3.042 I n h i b i t i o n of bovine pancreatic RNase A c a c t i v i t y by brain extracts Freshly prepared extracts i n h i b i t bovine pancreatic RNase A a c t i v i t y and the degree of i n h i b i t i o n i s proport i o n a l to the amount of extract added. Parachloromercuribenzoate prevented the i n h i b i t i o n of bovine pancreatic RNase A a c t i v i t y by f r e s h l y prepared extracts.  - 79 -  3.043 A c t i v a t i o n by pCMB o f RNase a c t i v i t y assayed a t pH 7»8 The  s u l f h y d r y l b l o c k i n g reagent, p a r a c h l o r o m e r c u r i -  benzoate (pCMB), a c t i v a t e s RNase a c t i v i t y i n f r e s h l y prepared e x t r a c t s assayed a t pH 7*8 and the degree o f a c t i v a t i o n i s p o s i t i v e l y c o r r e l a t e d with the e x t r a c t ' s bovine p a n c r e a t i c  RNase A a c t i v i t y .  decline i n the capacity  capacity  to i n h i b i t  The time-dependent  o f e x t r a c t s t o i n h i b i t bovine pan-  c r e a t i c RNase A a c t i v i t y was accompanied by a concomitant d e c l i n e i n the c a p a c i t y  o f pGMB t o s t i m u l a t e  the endogenous  pH 7.8 RNase a c t i v i t y . The  e f f e c t s o f pCMB are most l i k e l y explained  by ( l )  i t s i n a c t i v a t i o n o f a l a b i l e endogenous p r o t e i n i n h i b i t o r which depends on the i n t e g r i t y o f f r e e s u l f h y d r y l groups f o r i t s a c t i v i t y , and (2) the r e l e a s e a RNase-inhibitor  complex.  o f a c t i v e pH 7«8 RNase from  The p r o t e i n n a t u r e o f the i n h i -  b i t o r i s i n d i c a t e d by the f a c t s t h a t i t i s completely  inacti-  vated by h e a t i n g a t 100°, i t does not pass through d i a l y s i s membranes, and i t i s i n a c t i v a t e d by pGMB.  The i n s t a b i l i t y  of the i n h i b i t o r i s i n d i c a t e d by the time-dependent  increase  i n pH 7«8 RNase a c t i v i t y observed upon s t o r a g e o f e x t r a c t s and  the concomitant decrease i n pCMB s t i m u l a t i o n o f pH 7.8  RNase a c t i v i t y i n s t o r e d The during  extracts.  time-dependent a c t i v a t i o n o f pH 7»8 RNase a c t i v i t y  storage o f e x t r a c t s could be prevented by 1.0 mM EDTA  - 80 -  a p p a r e n t l y due t o the s t a b i l i z a t i o n t h i s reagent. stabilized  The i n h i b i t o r  o f RNase i n h i b i t o r by  a c t i v i t y c o u l d a l s o be  by B-mercaptoethanol  or d i t h i o t h r e i t o l .  These  s u l f h y d r y l r e d u c i n g agents were subsequently found t o have no d e t e c t a b l e e f f e c t activity  on the p a r t i a l l y p u r i f i e d  pH 7 . 8 RNase  (see Table VI, page 106). 3,04'+ E f f e c t o f EDTA on RNase a c t i v i t y assayed a t pH 7 . 8  The e f f e c t  o f removing  t r a c e s o f heavy metal i o n s was  examined by adding the c h e l a t i n g agent, EDTA, t o i n c u b a t i o n mixtures o f f r e s h l y  prepared e x t r a c t s .  produce near t o t a l i n h i b i t i o n  o f RNase a c t i v i t y assayed a t  pH 7 . 8 i n 50 mM T r i s - H C l b u f f e r . inhibition  EDTA was found t o  was due t o augmentation  To determine whether o f RNase i n h i b i t o r  or t o the b i n d i n g o f d i v a l e n t c a t i o n s more d i r e c t l y  this activity  essential  f o r RNase f u n c t i o n , pH 7 . 8 RNase a c t i v i t y o f e x t r a c t s was measured i n t h e presence o f both 1 . 0 mM EDTA and 0 . 2 mM pCMB (Table I I I ) . The l e v e l o f pH 7 . 8 RNase a c t i v i t y o b t a i n e d under these c o n d i t i o n s was s l i g h t l y g r e a t e r than t h a t measured i n t h e presence o f 0 . 2 mM pCMB a l o n e .  The f a c t  t h a t pCMB can completely r e s t o r e the pH 7 . 8 RNase a c t i v i t y inhibited  by EDTA, combined w i t h the o b s e r v a t i o n (Table VI)  t h a t n e i t h e r pCMB nor EDTA a l t e r e d the a c t i v i t y o f p a r t i a l l y purified  pH 7 . 8 RNase p r e p a r a t i o n s i n d i c a t e s t h a t the e f f e c t  of these reagents i s p r i m a r i l y upon t h e a c t i v i t y o f RNase inhibitor.  EDTA thus appears t o a c t by l i b e r a t i n g  RNase  -  81 -  i n h i b i t o r from i n a c t i v e complexes with t r a c e s o f metal i o n s , the f r e e i n h i b i t o r thereby becoming a v a i l a b l e t o combine w i t h f r e e pH 7 . 8 RNase.  A c o r o l l a r y o f t h i s e x p l a n a t i o n o f the  mechanism o f EDTA a c t i o n i s t h a t i n h i b i t i o n o f bovine panc r e a t i c RNase A by the i n h i b i t o r endogenous t o e x t r a c t s i s g r e a t e r i n the presence o f EDTA than i n i t s absence (Table I V ) . TABLE I I I . ACTIVATION OF EDTA-INHIBITED DEAE-CELLULOSE ELUATE pH 7 . 8 RNase ACTIVITY BY pCMB Enzyme p r e p a r a t i o n * Reagent p r e s e n t a t added (mis) f i n a l concentration  Increase i n A 2 6 0 p e r 30 minute i n c u b a t i o n  .05  .066  .05  EDTA ( 0 . 5 mM)  .010  .05  EDTA ( 5 . 0 mM)  .035  .05  pCMB ( 0 . 2 mM)  .928  .05  EDTA ( 0 . 5 mM)+ pCMB (0.2 mM)  .97**  .05  EDTA ( 5 . 0 mM)+ pCMB ( 0 . 2 mM)  1.110  •The pooled D E A E - c e l l u l o s e e l u a t e pH 7 * 8 RNase a c t i v i t y ( f r a c t i o n s 7 - 6 5 i n F i g . 8 ) was d i a l y z e d a g a i n s t 2 0 mM T r i s - H C l , pH 7 . 4 b u f f e r and a l i q u o t s o f t h e d i a l y s a t e were assayed i n 5 0 mM T r i s - H G l , pH 7 . 8 b u f f e r . k  -  82 -  TABLE IV. ENHANCEMENT GF RNase INHIBITOR ACTIVITY BY EDTA ~~ — Addition to assay 0.5  ng  pancreatic RNase A  0.5  ng  pancreatic RNase A  Increase i n A260 per 3 0 minute incubation .703  EDTA  (1.0  mM)  cytosol* cytosol* EDTA 0.5  ng  pancreatic RNase A  cytosol*  0.5  ng  pancreatic RNase A  cytosol* EDTA  .703 .043  (1.0  mM)  .009 .493  (1.0  mM)  .358  •Aliquots (0.15 ml) of a 1/10 d i l u t i o n ( i n i c e - c o l d .02 M Tris-HCl, pH 7 . 8 buffer) of a cytosol f r a c t i o n f r e s h l y prepared from adult r a t whole brain were assayed. Assay conditions were as described i n section 2.144 of Methods. 3.045 Comparison of the effects of pCMB on RNase a c t i v i t i e s i n l i v e r and brain A comparison of the l e v e l of inhibitor-bound RNase i n the brain and l i v e r of adult rats was made by measuring the capacity of pCMB to stimulate the RNase a c t i v i t y from each organ.  This date i s shown i n Figures 4 and 5»  A plot of  the RNase a c t i v i t y as a function of pH showed that i n the absence of pCMB the s p e c i f i c a c t i v i t y of acid RNase and a l k a l i n e RNase of l i v e r was 3-fold and 5-fold greater respectively than that of brain.  When t o t a l pH 7 . 8 RNase  - 83 200 h 180  L  160 140 c o u c o —  ~  120  ? 100 u  ro 80 CD  3  6 0  z  CO OC 40 20  0 _L  7  8  10  P H  FIGURE k. The s t i m u l a t i o n by pCMB o f RNase a c t i v i t y i n 0.1% T r i t o n X100 e x t r a c t s o f a d u l t r a t whole b r a i n ,  A l i q u o t s o f 0 , 5 ml o f e x t r a c t were incubated f o r 60 minutes i n 50 mM T r i s - H C l b u f f e r . Per cent s t i m u l a t i o n was c a l c u l a t e d as the d i f f e r e n c e i n A260 between samples assayed a t a g i v e n pH with and without 0 . 1 mM pCMB d i v i d e d by the c e n t r a l a c t i v i t y i n the absence of pCMB.  - 83a -  FIGURE 5. The effect of pCMB on RNase activity in 0.1% Triton X100 homogenate of adult rat l i v e r . Aliquots of 0.05 ml of a 5% (wet weight/ l i v e r / f i n a l volume) homogenate were incubated for 30 minutes. The RNase activity, expressed in units/mg protein, was assayed at each pH without pCMB (NH4-acetate buffer—A—, Tris-HCl b u f f e r — ) and in the presence of 0.1 mM pCMB (NH4-acetate buffer--o-- , Tris-HCl buffer —o ) . The difference curve, • — , is expressed as per cent stimulation and per cent inhibition by pCMB of control RNase a c t i v i t y .  - 84 -  a c t i v i t y was assayed i n t h e presence o f 0.1 mM pCMB, t h e s p e c i f i c a c t i v i t y was about 3 - f o l d g r e a t e r f o r l i v e r than brain.  The a c t i v i t y r e l e a s e d by pCMB was, however, 95$  greater  i n b r a i n than l i v e r .  I n both organs, pCMB a c t i v a -  t i o n was g r e a t e s t around pH 7.5.  The pH-dependent e f f e c t  of pCMB may be due t o changes i n t h e r e a c t i v i t y and/or a c c e s s i b i l i t y t o pCMB o f the c r i t i c a l  s u l f h y d r y l groups a t  d i f f e r e n t pH. Thus, i n a d d i t i o n t o s t i m u l a t i n g enzyme a c t i v i t y , pCMB produces a change i n t h e shape o f the pH-RNase a c t i v i t y curve. al.  T h i s may be r e l a t e d t o the o b s e r v a t i o n  of Colter et  who mixed v a r i o u s d i l u t i o n s o f RNase i n h i b i t o r with a  standard  amount o f bovine p a n c r e a t i c RNase A.  The p H - a c t i v i t y  curve o f the u n i n h i b i t e d enzyme had a sharp optimum a t pH 7.8. As t h e r a t i o o f RNase i n h i b i t o r t o p a n c r e a t i c RNase A i n creased,  the p o s i t i o n o f the optimum s h i f t e d t o more a l k a l i n e  pH v a l u e s . pH  Hence, the apparent a c t i v i t y optimum a t about  8.5 observed i n i s o t o n i c sucrose homogenates ( F i g . 1) and  0.1$ T r i t o n X100 extract©, ( F i g . 2) o f b r a i n assayed without pCMB may be due t o the pH-dependence o f R N a s e - i n h i b i t o r complex f o r m a t i o n — m o r e u n i n h i b i t e d RNase a c t i v i t y  being  expressed a t more a l k a l i n e pH due t o d i s s o c i a t i o n o f inhibitor-bound  pH 7.8 RNase.  Between pH 6 and pH 7, pCMB c o n s i s t e n t l y RNase a c t i v i t y i n e x t r a c t s by 20 t o 30$.  This  stimulates stimulation  -  85  -  i s not due t o a d i r e c t e f f e c t on a c i d RNase, nor i s i t due to r e l e a s e o f l a t e n t a c i d RNase from an i n h i b i t o r - b o u n d form, s i n c e a c i d RNase a c t i v i t y assayed a t a l l s t a g e s o f p u r i f i cation  subsequent t o i t s s e p a r a t i o n from a l k a l i n e  a c t i v i t y was i n h i b i t e d  RNase  3 0 % by the same c o n c e n t r a t i o n s o f  pCMB (see F i g u r e 6 a , page  9 2 ).  Rather, t h i s  activation  most l i k e l y r e p r e s e n t s the net outcome o f d i r e c t  inhibition  of a c i d RNase a c t i v i t y by pCMB and the r e l e a s e from i n h i b i t i o n of pH 7 « 8 RNase a c t i v i t y which r e t a i n s  6 8 % of i t s a c t i v i t y  when assayed a t pH 6 . 7 (see F i g .  9 c , page 1 0 4 ) . A r e l a t e d 2 Oiio b s e r v a t i o n has been r e p o r t e d by Rahman who observed a 1 5 t o 2 0 % i n h i b i t i o n of RNase a c t i v i t y i n the a c i d pH range upon r e c o n s t i t u t i n g culate fractions  the supernatant and sedimentable p a r t i -  of r a t l i v e r i s o t o n i c  sucrose homogenates.  The i n h i b i t o r y e f f e c t of pCMB above pH 8 . 7 i s a l s o l i k e l y t o be a composite r e s u l t . residual  The expected i n c r e a s e i n  t  pH 7 . 8 RNase a c t i v i t y n o r m a l l y expressed a t t h i s  pH upon r e l e a s e from i n h i b i t o r i s masked by the s t r o n g direct inhibitory  e f f e c t of the s u l f h y d r y l  upon some other a l k a l i n e  b l o c k i n g reagent  RNase a c t i v i t y i n t h i s pH range.  This i n h i b i t i o n i s s i g n i f i c a n t l y greater f o r l i v e r than f o r brain. Maximal a c t i v a t i o n  of RNase a c t i v i t y i n b r a i n  extracts  assayed i n the pH range between 7«Q and 8 , 5 was obtained a t pGMB; c o n c e n t r a t i o n s o f 0 . 2 mM.  Higher concentrations of  -  pCMB had no f u r t h e r e f f e c t , direct  86  -  s u g g e s t i n g t h a t pCMB has no  s t i m u l a t o r y or i n h i b i t o r y  RNase a c t i v i t y p e r se.  effect  on t h e pH 7 » 8  T h i s was subsequently  confirmed  by measuring t h e e f f e c t o f pCMB on p a r t i a l l y p u r i f i e d pH 7 . 8 RNase a c t i v i t y (see Table VI, page1o6 )• 3 . 0 5 Summary comment on the v a r i a b l e s i n f l u e n c i n g the d e t e r m i n a t i o n o f RNase a c t i v i t i e s i n crude e x t r a c t s The workers  1 9  l i t e r a t u r e shows a marked l a c k o f agreement between  3.202,214,300  w  h  Q  e s t  i  m a  ted  the l e v e l s  and pH 7 * 8 RNase a c t i v i t i e s i n r a t l i v e r .  of a c i d  RNase  I n t h i s organ, the  pH 7 . 8 RNase t o a c i d RNase a c t i v i t y r a t i o obtained by R o t h  3 0 0  214 was 0 , 2 ; Shortman obtained a r a t i o f o r frozen-thawed homo202  genates o f 0 . 0 5 , and De Lamirande and A l l a r d r a t i o of 2 . 3 .  obtained a  I n the p r e s e n t study a pH 7 « 8 RNase t o a c i d  RNase a c t i v i t y r a t i o o f 0 . 3 was obtained f o r b r a i n e x t r a c t s , as compared t o 0 . 7 when 0 . 1 mM pCMB was p r e s e n t i n the assay. I t i s apparent of r e s u l t s  from the present study t h a t a wide spectrum  c o u l d be obtained r a n g i n g from no RNase i n h i b i t o r  and h i g h pH 7 « 8 RNase a c t i v i t i e s t o h i g h i n h i b i t o r and no pH 7 * 8 RNase a c t i v i t y depending on t h e care taken t o a v o i d inhibitor inactivation t i o n and assay.  i n the course o f homogenate p r e p a r a -  A l s o , as p r e v i o u s l y mentioned, d i f f e r e n c e s  i n such parameters as b u f f e r system, i o n i c s t r e n g t h , and t h e extent o f r u p t u r e o f s u b e e l l u l a r o r g a n e l l e s do a f f e c t the  - 8? -  apparent l e v e l o f RNase a c t i v i t i e s measured i n v i t r o . another reason  f o r the marked d i s c r e p a n c i e s i n the  values and a c t i v i t y r a t i o s obtained  by d i f f e r e n t  g a t o r s i s the l a c k of s t a n d a r d i z a t i o n of the  Yet  absolute  investi-  RNA-precipitating  202 agent used to stop the r e a c t i o n .  Thus, i t i s o f t e n  c u l t to compare the r e s u l t s r e p o r t e d by d i f f e r e n t  diffi-  investi-  g a t o r s s i n c e i n the absence of common bases of r e f e r e n c e  no  r e l i a b l e b a s i s f o r comparison e x i s t s . 3.1 S e p a r a t i o n and B a r t i a l P u r i f i c a t i o n of the Components of the Multi-enzyiaeI n h i b i t o r System of A d u l t Rat Whole B r a i n 0.1% T r i t o n X100 E x t r a c t s The p r e c e d i n g  experiments r e v e a l e d the presence i n r a t  whole b r a i n homogenates and  e x t r a c t s of a t l e a s t two  ribonu-  c l e a s e s d i s t i n g u i s h a b l e by the pH a t which they e x h i b i t optimal activity.  The  a l k a l i n e RNase a c t i v i t y r e f e r r e d to as pH  RNase appears t o be present  7»8  l a r g e l y i n a l a t e n t form bound t o  a protein inhibitor. Although i t i s important  to c h a r a c t e r i z e enzyme a c t i -  v i t i e s under c o n d i t i o n s which most c l o s e l y approximate t h e i r i n s i t u s t a t e i n order to permit experimental  e x t r a p o l a t i o n of i n v i t r o  r e s u l t s to the i n v i v o c o n d i t i o n , the  g e n e i t y of RNase a c t i v i t i e s present b r a i n complicates  hetero-  i n crude e x t r a c t s o f r a t  the i n t e r p r e t a t i o n of r e s u l t s obtained  such a complex mixture of molecular  components.  An  using  attempt  - 88 -  was, t h e r e f o r e , made t o separate the components o f the RNase enzyme system from each o t h e r and from other p r o t e i n s by d i f f e r e n t i a l p r e c i p i t a t i o n with ammonium s u l p h a t e f o l l o w e d by a n i o n exchange column chromatography of t h e ammonium sulphate p r e c i p i t a b l e  fractions.  3«11 Ammonium sulphate f r a c t i o n a t i o n o f 0 . 1 % T r i t o n X100 e x t r a c t s o f a d u l t r a t whole b r a i n T r i t o n X100 ( 0 . 1 % ) e x t r a c t s o f a d u l t r a t whole b r a i n were f r a c t i o n a t e d with powdered ammonium sulphate a c c o r d i n g to t h e procedure  d e s c r i b e d i n s e c t i o n 2 . 1 3 1 of Methods.  The  ammonium sulphate f r a c t i o n a t i o n data recorded i n T a b l e V shows t h a t t h i s procedure  effected a clear separation of  a c i d and a l k a l i n e RNase a c t i v i t i e s . A c o n s i d e r a b l e l o s s of pH 6 . 7 RNase a c t i v i t y  occurs  d u r i n g the ammonium sulphate f r a c t i o n a t i o n and subsequent removal o f the s a l t by d i a l y s i s .  T o t a l per cent r e c o v e r y o f  pH 6 . 7 RNase a c t i v i t y i n a l l the d i a l y z e d ammonium f r a c t i o n s was c o n s i s t e n t l y about 6 0 % .  The p r o t e i n f r a c t i o n p r e c i p i -  t a t i n g between 7 5 and 100% ammonium sulphate s a t u r a t i o n c o n t a i n e d the h i g h e s t s p e c i f i c a c t i v i t y and accounted f o r n e a r l y 7 0 % o f the t o t a l r e c o v e r e d pH 6 . 7 RNase a c t i v i t y .  TABLE V. RECOVER*OF RNase ACTIVITIES IN AMMONIUM SULPHATE PRECIPITABLE FRACTIONS*  Enzyme preparation :  0.1% Triton X100 extract 0-25% saturated (NHJ^)2S0i». fraction  RNase activity assayed at pH 7.8 in the RNase activity RNase activity RNase activity presence of assayed at pH 6 . 7 assayed at pH 9 . 5 assayed at pH 7 . 8 0.2 mM pCMB Total % Re"Total % ReTotal % ReTotal % Reactivity covery activity covery activity covery activity covery 1,104.0  13.2  25-55% saturated (NH/f)2S0^ fraction 157.5 55-75% saturated (NH4)2S0/| fraction 65.4 75-100% saturated (NHif)2S04 fraction 424.2 % of i n i t i a l activity recovered in a l l (NHif)2S04 fractions ,  100  1.2  591.1  15.4  12.5  687.8  5.9  28.9  38.4  5,8.0  69.8  100  2.6  116.4 4.9  11.8  1.35»7,  4l4.0  100  920.0  100  Calculated latent RNase activity at pH 7 . 8 Total fo Reactivity covery 506.0  11.7  2.8  389.6  94.1  948.6  2.7  0.6  42.4  4.6  39.7  7.8  2.0  0.4  51.5  5.5  49.5  9.8  97.9,  10.4  ,  0.0  0.0  103.1 559.0  110.5  1.1  114.3  |  128.1  •An i n i t i a l volume of 230 ml 0.1% Triton X100 extract of adult rat whole brain was fractionated with (NHJ-JoSOi-. according to the procedure described in section 2.131 of Methods. After removal of the salt By dialysis against .02 M Tris-HCl, pH 7.4 buffer the fractions obtained were assayed in . 0 5 M Tris-HCl buffer for RNase activity at pH 7.8 with and without 0.2 mM pCMB, and at pH 6 . 7 and pH 9 . 5 . Latent pH 7 . 8 RNase activity is the difference in RNase activity at pH 7 . 8 assayed with and without pCMB. RNase activity is expressed in units as defined in section 2.141 of Methods  00  CD  -  90  -  T o t a l r e c o v e r y o f pH 9 . 5 RNase a c t i v i t y was g e n e r a l l y h i g h e r but somewhat more v a r i a b l e , r a n g i n g from  75  to  135$•  About 8 5 $ ' o f the t o t a l recovered pH 9 . 5 RNase a c t i v i t y as w e l l as the h i g h e s t s p e c i f i c ammonium sulphate f r a c t i o n  a c t i v i t y was found i n the  precipitating  between 2 5 and 3 5 $  saturation. Most o f the f r e e pH 7 . 8 RNase a c t i v i t y without pCMB) as w e l l as the l a t e n t , RNase a c t i v i t y  (assayed  i n h i b i t o r - b o u n d pH 7 « 8  ( t h e d i f f e r e n c e between the t o t a l pH 7 . 8 RNase  a c t i v i t y assayed i n the presence o f 0 . 2 mM pCMB and the f r e e pH 7 . 8 RNase a c t i v i t y assayed without pCMB) was a l s o r e covered i n t h e 2 5 t o 5 5 $ s a t u r a t e d fraction.  ammonium sulphate  T o t a l r e c o v e r i e s o f both f r e e and t o t a l pH 7 . 8  RNase a c t i v i t y ranged between 7 0 and 1 0 0 $ . Because the c a p a c i t y o f d i f f e r e n t ammonium sulphate f r a c t i o n  amounts of each  t o i n h i b i t the a c t i v i t y o f a  standard amount o f bovine p a n c r e a t i c RNase A was n o t measured, no q u a n t i t a t i v e statement can be made as t o t h e amount o f f r e e RNase i n h i b i t o r ammonium sulphate f r a c t i o n .  a c t i v i t y r e c o v e r e d i n each  However, f r e e RNase  a c t i v i t y appears t o be d i f f u s e l y fractions  25-55,  55-75,  and  distributed  75-100$  of these was capable o f i n h i b i t i n g A a c t i v i t y and t h i s i n h i b i t i o n  inhibitor  throughout  s a t u r a t i o n s i n c e each bovine p a n c r e a t i c RNase  c o u l d be prevented by 0 . 2 mM  - 91 pCMB.  T h i s o b s e r v a t i o n i s c o n s i s t e n t with Roth's r e p o r t  t h a t f r e e RNase i n h i b i t o r o f r a t l i v e r i s p r e c i p i t a t e d mostly  35-55$  between  ammonium sulphate s a t u r a t i o n w i t h some i n h i -  b i t o r a c t i v i t y p r e c i p i t a t i n g a t g r e a t e r than 60% s a t u r a t i o n . The  f a c t t h a t the t o t a l r e c o v e r y  of f r e e pH 7.8 RNase  a c t i v i t y was never g r e a t e r than 100% suggests t h a t the endogenous pH 7.8 R N a s e - i n h i b i t o r  complex was not d i s s o c i a t e d  d u r i n g ammonium sulphate f r a c t i o n a t i o n .  T h i s i n d i c a t e s the  f i r m n e s s o f the b i n d i n g between i n h i b i t o r and RNase. has r e p o r t e d  2 1 3  Shortman  t h a t s a l t c o n c e n t r a t i o n s up t o 0.3 M d i d not  s i g n i f i c a n t l y weaken the i n t e r a c t i o n between r a t l i v e r RNase i n h i b i t o r and bovine p a n c r e a t i c RNase A. I n summary, ammonium sulphate f r a c t i o n a t i o n does n o t e f f e c t any r e s o l u t i o n o f t h e f o l l o w i n g molecular  species 1  f r e e pH 7.8 RNase, f r e e RNase i n h i b i t o r , pH 7.8 RNasei n h i b i t o r complex, and pH 9«5 RNase.  However, i t succeeds  i n removing c o n s i d e r a b l e amounts o f i n a c t i v e p r o t e i n and i n s e p a r a t i n g the above components from t h e a c i d RNase activity  (see P i g s . 6a and 6 b ) .  FIGURE 6 a . T h e e f f e c t o f pH on the RNase a c t i v i t y p r e c i p i t a b l e between 7 5 and 100 per cent s a t u r a t i o n w i t h ammonium s u l p h a t e .  An i n i t i a l volume o f 160 ml o f a 0.1$ T r i t o n X100 e x t r a c t o f a d u l t r a t whole b r a i n was f r a c t i o n a t e d a c c o r d i n g to the procedure d e s c r i b e d i n M a t e r i a l s and Methods. The p r o t e i n f r a c t i o n p r e c i p i t a t i n g between 75-100S s a t u r a t i o n o f ammonium s u l p h a t e was d i s s o l v e d i n and d i a l y z e d a g a i n s t 20mM T r i s - H C l , pH 7 . b u f f e r and 20 u l a l i q u o t s o f the d i a l y s a t e were assayed i n 5 0 mM b u f f e r i n the absence (NH4acetate • { T r i s - H C l — • — ) and i n the oresence (NH4-acetate • s TrisHCl --O- ) o f 0.1 mM pCMB. k  - 93 -  1.3 1.2 c  0 o _o  1.1  V  1.0  **)  c  a  .9  C  Figure  i .8 o CO \ o •o CM  <  6 b  .7  .6 .5 .4 '  L  I  I  I  _  7  l  L 8  10  p H  FIGURE 6b.The e f f e c t of pH on the RNase a c t i v i t y p r e c i p i t a b l e between 25 and 55 p e r cent s a t u r a t i o n with ammonium sulphate.  The procedure f o r the p r e p a r a t i o n and assay of t h i s f r a c t i o n i s the same as t h a t d e s c r i b e d i n the legend t o F i g u r e 6 a . with the e x c e p t i o n t h a t 50 >uls o f the d i a l y s a t e was used f o r assay and i n c u b a t i o n was f o r 30 minutes. RNase a c t i v i t y was assayed i n 50 mM T r i s - H C l b u f f e r i n the absence — « — , and i n the presence --o---of 0.1 mM pCMB.  - 94 -  3 . 1 2 D E A E - c e l l u l o s e column chromatography of the 2 5 * 5 5 $ and the 7 5 - 1 0 0 $ s a t u r a t e d ammonium sulphate f r a c t i o n s An attempt was made t o achieve f u r t h e r s e p a r a t i o n and p u r i f i c a t i o n o f the c o n s t i t u e n t molecular  species of  the 7 5 t o 1 0 0 $ and the 2 5 t o 5 5 $ ammonium s u l p h a t e f r a c t i o n s by chromatography o f each o f these f r a c t i o n s on c columns o f D E A E - c e l l u l o s e a c c o r d i n g t o the procedure des c r i b e d i n s e c t i o n 2 . 1 3 2 o f Methods. F i g u r e 7 shows a t y p i c a l e l u t i o n p r o f i l e o f p r o t e i n and pH 6 . 7 RNase a c t i v i t y upon chromatography on DEAEcellulose of a tion. and  75-100$  Only one  s a t u r a t e d ammonium s u l p h a t e  frac-  RNase a c t i v i t y peak, e l u t i n g between 0 . 1  0 . 2 M NaCl, was d e t e c t e d i n the e l u a t e f r a c t i o n s . When the 2 5 t o 5 5 $  s a t u r a t e d ammonium sulphate  f r a c t i o n was chromatographed on a column o f D E A E - c e l l u l o s e two peaks o f RNase a c t i v i t y were detected i n t h e e l u a t e f r a c t i o n s ( F i g . 8 ) . The f i r s t enzyme peak c o i n c i d e s w i t h the unadsorbed p r o t e i n washed through equilibrating buffer.  the column with  The second enzyme peak was e l u t e d  at a NaCl c o n c e n t r a t i o n between 0,15 and 0 , 2 5 M„ e l u a t e f r a c t i o n s were assayed  When t h e  f o r t h e i r capacity to i n h i b i t  bovine p a n c r e a t i c RNase A a c t i v i t y , a broad peak o f i n h i b i t o r a c t i v i t y was d e t e c t e d e l u t i n g between 0 , 1 5 and 0 . 5 M NaCl and o v e r l a p p i n g the f r e e pH 7 , 8 RNase a c t i v i t y  O  20  40  60  Fraction  FIGURE 7.  80  IOO  120  Number  E l u t i o n p r o f i l e o f 75-100% s a t u r a t e d ammonium s u l p h a t e p r e c i p i t a b l e f r a c t i o n chromatographed on D E A E - c e l l u l o s e .  - 95 -  -  FIGURE  96  -  E l u t i o n p r o f i l e o f 7 5 - 1 0 0 $ s a t u r a t e d ammonium s u l p h a t e p r e c i p i t a b l e f r a c t i o n chromatographed on D E A E - c e l l u l o s e . 7.  The p r o t e i n f r a c t i o n p r e c i p i t a t i n g between 7 5 and 1 0 0 per cent s a t u r a t i o n with ammonium sulphate was d i a l y z e d a g a i n s t . 0 2 M T r i s - H C l , pH 6 . 9 b u f f e r , and 4 . 0 mis of the d i a l y s a t e c o n t a i n i n g 5 6 . 8 mg p r o t e i n and 1 5 2 u n i t s of RNase a c t i v i t y assayed a t pH 6 . 7 was loaded on a 1 cm by 1 5 cm column o f D E A E - c e l l u l o s e p r e - e q u i l i b r a t e d with 5 0 0 ml o f . 0 2 M T r i s - H C l , pH 6 . 9 b u f f e r . The sample was e l u t e d with a l i n e a r l y i n c r e a s i n g s a l t g r a d i e n t ( 2 5 0 ml T r i s - H C l , pH 6 . 9 t o 2 5 0 ml 1 . 0 M NaCl i n . 0 2 M T r i s - H C l , pH 6 . 9 ) . F r a c t i o n s o f 4 . 4 ml were collected. A l i q u o t s ( 0 . 2 ml) of the u n d i a l y z e l u a t e f r a c t i o n s were assayed f o r RNase a c t i v i t y a t pH 6 . 7 i n 5 0 mM T r i s - H C l b u f f e r . I n c u b a t i o n was f o r 6 0 minutes.  Absorbance  at  2 8 0 nm.  p •  o b  Ln  T  1  M  Q G  O  » W 00  -a w •1  M  a C o rt  o  H* H•tj O  t~ 3 <+  f» V cr 1 H* o n w> Ml M P  o o  01  0  Q o  O  Q  3 o  rt  zr  <D  * 1 Ox O 1 3 p) vj\  rt o cnn pCD V> rt CD P  •  .v.  -n  «+ •"•>  H-  A  ^  a o  3  Z  C  3  cr  o o  n  _ M O  a. c+  > o n o.  3 D  P  3  51 W3  —  o  1 t— O C O. S I- CO 1  e  c  o >d CQ ET co (a • rt  •  o  S o  U NaCl  °/o —J  o b  (M)  concentration I  •• L  inhibition of pancreatic RNase A activity —*1 l ro  'o  RNase  activity  -16  ( units / ml. )  -  o  -  98 -  FIGURE 8. E l u t i o n p r o f i l e of the 2 5 - 5 5 % s a t u r a t e d ammonium s u l p h a t e p r e c i p i t a b l e f r a c t i o n chromatographed on DEAE-cellulose. The p r o t e i n f r a c t i o n p r e c i p i t a t i n g between 2 5 and 5 5 p e r cent s a t u r a t i o n with ammonium sulphate was d i a l y z e d a g a i n s t . 0 2 M T r i s - H C l . pH 6 . 9 b u f f e r and 1 0 ml of the d i a l y s a t e c o n t a i n i n g 3 9 3 mg o f p r o t e i n was loaded on a 1 cm by 1 9 , 5 cm column o f D E A E - c e l l u l o s e p r e - e q u i l i b r a t e d w i t h . 0 2 M T r i s - H C l , pH 6 . 9 buffer. The sample was e l u t e d w i t h a l i n e a r l y i n c r e a s i n g s a l t g r a d i e n t (400 ml . 0 2 M T r i s HCl, pH 6.9 to 400 ml 1.0 M NaCl i n . 0 2 M T r i s - H C l , pH 6 . 9 ) . F r a c t i o n s o f 4,8 ml were c o l l e c t e d , A l i q u o t s (0.2 ml) o f the u n d i a l y z e d e l u a t e f r a c t i o n s were assayed immediately f o r RNase a c t i v i t y a t pH 9 * 5 —° and f o r f r e e RNase i n h i b i t o r a c t i v i t y . — • — , and on t h e f o l l o w i n g day f o r f r e e and t o t a l (with 0 . 2 mM pCMB)— - RNase a c t i v i t y a t pH 7.8. I n c u b a t i o n s i n a l l cases were f o r 6 0 minutes. Other d e t a i l s of the assay procedure were as d e s c r i b e d f o r t h e standard assay c o n d i t i o n i n M a t e r i a l s and Methods.  -  99  -  assayed i n the absence o f pCMB as w e l l as t h e t o t a l pH 7 . 8 RNase a c t i v i t y assayed i n t h e presence o f 0 . 2 mM pCMB. The wash-through RNase a c t i v i t y was found t o be v e r y unstable  and r a p i d l y l o s t a c t i v i t y upon storage i n t h e c o l d  room ( 4 ° ) .  T h i s a c t i v i t y was i n h i b i t e d  at pH 7 . 8 o r pH 9*5*  A t a f i n a l c o n e n t r a t i o n o f 0 . 2 mM,  pCMB p r o d u c e d 0 $ and 7 0 $ i n h i b i t i o n k  respectively.  by pCMB when assayed  The f a c t  a t pH 7 . 8 and pH 9 . 5  t h a t t h i s RNase a c t i v i t y  adsorbed on D E A E - c e l l u l o s e  i s not  columns e q u i l i b r a t e d a t pH 7 . 4  i n d i c a t e s t h a t i t has no n e t n e g a t i v e  charge a t t h i s pH and  i s hence a b a s i c p r o t e i n . f r e e pH 7 « 8 RNase a c t i v i t y recovered  The  i n the eluate  f r a c t i o n s was up t o 300% g r e a t e r than the amount loaded on the column.  This i s inferred  t o be due t o a  inactivation  o f the i n h i b i t o r  component o f pH 7 . 8 RNase-  inhibitor  preferential  complexes, as w e l l as t o t h e d i s s o c i a t i o n o f such  complexes and t h e p a r t i a l  s e p a r a t i o n o f f r e e pH 7 « 8 RNase  a c t i v i t y from f r e e i n h i b i t o r  a c t i v i t y d u r i n g the chroma-  tography. The  recovery  o f t o t a l pH 7 . 8 RNase a c t i v i t y  (assayed  i n t h e presence o f 0 . 2 mM pCMB) i n t h e e l u a t e f r a c t i o n s ranged between 7 5 and 1 0 0 $ o f t h e amount The  f r e e RNase i n h i b i t o r  f r a c t i o n s was very u n s t a b l e .  loaded.  a c t i v i t y i n the e l u a t e  Upon storage i n t h e c o l d room,  - 100  i t exhibited a rapid loss i n i t s capacity to i n h i b i t p a n c r e a t i c RNase A a c t i v i t y .  The p e r cent i n h i b i t i o n o f  bovine p a n c r e a t i c RNase A a c t i v i t y was h i g h e s t  a t pH 7*5»  thus i n d i c a t i n g t h a t the f r e e RNase i n h i b i t o r a c t i v i t y has a pH optimum o f  7»5»  3.2 P r o p e r t i e s o f the Three Separated RNase A c t i v i t i e s Three a p p a r e n t l y were thus separated  d i s t i n g u i s h a b l e RNase a c t i v i t i e s  from 0.1$ T r i t o n X100 e x t r a c t s by means  of ammonium sulphate  f r a c t i o n a t i o n followed  column chromatography.  by  DEAE-cellulose  I n order t o c o n c l u s i v e l y determine  whether these three enzyme f r a c t i o n s were d i s t i n c t enzymes and  t o f u r t h e r c h a r a c t e r i z e t h e i r p r o p e r t i e s , t h e e f f e c t of  v a r i o u s reagents on the a c t i v i t y o f the D E A E - c e l l u l o s e enzyme f r a c t i o n s was s t u d i e d . pH 7.8 RNase r e p o r t e d  eluate  The c h a r a c t e r i s t i c s o f the  i n the f o l l o w i n g s e c t i o n s  T a b l e s VI and V I I ) were determined u s i n g  ( P i g . 9c,  DEAE-cellulose  e l u a t e f r a c t i o n s which had been s t o r e d f o r some time and hence e x h i b i t e d no RNase i n h i b i t o r a c t i v i t y .  The absence  of RNase i n h i b i t o r a c t i v i t y was i n d i c a t e d by the f a i l u r e o f pCMB t o s t i m u l a t e the RNase a c t i v i t y i n these f r a c t i o n s and the i n a b i l i t y o f these enzyme f r a c t i o n s t o i n h i b i t bovine p a n c r e a t i c RNase A a c t i v i t y .  -  10.1 -  3.21 E f f e c t o f pH D E A E - c e l l u l o s e e l u a t e f r a c t i o n s with peak RNase a c t i v i t i e s were assayed a t v a r i o u s hydrogen i o n concent r a t i o n s i n 0,05 M b u f f e r . The pH optimum o f the D E A E - c e l l u l o s e e l u a t e a c i d RNase a c t i v i t y was found t o have s h i f t e d t o a more a c i d pH, with maximal a c t i v i t y now o c c u r r i n g a t pH 6.4 w i t h T r i s - H C l and a t pH 6 . 0 with NH*-. a c e t a t e b u f f e r s ( F i g . 9 a ) . T h i s a c t i v i t y was f r e e o f contamination by pH 7.8 RNase as i n d i c a t e d by the f a c t t h a t t h i s enzyme f r a c t i o n  exhibits  no a c t i v i t y when assayed a t pH 7 . 8 . The D E A E - c e l l u l o s e washwthrough RNase a c t i v i t y e x h i b i t s maximal a c t i v i t y above pH 9 and r e t a i n s 56% o f a c t i v i t y when assayed a t pH 7 . 8 .  The shoulder i n the pH  curve between pH 8 . 0 and 8.5 suggests t h a t t h i s enzyme f r a c t i o n may c o n s i s t o f a mixture enzyme s p e c i e s .  o f more than one d i s t i n c t  Further p u r i f i c a t i o n i s required to test  this possibility. The pH curve o f the D E A E - c e l l u l o s e e l u a t e pH 7.8 RNase a c t i v i t y i s shown i n F i g u r e 9 c .  T h i s enzyme e x h i b i t s  a broad but symmetrical pH p r o f i l e with a pH optimum o f about 7.7. pH 6 . 7 .  f f t ^ r e t a i n s 68% o f i t s a c t i v i t y when assayed a t  - 102 -  FIGURE 9a.The e f f e c t of pH on D E A E - c e l l u l o s e RNase a c t i v i t y .  eluate  acid  Peak a c t i v i t y f r a c t i o n s e l u t i n g between 0.1 and 0.25 M NaCl i n F i g u r e 7 were pooled, d i a l y z e d a g a i n s t .02 M T r i s - H C l , pH 7.4 b u f f e r , and the d i a l y s a t e was brought t o 30$ (v/v) g l y c e r o l . Aliquots (0.2 ml) of t h i s f i n a l p r e p a r a t i o n were assayed a t v a r i o u s pH i n .05 M N H 4 - a c e t a t e — , o r i n .05 M T r i s - H C l — — buffers. I n c u b a t i o n was f o r 60 minutes.  - 103 -  FIGURE 9 b . E f f e c t o f pH on D E A E - c e l l u l o s e wash-through RNase a c t i v i t y .  A l i q u o t s (0.2 ml) from f r a c t i o n number 4 i n F i g u r e 8 were assayed a t v a r i o u s pH i n .05 M T r i s - H C l buffer. I n c u b a t i o n was f o r 60 minutes.  - 104 -  FIGURE 9 c . E f f e c t o f pH on D E A E - c e l l u l o s e e l u a t e pH RNase a c t i v i t y .  7.8  Free pH 7.8 RNase a c t i v i t y e l u t e d between 0 . 1 and 0 . 2 M NaCl ( e l u a t e f r a c t i o n s 4 3 - 6 5 i n F i g u r e 8) were pooled and s t o r e d undial y z e d f o r one month a t 4 ° C p r i o r t o assay. A l i q u o t s o f 0 , 2 ml were assayed a t v a r i o u s pH i n . 0 5 M T r i s - H C l b u f f e r . I n c u b a t i o n was f o r 6 0 minutes.  - 105  3 . 2 2 E f f e c t of NaCl The effect of NaCl and buffer concentration on the DEAE-cellulose  eluate acid RNase a c t i v i t y was not de-  termined. DEAE-cellulose  eluate pH 9 . 5 RNase a c t i v i t y was  markedly i n h i b i t e d by NaCl as well as by increasing buffer concentration.  The i n h i b i t o r y effect of NaCl was much  greater than could be accounted f o r i n terms of an equivalent increase i n i o n i c strength. DEAE-cellulose  eluate pH 7.8 RNase a c t i v i t y was  stimulated by NaCl with maximal a c t i v i t y occurring at the NaCl concentration of 1 3 5 mM. than 180 mM were i n h i b i t o r y .  NaCl concentrations greater The stimulatory e f f e c t of  NaCl on pH 7.8 RNase a c t i v i t y can probably be completely accounted f o r i n terms of ionic strength since the increment i n enzyme a c t i v i t y i s nearly the same f o r both a 5 0 mM increase i n NaCl and a 5 0 mM increase i n buffer concentration. 3 . 2 3 E f f e c t of MgCl  2  Divalent cations were found to strongly i n h i b i t the a c t i v i t y of a l l three RNases.  pH 6.7 and pH 9 . ^ RNase  a c t i v i t i e s were completely i n h i b i t e d and pH 7.8 RNase h a l f i n h i b i t e d at a MgCl2 concentration of 5 mM.  However,  within a very narrow concentration range around 1 mM, Mg++  10& TABLE V I . EFFECT OF VARIOUS REAGENTS UPON DEAE' CELLULOSE ELUATE RNase ACTIVITIES F i n a l Concentration Reagent Added (mM)  j° o f C o n t r o l RNase A c t i v i t y Assayed i n 50 m T r i s - H C l buffer pH 6.7 RNase  Tris-HCl  25 50  100  100 -  124 100 66 68 45 24  5 10 25  40 100  NaCl  pH 9.5 RNase  180  75 46 16 23  7 1  9  4 2  25.0  EDTA  pCMB  0.5 1.0 2.0 2.5 10.0 .05  .1 .2  1.0  13 123  105  88 13  84 72 61  36  112  98  104  120  1.25  2.5 5.0 10.0 15.0  55 40 29 15  .3  Dithiothreitol  118  123  315  2  135  149 186 164  225 270  MgCl  64 100  129  135  0.5 1.0 2.5 5.0 10.0 15.0  pH 7.8 RNase  120  187 190 181  95  90 87  I  - 10? -  »ay  found t o s t i m u l a t e pH 9«5 RNase a c t i v i t y  (see s e c t i o n  i n f l u e n c e o f MgGl2 on the r e a c t i o n i s l i k e l y t o  The be complex.  Mg++ may a c t by d i r e c t l y a l t e r i n g the conforma-  t i o n o f the enzyme molecules o r by forming a c a t i o n  bridge  between enzyme and other p r o t e i n s , o r between p r o t e i n s and RNA.  I ti s known  3 0 1  "  3 0 3  that divalent eations  can con-  s i d e r a b l y a l t e r the s t r u c t u r e o f RNA molecules and thus a f f e c t the a c c e s s i b i l i t y o f s u b s t r a t e o f RNases. ** 30  bonds t o the a c t i o n  Thus, the mechanism o f Mg++ i n h i b i t i o n o f  RNase a c t i v i t y may be r e l a t e d t o the f a c t t h a t  Mg  + +  s t a b i l i z e s the secondary s t r u c t u r e o f RNA and may thereby maintain a substrate  c o n f i g u r a t i o n u n f a v o r a b l e t o enzymatic  attack. 3.24  E f f e c t o f EDTA  One mM EDTA a c t i v a t e d a c i d RNase a c t i v i t y assayed a t pH 6 . 7 by 2 3 % , whereas a t a c o n c e n t r a t i o n  o f 2 mM t h i s  reagent was s l i g h t l y i n h i b i t o r y . As  l i t t l e as 0.5 mM.EDTA produced near complete  s u p p r e s s i o n o f pH 9.5 RNase a c t i v i t y . reversed  T h i s e f f e c t c o u l d be  by r e s t o r i n g MgCl2 t o the r e a c t i o n mixture a t a  suitable concentration.  MgCl2 was then found t o s t i m u l a t e  t h i s enzyme a c t i v i t y by up t o 40% (see P i g . 1 0 ) .  This  enzyme thus appears t o have a d i v a l e n t c a t i o n requirement  - 108 -  160  0.5  1.0 MgCl2  1.5 (mM)  FIGURE 10. R e a c t i v a t i o n o f E D T A - i n h i b i t e d a c t i v i t y by Mg++.  N  2.0  pH 9 . 5 RNase  A DEAE-cellulose eluate f r a c t i o n containing peak pH 9 . 5 RNase a c t i v i t y ( f r a c t i o n number 5 i n F i g u r e 8 ) was assayed i n 5 0 mM T r i s - H C l , pH 9 . 5 b u f f e r . Various c o n c e n t r a t i o n s of MgC12 were added t o i n c u b a t i o n m i x t u r e s cont a i n i n g 1 . 0 mM EDTA and the enzyme f r a c t i o n . Reagents were added t o the i n c u b a t i o n mixture i n the f o l l o w i n g o r d e r i b u f f e r , EDTA, enzyme f r a c t i o n , MgC12, sRNA. I n c u b a t i o n was f o r 6 0 minutes. C o n t r o l pH 9 . 5 RNase a c t i v i t y was t h a t assayed i n the absence o f both EDTA and MgC12.  - 109 which can be met by Mg++. Hence, the previously observed i n h i b i t i o n by Mg++ of pH 9*5 RNase a c t i v i t y assayed without EDTA was due to the f a c t that t h i s requirement was already met by traces of divalent cations present i n the reaction mixture.  The addition of MgCl2 produced super-  optimal concentrations of divalent cation which resulted i n strong i n h i b i t i o n . EDTA has no s i g n i f i c a n t effect on free pH 7»8 RNase activity.  This supports the conclusion that the i n h i b i t o r y  effect of EDTA on RNase a c t i v i t y i n crude enzyme extracts assayed at pH 7.8 i s due to the s t a b i l i z i n g influence of t h i s chelating agent on RNase i n h i b i t o r a c t i v i t y . 3.25 E f f e c t of pCMB Parachloromercuribenzoate inhibited acid RNase a c t i v i t y about 40% and pH 9»5 RNase a c t i v i t y about 64% a t 0.2 mM concentration. Free pH 7.8 RNase a c t i v i t y was unaffected by up to 0.3 mM pCMB. 3.26 E f f e c t of 3-mercaptoethanol and d i t h i o t h r e i t o l D i t h i o t h r e i t o l stimulated acid RNase a c t i v i t y 20% at 1 mM.  Higher concentrations of either of these  reducing agents had no additional  effect.  sulfhydryl  The opposite  effects of pCMB and DTT on acid RNase a c t i v i t y are consistent  -  110 -  w i t h the c o n c l u s i o n t h a t t h i s enzyme c o n t a i n s f r e e h y d r y l group(s) which a r e r e q u i r e d f o r o p t i m a l  sulf-  enzymatic  activity, A s i m i l a r c o n c l u s i o n can be made with r e g a r d t o the pH 9*5 RNase a c t i v i t y .  The more s t r i k i n g e f f e c t s o f equal  c o n c e n t r a t i o n s of these reagents on the l a t e r enzyme i n d i c a t e s t h a t the c r i t i c a l s u l f h y d r y l groups i n v o l v e d a r e e i t h e r more r e a c t i v e and/or more e s s e n t i a l t o the a c t i v i t y o f t h i s enzyme i n comparison  w i t h a c i d RNase.  D i t h i o t h r e i t o l has no s i g n i f i c a n t e f f e c t on pH 7.8 RNase a c t i v i t y .  T h i s i s c o n s i s t e n t with i t s i n s e n s i t i v i t y  t o pCMB and suggests t h a t t h i s enzyme has no f r e e group requirements  sulfhydryl  or t h a t i t s s u l f h y d r y l groups a r e un-  r e a c t i v e and i n a c c e s s i b l e t o these reagents a t pH 7 « 8 . The l a c k of i n f l u e n c e o f DTT, pCMB and EDTA on p a r t i a l l y pH 7.8 RNase i n d i c a t e s t h a t the p r e v i o u s l y observed  purified effect  o f these reagents on RNase a c t i v i t y assayed i n crude enzyme p r e p a r a t i o n s a t pH 7-8 was mediated  through the a c t i o n o f  these reagents on RNase i n h i b i t o r . 3.27  E f f e c t o f detergents  A l l t h r e e RNase a c t i v i t i e s were almost i n h i b i t e d by the i n c l u s i o n of .005$ s u l p h a t e i n the assay mixtures  completely  (w/v) sodium l a u r y l  (Table V I I ) .  - I l l-  TABLE V I I . EFFECT OF VARIOUS DETERGENTS UPON DEAECELLULOSE ELUATE RNase ACTIVITIES Final Concentration {%}  Detergent Added  Sodium l a u r y l sulphatel Sodium desoxycholate T r i t o n XIOO  2  $ of C o n t r o l RNase A c t i v i t y assayed i n 50 mM T r i s - H C l buffer pH 7.8 RNase  pH 6.7 RNase  PH 9.5 RNase  .005  15  12  0  .025  18  4  0  .05  133  24  107  .10  43  11  113  .025  171  .10  153  106  95  108 108  F i n a l c o n c e n t r a t i o n g i v e n i n p e r cent  (weight/volume).  ^ F i n a l c o n c e n t r a t i o n g i v e n i n per cent  (volume/volume).  Sodium deoxycholate a t a f i n a l c o n c e n t r a t i o n o f . 9 5 $ (w/v) i n h i b i t e d  pH 9 . 5 RNase a c t i v i t y by 7 6 $ but s t i m u l a t e d  pH 6.7 RNase a c t i v i t y by 33$.  I n c r e a s i n g the sodium deoxy-  c h o l a t e c o n c e n t r a t i o n to 0 . 1 $ r e s u l t e d i n 57$ i n h i b i t i o n o f pH 6.7 RNase a c t i v i t y .  At these c o n c e n t r a t i o n s  c h o l a t e had no s i g n i f i c a n t  sodium deoxy-  e f f e c t on pH 7*8 RNase a c t i v i t y .  Both pH 7.8 RNase and pH 9.5 RNase a c t i v i t y was u n a f f e c t e d by 0 , 1 $ (v/v) T r i t o n X 1 0 0 , whereas pH 6.7 RNase a c t i v i t y was s t i m u l a t e d 71$ and 53$ by T r i t o n X100 a t conc e n t r a t i o n s of  .025$  (v/v) and  0.1%  (v/v) r e s p e c t i v e l y .  -  3.28  Effect  112  -  o f storage  Although crude enzyme p r e p a r a t i o n s r o u t i n e l y  stored  at 0° f o r up t o two weeks e x h i b i t e d no n o t i c e a b l e l o s s i n a c i d RNase a c t i v i t y , a t a l l subsequent stages o f p u r i f i c a t i o n t h i s enzyme was markedly more l a b i l e than pH 7.8 RNase activity. is  The l a b i l i t y o f pH 6.7 RNase a c t i v i t y from b r a i n  c o n s i s t e n t with o b s e r v a t i o n s o f t h i s enzyme a c t i v i t y i n 190 191 202  other organs by s e v e r a l i n v e s t i g a t o r s it  7  * -^ -* J  J  to be r e l a t i v e l y more heat- and a c i d - l a b i l e  RNase.  W  h o found  than pH 7.8  I n the p r e s e n t study, D E A E - c e l l u l o s e e l u a t e  fractions  c o n t a i n i n g peak pH 6.7 RNase a c t i v i t y l o s t Q$ o f t h e i r k  t i a l activity after HCl,  pH 7.  storage a t 0° f o r 1 1 days i n 20 mM T r i s -  b u f f e r c o n t a i n i n g 1 0 $ (v/v) g l y c e r o l .  k  ini-  However,  storage a t 0° f o r 1 3 days i n e i t h e r 20 mM NHk-acetate, pH 5 b u f f e r or i n 20 mM T r i s - H C l , (v/v)  glycerol  resulted  pH 6.7 b u f f e r c o n t a i n i n g 3 0 $  i n o n l y 8$ l o s s  in activity.  S t a b i l i z a t i o n o f t h i s enzyme a c t i v i t y c o u l d thus be achieved by  storage a t a c i d pH o r i n the presence o f 3 0 $ (v/v) g l y c e r o l . P a r t i a l l y p u r i f i e d pH 9 * 5 RNase a c t i v i t y was a l s o  found t o undergo r a p i d inactivation  inactivation  upon s t o r a g e .  This  appears t o be due t o the o x i d a t i o n o f f r e e  sulf-  h y d r y l groups s i n c e ,3-mereaptoethanol o r d i t h i o t h r e i t o l were a b l e t o r e a c t i v a t e t h i s enzyme and prevent i t s l o s s o f activity.  D E A E - c e l l u l o s e wash-through pH 9 « 5 RNase a c t i v i t y  - 113 stored at 4° f o r 14 days i n 20 mM Tris-HCl at pH 6.4, 7.0, 8.0 and 9.5 retained 94$, 93$, 68$ and 35$ respectively of its i n i t i a l activity.  Stored under the same conditions at  pH 9.5 i n the presence of 5mM B-merceptoeibhanol 66$ of the o r i g i n a l a c t i v i t y was retained. The pH 7.8 RNase a c t i v i t y was found to be comparat i v e l y stable and no attempt was made to determine optimal s t a b i l i z a t i o n conditions f o r t h i s enzyme. 3.3 DNase and Phosphodiesterase A c t i v i t i e s of DEAE-cellulose eluate enzyme f r a c t i o n s The p o s s i b i l i t y that the i d e n t i f i e d RNA-depolymerizing enzyme a c t i v i t i e s might be due to nucleases  or phosphodie-  sterases capable of cleaving phosphodiester bonds i n ribonuc l e i c acid molecules was tested by assaying f o r these a c t i v i t i e s using double-stranded c a l f thymus DNA or b i s - p - n i t r o phenyl phosphate as substrates. Table VIII shows that the DEAE-cellulose  eluate enzyme  fractions do not s i g n i f i c a n t l y degrade double-stranded DNA but exhibit p r e f e r e n t i a l s p e c i f i c i t y f o r the polynucleotide substrate containing ribose and u r a c i l moieties. Table IX shows that the p a r t i a l l y p u r i f i e d enzyme preparations exhibit no s i g n i f i c a n t  amounts of either  J'PQL-  or 5' POk-forming phosphodiesterase a c t i v i t y as compared to crude enzyme preparations of whole b r a i n .  - 114 -  TABLE V I I I . DNase ACTIVITY OF DEAE-CELLULOSE ELUATE ENZYME FRACTIONS  Enzyme Preparation  .Buffer &•&  J.  final  j.i  50 mM Tris-HCl, pH 6.7  Substrate*  Increase i n A260 per 60 minute incubation  sRNA  .815  ' Wk  .036  50 mM. DEAE-cellulose Tris-HCl, eluate f r a c t i o n pH 6.7 number 32 ( F i g . 7)  sRNA  .234  DNA  .025  DEAE-cellulose pooled e l u a t e fractions 55-70 ( F i g . 8 )  50 mM Tris-HCl, pH 7.8  sRNA  .860  DNA  .010  DEAE-cellulose pooled e l u a t e fractions 5-16 ( F i g . 8)  50 mM. Tris-HCl, pH 9.5  sRNA  .301  DNA  .011  75-100% saturated (NH4)2S0^ precipitable fraction  *0.5 ml o f a 0.1% (w/v) stock s o l u t i o n o f e i t h e r sRNA d i s s o l v e d i n d i s t i l l e d H2O o r double-stranded c a l f thymus DNA d i s s o l v e d i n 10 mM NaCl was added t o the i n c u b a t i o n mixture as s u b s t r a t e . Other d e t a i l s o f assay procedure a r e d e s c r i b e d i n s e c t i o n 2.142 o f Methods.  -  115  -  TABLE IX. PHOSPHODIESTERASE ACTIVITY OF DEAECELLULOSE ELUATE ENZYME FRACTIONS* I n c r e a s e i n A OO per 6 0 minute incubation k  Enzyme Preparation  Final buffer concentration  0 . 5 ml whole genate  h 0 m  °"  3 3 mM T r i s - H C l , pH 8 . 9 3  3  m  N H  4 - a c e t a t e , pH 5 . 0  ml whole brain extract  33  mM-Tris-HCl, pH  ml 7 5 - 1 0 0 $ saturated (NH ) S0k fraction  33  mMTris-HCl, p H . 6 . 7  0.5  .05  1.363 1.589  8.9  0.270  . 0  k  7  k  2  0 . 2 ml DEAEcellulose eluate fraction #32 (Fig. 7)  3 3 mM T r i s - H C l , pH 6 . 7  .000  0 . 2 ml DEAE3 3 mM T r i s - H C l , pH 7 . 8 cellulose e l u a t e pooled fractions  .017  55-70  (Fig.8)  0 . 2 ml DEAE3 3 mM.Tris-HCl, pH 9 . 5 cellulose eluate pooled fractions 5-16 (Fig. 8)  .000  • C o n d i t i o n s of assay a r e as d e s c r i b e d i n s e c t i o n 2 . 1 3 o f Methods. k  -  116  -  3.*+ I n t r a c e l l u l a r D i s t r i b u t i o n o f RNase A c t i v i t i e s and RNase I n h i b i t o r A c t i v i t y Isotonic  sucrose homogenates of a d u l t  were prepared and f r a c t i o n a t e d  r a t whole b r a i n  by d i f f e r e n t i a l c e n t r i f u g a t i o n  i n t o n u c l e a r , crude m i t o c h o n d r i a l , microsomal, f r a c t i o n s a c c o r d i n g t o t h e procedure of Methods.  Each s u b c e l l u l a r  a c t i v i t y i n 5 0 mM T r i s - H C l  and c y t o s o l  described i n section 2 . 1 2  f r a c t i o n was assayed f o r RNase  b u f f e r a t pH 6 . 7 , pH 9 . 5 , and a t  pH 7 . 8 w i t h and without 0 . 2 mM pCMB. The r e s u l t s recorded i n T a b l e X, , XI, and XII show t h a t f o r each RNase the sum o f the separate a c t i v i t i e s o f each s u b c e l l u l a r f r a c t i o n i s s i g n i f i c a n t l y g r e a t e r than t h e t o t a l a c t i v i t y expressed homogenate.  i n the i n i t i a l  isotonic  sucrose  T o t a l r e c o v e r y of each RNase a c t i v i t y i n a l l the  s u b c e l l u l a r f r a c t i o n s assayed a t pH 6 . 7 , pH 9 . 5 and pH 7 . 8 was 192$,  167$  and  205$  respectively  o f the a c t i v i t y expressed i n  the i s o t o n i c sucrose whole homogenate.  This a c t i v a t i o n of  RNase a c t i v i t i e s i s due t o the rehomogenisiation o f the separated s u b c e l l u l a r p a r t i c u l a t e f r a c t i o n s .  This  procedure  r e s u l t s i n the l i b e r a t i o n p f & c o n s i d e r a b l e RNase a c t i v i t y which was i n i t i a l l y  present i n the i s o t o n i c sucrose whole  homogenate i n a l a t e n t , n o n - f u n c t i o n a l form e i t h e r bound t o or compartmentalized  within  organelles.  The t o t a l r e c o v e r y  of each RNase a c t i v i t y i n a l l the s u b c e l l u l a r assayed  fractions  a t pH 6 . 7 , pH 9 . 5 , and pH 7 . 8 was, however, o n l y 5 6 $ ,  -  63%,  and 7 6 % r e s p e c t i v e l y  T r i t o n X100 homogenates.  117  -  o f the a c t i v i t y expressed i n 0.1% This indicates  t i o n o f the separated s u b c e l l u l a r isotonic  t h a t homogeniza-  particulate  fractions i n  sucrose does not l i b e r a t e a l l o f the t o t a l d e t e r g e n t -  e x t r a c t a b l e RNase a c t i v i t y . T a b l e X shows t h a t the pH 6 . 7 RNase a c t i v i t y which i s expressed under these c o n d i t i o n s i s predominantly i n the crude m i t o c h o n d r i a l and c y t o s o l  localized  fractions.  Of the  t o t a l r e c o v e r e d pH 6 . 7 RNase a c t i v i t y , 41% was p r e s e n t i n the crude m i t o c h o n d r i a l f r a c t i o n and 3 1 % i n t h e c y t o s o l  fraction.  activity The  s p e c i f i c ~ o f t h i s enzyme i n the c y t o s o l  f r a c t i o n was t h r e e -  f o l d g r e a t e r than t h a t o f any o f the o t h e r s u b c e l l u l a r  frac-  tions. Studies  2 9 5 , 3 0 5  o f the i n t r a c e l l u l a r d i s t r i b u t i o n o f  a c i d RNase i n r a t l i v e r by d i f f e r e n t i a l and sucrose d e n s i t y gradient centrifugation  have concluded t h a t the a c i d  RNase  a c t i v i t y a s s o c i a t e d w i t h the crude m i t o c h o n d r i a l f r a c t i o n i s l o c a l i z e d i n lysosomes.  I n view o f the s i m i l a r i t i e s o f the  pH 6 . 7 RNase a c t i v i t y r e p o r t e d i n the p r e s e n t study t o t h a t of A c i d RNase o f r a t l i v e r , i t seems l i k e l y t h a t the l a r g e amount o f pH 6 . 7 Rttase a c t i v i t y o f r a t b r a i n which i s r e c o v e r e d i n t h e crude m i t o c h o n d r i a l f r a c t i o n may a l s o be l o c a l i z e d w i t h i n the lysosomes o f t h i s  fraction.  TABLE X. INTRACELLULAR  Enzyme  Preparation  DISTRIBUTION OF pH 6 . 7 RNase ACTIVITY  Total Activity (units.)  Specifio aotivity (units/mg . protein)  pH 6 . 7 RNase A c t i v i t y % o f the sum o f T o activities re$> o f s u c r o s e covered i n a l l t h e homogenate a c t i - s u b c e l l u l a r f r a c v i t y recovered tions •  t a l activity (units) recovered p e r gram wet weight o f whole b r a i n  0,1# T r i t o n X100 homogenate  420.0  0.55  '347  181  70.0  I s o t o n i c sucrose homogenate  120.9  0.16  100  52  20.2  800 x g p e l l e t (nuclear f r a c t i o n )  40.0  >0.23  33  17  8,000 x g p e l l e t (crude m i t o c h o n d r i a l fraction)  94.8  0.29  78  41  15.8  105,000 x g p e l l e t (microsomal f r a c t i o n )  23.8  0.21  20  10  4.0  105,000 x g supernatant (cytosol fraction)  73.2  0.62  61  32  12.2  6.6  c  I n t h e e x p e r i m e n t s r e c o r d e d i n t h e above and f o l l o w i n g T a b l e s , 60 mis o f a 10% (wet wt. b r a i n / f i n a l volume) i s o t o n i c s u c r o s e homogenate o f a d u l t (4-month-old) r a t whole b r a i n s wa3 f r a c t i o n a t e d a c c o r d i n g t o t h e p r o c e d u r e d e s c r i b e d i n s e c t i o n 2.12 o f Methods. P e l l e t e d subc e l l u l a r f r a c t i o n s were resuspended by h o m o g e n i z a t i o n i n O.32 M s u c r o s e , brought t o f i n a l volume w i t h 0*32 M s u c r o s e , and a l i q u o t s o f each f r a c t i o n were assayed i n 50 mM T r i s - H C l b u f f e r a t t h e a p p r o p r i a t e pH.  -  119  -  Although the s t u d i e s on r a t l i v e r found t h a t l i t t l e  acid  RNase was present i n the c y t o s o l f r a c t i o n , De Lamirande and Allard  found t h a t a c i d RNase o f i n t e s t i n a l mucosa and  kidney, i n c o n t r a s t w i t h l i v e r , was present mainly i n the soluble f r a c t i o n .  I t i s not known t o what extent such  apparent t i s s u e - s p e c i f i c d i f f e r e n c e s i n the i n t r a c e l l u l a r d i s t r i b u t i o n o f t h i s RNase a c t i v i t y may r e p r e s e n t  differences  i n the f r a g i l i t y o f lysosomes or other s u b c e l l u l a r p a r t i c l e s and,  hence, i n the r e l e a s e of enzyme i n t o the s o l u b l e , f r a c t i o n  d u r i n g the i s o l a t i o n o f s u b c e l l u l a r p a r t i c l e s .  I t i s also  p o s s i b l e t h a t a l a r g e p o r t i o n o f t h i s enzyme, a t l e a s t i n b r a i n , may be a normal c o n s t i t u e n t  o f the c e l l sap and the  a c t i v i t y found i n other s u b c e l l u l a r f r a c t i o n s may  represent  enzyme which has been adsorbed onto sedimentable p a r t i c l e s during known  their isolation. 2 9 6  Such a d s o r p t i o n  phenomena are a w e l l  source o f a r t i f a c t s i n s u b c e l l u l a r d i s t r i b u t i o n  studies.  However, i n r a t l i v e r , Rahman  evidence o f the a d s o r p t i o n  *  found no  o f s o l u b l e RNases t o s u b c e l l u l a r  particles. Table XI shows t h a t pH 9 » 5 RNase a c t i v i t y i s a l s o found predominantly i n the crude m i t o c h o n d r i a l fractions.  and c y t o s o l  However, p r o p o r t i o n a l l y more o f t h i s enzyme  a c t i v i t y i s r e c o v e r e d i n t h e crude m i t o c h o n d r i a l (57$)  than i n t h e c y t o s o l ( 2 0 $ ) .  fraction  Both these f r a c t i o n s  have equal s p e c i f i c a c t i v i t i e s which were twice t h a t o f the n u c l e a r  and microsomal f r a c t i o n s .  120 TABLE XI. INTRACELLULAR DISTRIBUTION OF pH 9 . 5 RNase ACTIVITY pH 9.5 RNase Activity  Enzyme Preparation  Total activit; (units  I  Specific activity (units/mg protein)  i» of sucrose homogenate a c t i vity recovered  io of the sum?of activities recovered i n a l l the subcellular fractions  Total activity (units) recovered per gram wet weight of whole brain  0.1% Triton X100 homogenate  244.2  0.32  263  158  40.7  Isotonic sucrose homogenate  93*0  0.12  100  60  15.5  800 x g pellet (nuclear fraction)  21.8  0.13  23  14  3.6  8,000 x g pellet 93.6 (crude mitochondrial fraction)  0.29  101  60  15.6  10.1  0.09  11  29.4  0.28  32  105,000 x g pellet (microsomal fraction) 105,000 x g supernatant (cytosol fraction)  1.7  19  4.9  to O  -  The  121  -  i n t r a c e l l u l a r d i s t r i b u t i o n o f the recovered  pH 9 « 5 RNase  a c t i v i t y thus resembles t h a t o f pH 6 . 5 RNase except t h a t a l a r g e r p o r t i o n o f t h i s a c t i v i t y i s obtained  i n the crude  m i t o c h o n d r i a l f r a c t i o n and l e s s i n t h e microsomal and c y t o s o l fractions.  T h i s d i s t r i b u t i o n d i f f e r s from t h a t r e p o r t e d by f o r pH 9*5 RNase of, r a t l i v e r .  Rahman  more o f the t o t a l c e l l u l a r content was recovered  In l i v e r ,  relatively  o f pH 9 . 5 RNase a c t i v i t y  i n the microsomal f r a c t i o n and very l i t t l e  was  found i n the c y t o s o l f r a c t i o n . Table XII shows t h a t the l a r g e s t amount 6 3 % o f r e covered  f r e e pH 7 . 8 RNase a c t i v i t y and the h i g h e s t  a c t i v i t y o f t h i s enzyme was found i n the crude  •.  specific  mitochondrial  fraction. However, assuming t h a t 5 6 $ o f the o p t i m a l a c t i v i t y o f . pH 9 » 5 RNase i s r e t a i n e d i n assays a t pH 7 . 8 , 8 . 7 u n i t s ( p e r gram wet weight) o f the t o t a l RNase a c t i v i t y expressed  i n the  crude m i t o c h o n d r i a l f r a c t i o n assayed a t pH 7 . 8 ( 1 3 . 2 u n i t s / g wet  weight) can be accounted f o r i n terms o f pH 9 * 5 RNase  a c t i v i t y expressed 4.5  a t pH 7 * 8 .  T h i s l e a v e s a remainder o f  u n i t s ( ( p e r gram wet weight) which must r e p r e s e n t the  a c t u a l a c t i v i t y c o n t r i b u t e d by f r e e pH 7 « 8 RNase. It  i s u n l i k e l y t h a t i n t h e i n t a c t c e l l the s o l u b l e  c e l l sap c o n t a i n s any f r e e pH 7 * 8 RNase a c t i v i t y i n view o f the l a r g e excess o f f r e e RNase i n h i b i t o r found i n t h e c y t o s o l .  TABLE XII. INTRACELLULAR DISTRIBUTION OF FREE pH 7.8 RNase ACTIVITY Free pH 7.8 RNase Activity  Enzyme Preparation  Specific Total activity activity (units/mg [units, jrotein  0.1# Triton X100 homogenate Isotonic sucrose homogenate 800 x g pellet (nuclear fraction)  f<> of sucrose homogenate activity recovered  # of the sum of activities recovered i n a l l the subcellular fractions  Total activity (units) recovered per gram wet weight of whole brain  163.2  0.21  268  131  27.2  60.9  0.08  100  49  10.2  24.4  0.15  40  20  78.9  0.24  4.0  130  63  13.2  105,000 x g pellet 10.6 (microsomal fraction)  0.10  17  1.8  0.09  18  1.8  8,000 x g pellet (crude mitochondrial fraction)  105,000 x g supernatant (cytosol fraction) 11.0  ro ro  -  The  123  -  9 $ o f f r e e RNase a c t i v i t y assayed a t pH 7 . 8 which i s  recovered i n t h i s f r a c t i o n can be completely accounted f o r i n terms o f pH 9 » 5 RNase a c t i v i t y expressed a t pH 7 . 8 . Table XIII shows t h a t t o t a l pH 7 . 8 RNase a c t i v i t y assayed i n the presence o f 0 . 2 mM pCMB was mostly r e c o v e r e d i n the c y t o s o l f r a c t i o n , and t h e s p e c i f i c a c t i v i t y o f t h i s f r a c t i o n was 5 - t o 1 0 - f o l d g r e a t e r than t h a t o f the other subcellular and  fractions.  From t h e d i f f e r e n c e between  free  t o t a l pH 7 * 8 RNase a c t i v i t y i t can be c a l c u l a t e d  an apparent 8 8 $ of the l a t e n t ,  that  i n h i b i t o r - b o u n d pH 7 » 8 RNase  i s present i n the c y t o s o l (Table X I V ) . The  pH 7 * 8 RNase a c t i v i t y i n the crude m i t o c h o n d r i a l  f r a c t i o n assayed i n the presence o f 0 . 2 mM pCMB was 3 8 $ lower than t h a t assayed without pCMB, i n d i c a t i n g has  a net i n h i b i t o r y  fraction  t h a t pCMB  e f f e c t on pH 7 « 8 RNase a c t i v i t y i n t h i s  (see F i g . 1 2 a ) .  From t h e f a i l u r e o f pCNB t o stimu-  l a t e RNase a c t i v i t y assayed a t pH 7 * 8 i n the crude mitochondrial fraction, no  i t would appear t h a t t h i s f r a c t i o n  i n h i b i t o r - b o u n d pH 7 * 8 RNase d e s p i t e the f a c t t h a t  contains this  f r a c t i o n c o n t a i n s 6 3 $ o f the t o t a l r e c o v e r e d f r e e pH 7 « 8 RNase a c t i v i t y and 2 7 $ of the t o t a l r e c o v e r e d f r e e i n h i b i t o r a c t i v i t y (Table XV).  RNase  - 124 TABLE XIII. INTRACELLULAR DISTRIBUTION OF TOTAL pH 7.8 RNase ACTIVITY  Enzyme Preparation  Total activity (units)  O.ljfr..Triton X100 homogenate 244.2 Isotonic sucrose homogenate 192.6 800 x g pellet 38*0 (nuclear fraction)  Specific activity (units/mg protein)  Total pH 7.8 RNase Activity fo of the sum of activities recovered % of sucrose in a l l the subhomogenate acticellular fractions vity recovered  Total activity (units) recovered per gram wet weight of whole brain  0.32  127  107  40.7  0.25  100  S85  32.1  0.22  20  17  6.3  8,000 x g pellet 49.0 (crude mitochondrial fraction)  0.15  25  22  105,000 x g pellet (microsomal fraction)  «i,5  0.11  105,000 x g supernatant (cytosol fraction)  128.2  1.08  8.2 2.1  67  56  21.4  - 125 TABLE XIV. INTRACELLULAR DISTRIBUTION OF LATENT pH 7.8 RNase ACTIVITY Latent ,.P,H ,7.8 RNase A c t i v i t y * $> of'the sum of a c t i v i t i e s recovered i> of sucrose homogenate a c t i v i t y i n a l l the subcellular fractions recovered m i  Enzyme P f o n a r a t 1 on  Total activity (units)  Specific activity (units/mg protein)  0.1J6 T r i t o n X100 homogenate  81.0  0.11  62  Isotonic sucrose homogenate  131.7  0.17  100  800 x g p e l l e t 13*6 (nuclear f r a c t i o n )  0.08  10  8,000 x g p e l l e t  -29.9  61  13.5  99  21.9  10  2.3  0  a  chondrial f r a c t i o n ) 1.9  0.02  105,000 x g super- 117.2 natant ( c y t o s o l fraction)  0.98  105,000 x g p e l l e t (microsomal fraction)  Total a c t i v i t y (units) recovered per gram wet weight of whole brain  0  0.3  89  88  19.5  •Latent or inhibitor-bound pH 7.8 RNase a c t i v i t y i s defined as the a c t i v i t y released upon treatment with pCMB, and was calculated by subtracting the free pH 7.8 RNase a c t i v i t y (Table XII) from the t o t a l pH 7.8 RNase a c t i v i t y assayed i n the presence of 0.2 mM pCMB (Table X I I I ) . T h i s negative value r e f l e c t s the net i n h i b i t o r y e f f e c t of pCMB on RNase a c t i v i t y assayed at pH 7*8 i n the crude mitochondrial f r a c t i o n . This value was taken as zero i n c a l c u l a t i n g t o t a l recovery. a  ro  - 126 TABLE XV. INTRACELLULAR DISTRIBUTION OF FREE RNase INHIBITOR ACTIVITY  Enzyme Preparation  Total Activity (units)  Specific Activity (units/mg protein)  6,660  8.54  Isotonic sucrose homogenate 800 x g pellet (nuclear fraction)  217.8  Free RNase Inhibitor Activity* % of sura of % of total % of sucrose activities protein homogenate recovered in recovered in- activity a l l subfraction recovered cellular fractions 100 100 100  5.56  105,000 x g pellet (microsomal fraction)  315.0  2.84  105,000 x g supernatant4,317.5 (cytosol fraction)  36.34  1,110.0  3  22  27  42  5  14  5  52.5  -5  65  719.6  1.26  8,000 x g pellet 1,800.0 (crude mitochondrial fraction)  Total activity (units recovered per gram wet weight of whole brain  3  36.3  27  300.0  1  0 1  6  5  X  •Free RNase inhibitor activity was assayed according to the procedure described in section 2.144 of Methods. The incubation mixture contained 0.5 rig. bovine pancreatic RNase A, 1 mM EDTA, and various aliquots of tissue fractions suitably diluted with ice-cold .02 M Tris-HCl, pH 7.8 buffer so as to yield inhibitions of control bovine pancreatic RNase A activity between 0 and 80%. Incubation was for 30 minutes. A unit of free inhibitor activity ia defined as the amount of inhibitor which produces 50% inhibition of the activity of 0.5 ng.bovine pancreatic RNase A. Units of inhibitor activity were interpolated from standard curves expressing per cent inhibition of the control activity of 0.5 ng pancreatic RNase A as a function of the amount of tissue sample added.  - 12? -  I t I s u n l i k e l y t h a t these r e s u l t s can be e x p l a i n e d i n terms of d i f f e r e n t i a l compartmentalization  o f the f r e e pH 7»8  RNase a c t i v i t y and the f r e e RNase i n h i b i t o r a c t i v i t y s i n c e the rehoraogenization t o which t h i s f r a c t i o n was s u b j e c t e d and the hypotonic  c o n d i t i o n o f assay would be expected t o l a r g e l y  remove the c o n s t r a i n t s t o i n t e r a c t i o n between molecular  segregated  species.  The  data suggests t h a t the crude m i t o c h o n d r i a l  fraction  c o n t a i n s an a l k a l i n e RNase d i f f e r i n g from t h a t §f the c y t o s o l i n being  (1) i n h i b i t e d by pCMB, and (2) i n s e n s i t i v e t o i n h i -  b i t i o n fey the f r e e RNase i n h i b i t o r endogenous t o the crude mitochondrial  fraction.  I n r a t l i v e r , both R o t h  1 9 5  and S h o r t r a a n  213  found t h e  m i t o c h o n d r i a l pH 7.8 RNase a c t i v i t y t o be s e n s i t i v e t o i n h i b i t i o n by the f r e e RNase i n h i b i t o r present Roth  1 8 7  i n the c y t o s o l .  S@tfe  i n f e r r e d t h a t the predominant f r a c t i o n o f m i t o c h o n d r i a l  pH 7,8 RNase a c t i v i t y must hence be present He a l s o r e p o r t e d  1 9 0  evidence  i n t h e f r e e form.  f o r the presence o f some i n h i b i t o r -  bound pH 7.8 RNase i n the crude m i t o c h o n d r i a l f r a c t i o n o f r a t liver. The  s i m p l e s t e x p l a n a t i o n f o r the apparent absence o f  d e t e c t a b l e i n h i b i t o r - b o u n d pH 7«8 RNase i n the crude mitoc h o n d r i a l f r a c t i o n o f b r a i n l i e s i n the f a c t t h a t bound pH 7.8 RNase i s normally  inhibitor-  i n d i r e c t l y i n f e r r e d from t h e  - 128 -  a c t i v a t i n g e f f e c t of pCMBf but due to the high pH 9.5 RNase content of the crude mitochondrial f r a c t i o n , pCMB a c t i v a t i o n of inhibitor-bound pH 7*8 RNase i s more than  counterbalanced  by the i n h i b i t o r y effect of pCMB on the residual pH 9.5 RNase a c t i v i t y expressed i n assays at pH 7 . 8 . The r e s u l t s thus represent a composite effect r e f l e c t i n g the i n a b i l i t y of the assay conditions to c l e a r l y d i s criminate between the RNase a c t i v i t i e s present i n the crude mitochondrial f r a c t i o n .  The RNase a c t i v i t y measured at a  given pH i s not due s o l e l y to a single  enzyme  but i s the  r e s u l t of the combined actions of the multiple enzyme species present i n t h i s subcellular  fraction.  Table XV shows that free RNase i n h i b i t o r a c t i v i t y assayed as described i n section 2.144 of Methods was found predominantly  i n the cytosol (66% of t o t a l recovered a c t i v i t y )  and crude mitochondrial f r a c t i o n (27% of t o t a l recovered activity).  The small amount of i n h i b i t o r a c t i v i t y recovered  i n the nuclear f r a c t i o n probably represents the degree to which t h i s f r a c t i o n i s contaminated with adhering cytoplasmic material. The i n t r a c e l l u l a r d i s t r i b u t i o n of free RNase i n h i b i t o r a c t i v i t y i n brain closely resembles that reported f o r r a t liver.  Roth ® 1  7  found RNase i n h i b i t o r a c t i v i t y to be high i n  the cytosol and low i n the microsomal and nuclear f r a c t i o n s  129 -  of r a t l i v e r .  Roth " ' has more r e c e n t l y £  L5  r e p o r t e d the  absence o f any d e t e c t a b l e f r e e RNase i n h i b i t o r i n p u r i f i e d n u c l e i from r a t l i v e r . I t i s of special interest that  the sum o f the f r e e  RNase i n h i b i t o r a c t i v i t i e s o f the s u b c e l l u l a r  fractions  was  founfl t o be equal t o the t o t a l f r e e RNase a c t i v i t y i n the isotonic  sucrose whole homogenate.  T h i s component o f the  RNase enzyme system thus e x h i b i t s no s u b c e l l u l a r l i n k e d l a t e n c y and t h i s may be c r i t i c a l t o the of t h i s p r o t e i n  functioning  i n t i t r a t i n g the l e v e l o f a c t i v i t y o f pH  RNase i n the i n t a c t 3.5  structure-  7.8  cell.  C h a r a c t e r i z a t i o n o f RNase a c t i v i t i e s i n separated s u b c e l l u l a r f r a c t i o n s In view o f the i n a b i l i t y o f the assay c o n d i t i o n s used  to c l e a r l y d i f f e r e n t i a t e the s p e c i f i c c o n t r i b u t i o n the  o f each o f  m u l t i p l e enzyme s p e c i e s to the RNase a c t i v i t y measured a t  a g i v e n pH, a more r e l i a b l e d e t e r m i n a t i o n o f the a c t i v i t y contributed  by each component RNase seemed t o r e q u i r e t h e i r more  thorough c h a r a c t e r i z a t i o n particular subcellular  and s e p a r a t i o n s t a r t i n g from a  fraction.  A l s o , i n view o f the r a t h e r  unremarkable s i m i l a r i t i e s o f the t h r e e RNases d e t e c t e d i n b r a i n t o those p r e v i o u s l y r e p o r t e d i n r a t l i v e r , a more detailed  study o f the RNase a c t i v i t i e s o f the i n d i v i d u a l  sub-  c e l l u l a r f r a c t i o n s was made i n an attempt t o d e t e c t n o v e l RNase a c t i v i t i e s or d i s t i n c t i v e c h a r a c t e r i s t i c s not d e t e c t a b l e  -  i n previous studies  130  -  o f whole homogenates and e x t r a c t s .  The  RNase a c t i v i t y o f each s u b c e l l u l a r f r a c t i o n , i s o homogenates , , l a t e d from i s o t o n i c s u c r o s e ^ o f a d u l t r a t b r a i n , was assayed over a range o f hydrogen i o n c o n c e n t r a t i o n s w i t h and without 1 mM EDTA, 0 . 2 mM pCMB o r 2 . 0 M u r e a .  The c o n t r o l pH curves  of a l l f o u r s u b c e l l u l a r f r a c t i o n s i n the absence o f any added reagents e x h i b i t e d h i g h e r a c t i v i t y i n the a c i d pH r e g i o n than i n t h e a l k a l i n e r e g i o n .  The c o n t r o l pH optimum f o r a c i d  RNase a c t i v i t y was pH 6 . 7 i n T r i s - H C l b u f f e r and pH 6 . 3 i n NH14, a c e t a t e b u f f e r as f o r the whole c e l l homogenate.  However,  each s u b c e l l u l a r f r a c t i o n d i f f e r e d from the whole homogenate i n the a l k a l i n e r e g i o n  o f the c o n t r o l a c t i v i t y versus pH  profile. The  c o n t r o l pH curve o f the n u c l e a r f r a c t i o n has a  bimodal appearance suggestive o f a c t i v i t y maximae a t pH 8 and 8.6.  The crude m i t o c h o n d r i a l  plateau  f r a c t i o n e x h i b i t s a broad  o f a c t i v i t y between pH 8 and 9 « 5 . and the microsomal  f r a c t i o n has a d i s t i n c t a c t i v i t y maxima a t pH 8 „ 9 .  The pH  curve o f f r e s h l y prepared c y t o s o l f r a c t i o n e x h i b i t s a deep trough between pH 7 and 8 . 5 with a c t i v i t y minima a t pH  7«5»  Above pH  8.5  broad a c t i v i t y p l a t e a u on page  / i f f )•  occurring  the c y t o s o l f r a c t i o n e x h i b i t s a  with no d i s t i n c t maxima (see F i g . 14  - 131 The  e f f e c t o f 0.2  mM pCMB on the RNase a c t i v i t i e s  of the n u c l e a r and m i t o c h o n d r i a l f r a c t i o n s i s shown i n F i g u r e s 11a and 12a r e s p e c t i v e l y .  This sulfhydryl blocking  agent s t i m u l a t e d a c t i v i t y i n the n u c l e a r f r a c t i o n between pH 7 and 8.5 with a maximal s t i m u l a t i o n o f at the pH minima o f c o n t r o l a c t i v i t y . of a c t i v i t y was observed above pH 8.6  53$  occurring  A slight  inhibition  and below pH 6.7*  The m i t o c h o n d r i a l f r a c t i o n RNase a c t i v i t y , however, showed a c o n s i s t e n t i n h i b i t i o n o f about kQ% by 0.2 out the e n t i r e pH range t e s t e d .  mM pCMB through-  The magnitude .of the  measured a c t i v i t y i s the n e t e f f e c t r e s u l t i n g from d i f f e r e n t i a l a c t i v a t i o n and i n h i b i t i o n by pCMB o f t h e v a r i o u s enzyme s p e c i e s present  i n t h i s f r a c t i o n , and i s i n t e r p r e t e d  to i n d i c a t e t h a t the pH 9*5 RNase/inhibitor-bound  pH 7.§ RNase  r a t i o f o r t h i s f r a c t i o n i s c o n s i d e r a b l y h i g h e r than t h a t f o r n u c l e a r and c y t o s o l f r a c t i o n s .  More d i r e c t v e r i f i c a t i o n o f  t h i s putative explanation w i l l require the separation o f these enzyme components from each other s t a r t i n g from t h e crude m i t o c h o n d r i a l The  fraction^.  e f f e c t o f 1 mM EDTA on RNase a c t i v i t y i n n u c l e a r  and m i t o c h o n d r i a l f r a c t i o n s i s shown i n F i g u r e s llb'o and 12b r e s p e c t i v e l y .  T h i s c o n c e n t r a t i o n o f EDTA  completely  a b o l i s h e s m i t o c h o n d r i a l a l k a l i n e RNase a c t i v i t y whereas about 50$ o f the c o n t r o l a l k a l i n e RNase a c t i v i t y i s r e t a i n e d i n the n u c l e a r  fraction.  -  Fiqure  132  -  11 a  .5 ^  E  4  /7\  o .3  FIGURE 11. The RNase a c t i v i t y i n the n u c l e a r f r a c t i o n i s o l a t e d from a d u l t r a t whole b r a i n (a) i n t h e presence o f 2 . 0 M urea and 0 . 2 mM pCMB, and (b) i n t h e presence o f 1 . 0 mM EDTA. A l i q u o t s o f 0 . 2 ml o f t h e n u c l e a r f r a c t i o n were assayed i n 50 mM b u f f e r i n the absence o f any added reagents ( N H 4 - a c e t a t e — * — ; T r i s - H C l — • — ), and i n the presence o f e i t h e r 2 . 0 M urea (NH4-acetate - v - - ; T r i s - H C l " - o - - ) , o r 0.2 mM pCMB ( T r i s - H C l ), or 1.0 mM EDTA (NH4-acetate --v-- i T r i s - H C l - - o - - ) . I n c u b a t i o n was f o r 60 minutes.  -  133  -  FIGURE 12. The RNase a c t i v i t y i n the crude m i t o c h o n d r i a l f r a c t i o n i s o l a t e d from a d u l t r a t whole b r a i n i (a) e f f e c t of 0.2 mM pCMB, and (b) e f f e c t o f 1.0 mM EDTA and 0.5 mM MgC12. A l i q u o t s o f 0.1 ml o f the crude m i t o c h o n d r i a l f r a c t i o n were assayed i n 50 mM b u f f e r i n the absence o f any added reagents ( N H 4 - a c e t a t e — * — j T r i s - H C l — • — ) and i n the presence o f e i t h e r 0.2 mM pCMB ( T r i s - H C l ••+••), or 1.0 mM EDTA (NH4-acetate — v — ; T r i s - H C l - - 0 - - ) or 0.5 mM MgC12 ( T r i s - H C l — • ). I n c u b a t i o n was f o r 60 minutes. f  - 13 k  acetate In ammonium buffer, both mitochondrial and nuclear A  acid RNase a c t i v i t y i s s l i g h t l y stimulated by 1 mM EDTA, with maximal stimulation occurring above pH 6.0. I n T r i s HCl buffer, however, the acid RNase a c t i v i t y assayed below pH 6.7 i s s l i g h t l y i n h i b i t e d by 1 mM EDTA i n both mitochond r i a l and nuclear f r a c t i o n s . In Tris-HCl buffer above pH 6.7, acid RNase a c t i v i t y i s stimulated i n the nuclear f r a c t i o n and unaffected i n the mitochondrial  fraction.  The effect of 2.0 M urea on RNase a c t i v i t y i n the nuclear and microsomal f r a c t i o n s i s shown i n Figures 11a and 13 respectively.  Acid RNase a c t i v i t y i s markedly  stimulated 140$ and 100$ i n nuclear and microsomal f r a c t i o n s respectively and the pH optimum of t h i s enzyme a c t i v i t y i s shifted to pH 5.8. In the a l k a l i n e region of the pH curve, 2.0 M urea stimulates RNase a c t i v i t y maximally (193$) at pH 8 i n the nuclear f r a c t i o n .  Urea-activation of RNase a c t i v i t y  assayed at t h i s pH cannot be completely  accounted f o r i n terms  of the release of inhibitor-bound pH 7.8 RNase a c t i v i t y since the l e v e l of a c t i v i t y evoked i n the presence of urea i s 2-fold greater than that obtained i n the presence of an optimal concentration (0.2 mM) of pCMB. Hence, the a c t i v a t i o n by urea and pCMB of RNase a c t i v i t y assayed at pH 7.8 must be effected at least i n part by d i f f e r e n t mechanisms.  - 135 -  >>  c  -— •'  o  CO  CD CO  Figure 1 3  CD  > CO  .5  i_  O a  £ \  .4 .3 .2  C/J  co •• < z— E .1 CC 3  j  I  i  L  7 p H  FIGURE 1 3 . E f f e c t o f 2 . 0 M urea on the RNase a c t i v i t y o f the microsomal f r a c t i o n . A l i q u o t s o f 0 . 2 ml o f the microsomal f r a c t i o n were assayed i n the absence of any added r e a g e n t s (NH4-acetate — • — ; T r i s - H C l — • — ) and i n the presence of 2 . 0 M urea (NH4-acetate - - v - - j T r i s - H C l —o—). I n c u b a t i o n was f o r 60 minutes.  10  - 136  -  Urea s t i m u l a t e s a l k a l i n e RNase a c t i v i t y i n the microsomal f r a c t i o n to a l e s s e r extent (100%) than i n the n u c l e a r fraction.  I n a d d i t i o n to the a c t i v i t y maxima a t pH 8, a  second a c t i v i t y maxima a t pH 8.6 tion.  T h i s a c t i v i t y may  J  " * *  and known to be  tD  Thus, both a c i d and a l k a l i n e RNase  a c t i v i t e s a r e markedly s t i m u l a t e d i n the presence o f M urea.  The  frac-  correspond to the microsome-bound ooo oo'c oftA  RNase r e p o r t e d by other workers a c t i v a t e d by u r e a .  i s observed i n t h i s  2.0  s t i m u l a t i n g e f f e c t of urea on pH 9»5 RNase i s  l e a s t marked.  The o p t i m a l c o n c e n t r a t i o n o f urea was  not  determined. The i n c r e a s e d y i e l d of a c i d - s o l u b l e  oligoribfonuc-  l e o t i d e s i n the presence of ureail&spprb&ably nut dueato a d i r e c t s t i m u l a t i o n o f the RNases s i n c e the d i r e c t a c t i o n o f urea on these enzymes would be expected t o u n f o l d s t r u c t u r e and i n h i b i t t h e i r a c t i v i t y . l a t i n g e f f e c t o f urea may  their  Rather, the stimu-  be due to ( l ) ; d i s s o c i a t i o n of  n o n - s p e c i f i c protein-RNase and protein-RNA  complexes and  aggregates thereby f a c i l i t a t i n g enzyme-substrate  interaction!  (2) d i s r u p t i o n of the H-bonded secondary s t r u c t u r e of the RNA  thereby making the s u b s t r a t e more v u l n e r a b l e t o enzyme  a c t i o n , and/or  (3) s i n c e RNases may  endonueteolytic breaks i n RNA,  produce  single-stranded  leaving oligoribonucleotide  sequences H-bonded to the i n t a c t s t r a n d , urea may  facilitate  - 137 -  the r e l e a s e of those products of the enzyme a c t i o n which remain H-bonded.  E l u c i d a t i o n of the p r e c i s e  contribution  of each of these mechanisms t o theobserved composite of urea r e q u i r e s f u r t h e r experiments.  effect  A s i m i l a r enhancement  of p a n c r e a t i c RNase A a c t i v i t y by urea has been r e p o r t e d by 297  K a l n i t s k y et a l . '  These workers found t h a t o n e - t h i r d o f  the i n c r e a s e c o u l d be accounted f o r by the  solubilizing  e f f e c t of urea on the p r o d u c t s of the r e a c t i o n ; i . e . , f a c i l i t a t i o n of the r e l e a s e of H-bonded base p a i r s i n t o a c i d soluble  form. T h i s study of the RNase a c t i v i t i e s of i n d i v i d i u a l  s u b c e l l u l a r f r a c t i o n s suggests the presence of an a d d i t i o n a l a l k a l i n e RNase a c t i v i t y d i s t i n c t from those p r e v i o u s l y t e c t e d i n the whole c e l l homogenates and e x t r a c t s .  de-  T h i s micro-  somal a l k a l i n e RNase a c t i v i t y with an apparent pH optimum between pH 8.6  and 8,9  dominance o f pH 7.8  was  and pH 9«5  whole c e l l homogenates and I t may  presumably  masked by the p r e -  RNase a c t i v i t i e s i n assays of  extracts.  be p o s s i b l e t o take advantage  o f the d i f f e r e n t i a l  i n t r a c e l l u l a r l o c a l i z a t i o n of the v a r i o u s RNases and reduce the complexity of the system under o b s e r v a t i o n by attempting t o separate and p u r i f y the v a r i o u s enzyme a c t i v i t i e s present w i t h i n a p a r t i c u l a r s u b c e l l u l a r f r a c t i o n , r a t h e r than s t a r t i n g the whole c e l l homogenate.  from  -  138  -  i  I  D)  E  J 5  1  1 6  7  i  ! 8  i  I  I  9  1_ 10  P H  FIGURE 14. RNase a c t i v i t y as a f u n c t i o n o f pH i n f r e s h l y prepared and aged c y t o s o l f r a c t i o n . A l i q u o t s o f 0.2 ml o f c y t o s o l were assayed immediately upon p r e p a r a t i o n (NH4-acetate —f—~ j T r i s - H C l — • — ) and a f t e r two weeks s t o r a g e a t 0° ( T r i s - H C l - - * - — ). I n c u b a t i o n was f o r 60 minutes.  -  139  -  3 . & Developmental Changes i n RNase A c t i v i t i e s and i n RNase I n h i b i t o r A c t i v i t y i n Rat Whole B r a i n F i g u r e s 1 5 , 1 6 and 1 7 show the s p e c i f i c levels  o f RNase assayed a t pH 6 . 7 .  activity  9 . 5 and 7 . 8 i n 0 . 1 $  T r i t o n X100 whole b r a i n homogenates o f r a t s a t v a r i o u s stages o f p o s t n a t a l m a t u r a t i o n . The  specific  a c t i v i t y o f RNase assayed a t pH 6 . 7 i s  h i g h e s t a t b i r t h , begins t o d e c l i n e g r a d u a l l y a t about day 7,  then more r a p i d l y a f t e r  day 2 2 u n t i l about day 3 2 a f t e r  which i t remains r e l a t i v e l y  constant.  at day 3 2 i s 6 0 $ o f t h a t a t day 1 . wet  The s p e c i f i c  activity  However, on a p e r u n i t  weight of b r a i n b a s i s the a c t i v i t y p r o g r e s s i v e l y i n -  creases from b i r t h , peaks a t day 2 2 ( 1 2 0 $ of day 1 l e v e l ) and  f a l l s rapidly thereafter  adult levels  decrease) t o reach  by day 3 2 ( 8 7 $ o f day 1 l e v e l ) .  Both the s p e c i f i c wet  (1.5-fold  a c t i v i t y and the a c t i v i t y per gram  weight b r a i n of RNase assayed a t pH 9 . 5 are minimal a t  b i r t h , r i s e r a p i d l y from day 7 t o peak a t day 2 2 , and subsequently e x h i b i t a g r a d u a l but d i s t i n c t d e c l i n e throughout adulthood.  Specific  a c t i v i t i e s a t day 2 2 and 8 months are  224$ and 1 5 2 $ r e s p e c t i v e l y o f t h a t a t day 1 . The i s similar  developmental p r o f i l e o f f r e e pH 7 . 8 RNase a c t i v i t y to t h a t o f RNase a c t i v i t y assayed a t pH 9 * 5 except  t h a t the l a g i n the i n c r e a s e up t o age 7 days i s not observed  - 139a-  -I  1  1  5  10  15  1  1  20 25  1  1  ft  30 days Post-natal  I  3 months  I  L  4  5  Age  FIGURE 15, Changes i n a c i d RNase a c t i v i t y d u r i n g development,  postnatal  RNase a c t i v i t y i n 0.1% T r i t o n X100 homogenates of a d u l t r a t whole b r a i n was assayed a t pH 6 , 7 i n 50 mM T r i s - H C l buffer. A l i q u o t s of 0 , 0 5 ml of homogenate were used f o r a s s a y . I n c u b a t i o n was f o r 30 minutes.  -139b -  i i ? i  i  I  o  Figure 16  60  o a  H  E  cf)  \  -— *CO• 50Y  .5 c>  "0-  "c 3  c  -A.  / A  40  3  30  .3 .t: >  2o[*-'  .2  •4-»  o  CO  CD CO CO  z rr  1  i c  o-  -N  10h  -— *• ° (0  CU CO CO  Z DC  5  10  15  20  25  3 0 days Post natal  3 months 4 age  FIGURE 1 6 . P o s t n a t a l developmental RNase a c t i v i t y .  changes i n pH 9 * 5  I n c u b a t i o n was f o r 6 0 minutes. A l l other c o n d i t i o n s were as i n legend t o F i g u r e 15» except the assay was conducted a t pH 9 . 5 .  - T39c-  z5  I  I  I  0  5  10 15 20 25 30 days  I  I  1  I  1  ff.  I  L  3 months 4  Post-natal Age FIGURE 17. P o s t n a t a l developmental changes i n pH 7.8 RNase a c t i v i t y . Free pH 7»8 RNase a c t i v i t y assayed i n t h e absence o f pCMB expressed i n units/mg p r o t e i n - - ^ — a n d i n u n i t s / g r a m wet weight brain — o — , T o t a l pH 7«8 RNase a c t i v i t y assayed i n the presence o f 0.2 mM pCMB expressed i n units/mg p r o t e i n — ¥ — and i n units/grate wet weight b r a i n • — » — . I n c u b a t i o n was f o r oO minutes. A l l o t h e r c o n d i t i o n s were as i n legend t o F i g u r e 15*  -  140  -  and the rate of increase i s more gradual.  The magnitude  of the net increase, however, i s nearly the same. The s p e c i f i c a c t i v i t y observed at age 22 days and 5 months i s 221% and 150% respectively of that at day 1.  Free  pH 7 . 8 RNase s p e c i f i c a c t i v i t y also undergoes a similar gradual decline throughout adulthood with the s p e c i f i c a c t i v i t y at 5 months being 6 8 % of the peak s p e c i f i c a c t i v i t y at age 22 days. Total pH 7 * 8 RNase a c t i v i t y (assayed i n the presence of 0.2 mM pCMB) exhibits a rapid 2 . 3 - f o l d increase i n s p e c i f i c a c t i v i t y between day 10 and day 22 a f t e r which i t remains r e l a t i v e l y constant throughout adulthood.  Parachloro-  mercuribenzoate i s inhibitory at a l l ages prior to day 2 0 . The shape of the difference curve between <t'he.specific a c t i v i t i e s pH 7.8 RNase ©f  f r e e , and t o . t a l A i s  interpreted as an index of the i n h i b i t o r -  bound pH 7 . 8 RNase/pH 9.5 RNase a c t i v i t y r a t i o .  Thus, t h i s  ratio i s r e l a t i v e l y low at b i r t h and decreases to a minimum at 10 days due to a proportionally greater increase i n pH 9.5 RNase s p e c i f i c a c t i v i t y during t h i s period.  I t then increases  and attains day 1 values by age 1 8 days due to a proport i o n a l l y greater increase during this time i n free RNase i n h i b i t o r and hence i n inhibitor-bound pH 7 . 8 RNase. The fact that after age 20 days the net effect of pCMB i s a stimul a t i o n of RNase a c t i v i t y assayed at pH 7 . 8 indicates that t h i s r a t i o has become s u f f i c i e n t l y large so that the i n h i b i t o r y  - 141 -  effect  o f pCMB on pH 9 * 5 RNase a c t i v i t y expressed i n assays  a t pH 7 . 8 i s masked by the o v e r r i d i n g s t i m u l a t o r y e f f e c t o f pCMB due t o i t s i n a c t i v a t i o n o f i n h i b i t o r i n h i b i t o r - b o u n d pH 7 . 8 RNase a c t i v i t y . between the s p e c i f i c  and r e l e a s e o f  The d i f f e r e n c e  a c t i v i t y curves o f RNase assayed a t  pH 7 . 8 i n t h e absence o r presence o f pCMB does n o t r e p r e s e n t the a b s o l u t e activity. specific  amount o f i n h i b i t o r - b o u n d , l a t e n t pH 7 . 8 RNase  Whereas from day 2 2 onward, the t o t a l pH 7 . 8 RNase a c t i v i t y remains constant,  the amount o f i n h i b i t o r -  bound pH 7 . 8 RNase a c t i v i t y per mg p r o t e i n a p p a r e n t l y i n creases p r o g r e s s i v e l y from 0% a t day 2 0 t o 6 $ a t day 2 2 , t o 3 0 $ a t 3 months, o f the t o t a l pH 7 . 8 RNase a c t i v i t y .  This  p r o g r e s s i v e i n c r e a s e i n pCMB a c t i v a t i o n o f RNase a c t i v i t y assayed a t pH 7 * 8 would appear t o i n d i c a t e an i n c r e a s e i n the c o n c e n t r a t i o n o f RNase i n h i b i t o r - p H 7 . 8 RNase complexes and such an i n t e r p r e t a t i o n i s c o n s i s t e n t with  the observed con-  c u r r e n t d e c l i n e i n f r e e pH 7 . 8 RNase s p e c i f i c However, no s i m i l a r concurrent of f r e e RNase i n h i b i t o r f r e e RNase i n h i b i t o r  decline i n specific  i s observed.  activity  Hence, the amount o f  must i n c r e a s e a t such a r a t e as t o  r e p l e n i s h t h a t u t i l i z e d i n the f o r m a t i o n f r e e pH 7 * 8 RNase i n order t o m a i n t a i n f r e e RNase i n h i b i t o r  activity.  o f complexes  a constant  throughout adulthood.  f a v o r e d i n t e r p r e t a t i o n o f the p r o g r e s s i v e  with  amount o f  Howeyer, t h e i n c r e a s e i n pGMB a  a c t i v a t i o n o f RNase a c t i v i t y assayed a t pH 7 . 8 i s t h a t i t i s  142  -  due to a further increase i n the inhibitor-bound pH  7.8  RNase/pH 9 » 5 RNase r a t i o which i s due not to an increase i n the amount of inhibitor-bound pH 7.8 RNase but, rather, an age-dependent decline i n pH 9 « 5 RNase a c t i v i t y and hence a concurrent decline i n the contribution of the i n h i b i t o r y e f f e c t of pCMB to the t o t a l RNase a c t i v i t y assayed at pH  7.8.  I t follows from t h i s i n t e r p r e t a t i o n that the amount of i n h i bitor-bound pH 7«8 RNase remains constant throughout adulthood, whereas the amount of free pH 7«8 RNase a c t i v i t y  and  pH 9 . 5 RNase a c t i v i t y concurrently decline a f t e r 2 2 days. In summary, on a per gram wet weight basis a l l three RNase a c t i v i t i e s a t t a i n t h e i r maximal values at age 2 2 days and subsequently decline.  This decline i s gradual and con-  tinuous throughout adulthood i n the case of pH 7.8 RNase a c t i v i t i e s .  and pH 9 . 5  Acid RNase a c t i v i t y , however, exhibits a  rapid decline i n a c t i v i t y between age 2 2 and 3 2 days and, subsequently, remains comparatively constant throughout adulthood. The s p e c i f i c a c t i v i t y of free pH 7«8 RNase i n h i b i t o r i n the whole brain of newborn rats i s about 5 0 $ that of adults.  There i s a rapid increase i n free RNase i n h i b i t o r  a c t i v i t y between age 10 and 18 days and the l e v e l of a c t i v i t y attained by 18 days remains constant throughout adulthood. Comparison of the developmental p r o f i l e s of free RNase  - 143 -  ~ 100 h o 90 CO  <  h  80  h  CD CO  *  Figure  is  70  CC o 60 h  co ^ 50 o  2  40  °  30  Q.  c o  '£ 20 JO  £  10 JL  10  15 20  30 days  25  Post-natal  3 months 4 Age  FIGURE 18. P o s t n a t a l developmental changes i n f r e e RNase inhibitor activity. Homogenates of 0 . 1 % T r i t o n X 1 0 0 were d i l u t e d with i c e - c o l d . 0 2 M T r i s - H C l , pH 7«8 b u f f e r to a p r o t e i n c o n c e n t r a t i o n of 2 . 0 mg/ml and 0 . 2 ml a l i q u o t s c o n t a i n i n g 0 r 2 mg p r o t e i n were assayed f o r i n h i b i t i o n o f the a c t i v i t y of 0 . 5 ng. p a n c r e a t i c RNase A i n the presence of 1 . 0 mM EDTA. I n c u b a t i o n was f o r 60 minutes. t  - 144 -  i n h i b i t o r a c t i v i t y and f r e e pH 7»8 RNase a c t i v i t y a rapid increase  i n the f r e e RNase i n h i b i t o r  indicates  activity/free  pH 7.8 RNase a c t i v i t y r a t i o between day 10 and day 18. There i s a s l i g h t decrease i n t h i s r a t i o between day 18 and day  22 s i n c e f r e e RNase i n h i b i t o r a c t i v i t y has a t t a i n e d i t s  a d u l t l e v e l by 18 days whereas f r e e pH 7»8 RNase a c t i v i t y continues to increase  up t o 22 days.  r a t i o again increases  and c o n t i n u e s to i n c r e a s e  adulthood due t o the g r a d u a l d e c l i n e  A f t e r age  22 days t h i s throughout  i n f r e e pH 7.8 RNase  activity. 285 Suzuki and Takahashi  have s t u d i e d  the developmental  change i n f r e e RNase i n h i b i t o r a c t i v i t y i n the c y t o s o l t i o n of r a t c e r e b r a l cortex.  frac-  These i n v e s t i g a t o r s found  that  t h i s component o f the RNase enzyme system e x h i b i t s a sharp peak i n s p e c i f i c a c t i v i t y between the 5th birth, falls  and 10th  day a f t e r  t o near n e o n a t a l l e v e l s by day 13 and remains  r e l a t i v e l y constant t h e r e a f t e r .  However, i n the p r e s e n t  study u s i n g whole c e l l homogenates o f whole b r a i n , f r e e RNase inhibitor s p e c i f i c a c t i v i t y exhibited days a f t e r which i t i n c r e a s e d l e v e l s by 18 days.  low l e v e l s up t o age 10  r a p i d l y and plateaued a t a d u l t  Two o f the most obvious e x p l a n a t i o n s f o r  t h i s apparent d i s c r e p a n c y are t h a t  (1)  dependent i n t r a c e l l u l a r r e d i s t r i b u t i o n  t h e r e occurs an ageo f f r e e RNase  inhibitor  such t h a t the developmental p r o f i l e o f t h i s a c t i v i t y i n the  - 145  c y t o s o l i s n o t c h a r a c t e r i s t i c o f the developmental  profile  of f r e e RNase i n h i b i t o r s p e c i f i c a c t i v i t y i n the whole c e l l homogenate, and/or  (2) t h e r e a r e r e g i o n - s p e c i f i c  i n the developmental p a t t e r n  differences  o f f r e e RNase i n h i b i t o r a c t i v i t y .  The composite r e s u l t s obtained u s i n g whole r a t b r a i n may not be r e p r e s e n t a t i v e  o f the developmental changes i n f r e e RNase  i n h i b i t o r a c t i v i t y i n d i f f e r e n t brain regions. explanation  I f t h i s second  i s the c o r r e c t one, then t h e r e i s a s t r i k i n g and,  no doubt, f u n c t i o n a l l y s i g n i f i c a n t d i f f e r e n c e between the cerebral cortex  and the r e s t o f the b r a i n i n the developmental  p r o f i l e o f f r e e RNase i n h i b i t o r s p e c i f i c a c t i v i t y .  A study o f  developmental changes i n t h i s component o f the RNase enzyme system i n d i f f e r e n t r e g i o n s of the b r a i n such as the c e r e b r a l cortex,  c e r e b e l l u m and brainstem may y i e l d f u r t h e r i n s i g h t  i n t o the r o l e o f RNase i n h i b i t o r i n RNA metabolism and i n t o the f u n c t i o n a l importance o f i t s presence i n such h i g h tration i n brain.  Takabashi and S u z u k i ^ - * have  concen-  reported  s i g n i f i c a n t d i f f e r e n c e s i n t h e s p e c i f i c a c t i v i t y o f f r e e RNase i n h i b i t o r i n d i f f e r e n t r e g i o n s o f the a d u l t r a b b i t I t must be emphasized  brain.  t h a t a developmental i n c r e a s e i n  enzymatic a c t i v i t y measured i n v i t r o does not prove  increased  enzyme f u n c t i o n i n v i v o where enzyme a c t i v i t y may be by s u b s t r a t e  a c c e s s i b i l i t y and o t h e r c o n t r o l f a c t o r s .  mental i n c r e a s e s  regulated Develop-  i n enzyme a c t i v i t y do . n o t n e c e s s a r i l y  146 -  i n d i c a t e a s p e c i f i c increase  i n enzyme amount s i n c e  increased  a c t i v i t y could a l s o r e s u l t from a n o n - s p e c i f i c i n c r e a s e i n the r a t e o f p r o t e i n s y n t h e s i s , a c t i v a t i o n o f p r e c u r s o r s ( a c t i v a t i o n o f bound o r l a t e n t formsoof the enzyme) o r t o a decrease i n the r a t e o f d e g r a d a t i o n o f the enzyme. the  observed changes i n enzyme a c t i v i t y d u r i n g  s i g n i f y an a c t u a l i n c r e a s e  Whether  development  i n enzyme p r o t e i n due t o an  enhanced r a t e o f de novo enzyme s y n t h e s i s ,  o r t o developmental  changes i n f a c t o r s r e g u l a t i n g the a c t i v i t y o f a constant amount of enzyme, remains t o be shown.  Also,  i t must be  s t r e s s e d t h a t l e v e l s o f RNase a c t i v i t y expressed i n 0,1$ T r i t o n X100 homogenates are an index o f the t o t a l RNase of the t i s s u e .  Functional  capacity  l e v e l s o f RNase a c t i v i t y i n the c e l l  may be more c l o s e l y approximated by the l e v e l o f a c t i v i t y exp r e s s e d i n i s o t o n i c sucrose homogenates,  «*»*»**  IV. DISCUSSION  The presence d e g r a d a t i o n of RNA  of m u l t i p l e enzymes r e s p o n s i b l e f o r the i n b r a i n i s c o n s i s t e n t with the h e t e r o -  g e n e i t y of t h i s enzyme system i n other mammalian t i s s u e s and i n p r o c a r y o t e s where i n the case of w i l d - t y p e E. a t l e a s t seven d i s t i n c t RNases have been r e p o r t e d .  coli The  t r u e complexity of t h i s enzyme system i n mammalian t i s s u e s has yet t o be e l u c i d a t e d .  I t i s l i k e l y t h a t the RNases  c h a r a c t e r i z e d i n the present study, as w e l l as  those  c h a r a c t e r i z e d by other i n v e s t i g a t o r s i n other mammalian t i s s u e s , are the most r e a d i l y d e t e c t a b l e components of an enzyme system c o n s i s t i n g of many other a d d i t i o n a l enzymes of v a r i o u s s p e c i f i c i t i e s capable of c l e a n i n g phosphodiester bonds i n RNA.  A l l o f the enzymes i d e n t i f i e d i n the present  i n v e s t i g a t i o n p r o b a b l y f u n c t i o n i n the exo- and/or endonuc l e o l y t i c cleavage of RNA. which are thought  Highly specific  endoribonucleases  t o p a r t i c i p a t e i n the m a t u r a t i o n a l p r o c e s -  s i n g of p r e c u r s o r t r a n s c r i p t i o n products i n t o mature RNA  functionally  molecules would not c o n t r i b u t e t o the  findings  r e p o r t e d here s i n c e such a c t i v i t i e s would not be expected y i e l d s u f f i c i e n t a c i d - s o l u b l e products t o be d e t e c t a b l e by the assay employed i n the present  147  study.  to  - 148 -  4.0 R e g u l a t i o n o f the I M J Vivo o f RNases i n b r a i n  Function  The f a c t t h a t a l a r g e f r a c t i o n o f the d e t e r g e n t e x t r a c t a b l e RNase a c t i v i t y i s n o t expressed  i n assays o f  i s o t o n i c sucrose homogenates i n d i c a t e s t h a t c o n s i d e r a b l e amounts o f the maximal l e v e l s o f a l l three RNases a r e present i n the c e l l s o f the b r a i n i n a l a t e n t , n o n - f u n c t i o n a l state.  I n s i t u , l a t e n t RNase may be present bound t o and/or  compartmentalized w i t h i n s u b c e l l u l a r o r g a n e l l e s thus  rendering  the enzymes i n a c c e s s i b l e t o and i n a c t i v e a g a i n s t s u b s t r a t e * T h i s i s supported  by i n t r a c e l l u l a r d i s t r i b u t i o n s t u d i e s which  have shown t h a t while t h e crude m i t o c h o n d r i a l f r a c t i o n only 1 6 $ o f the t o t a l c e l l u l a r RNA,-^  11  contains  5 2 $ o f the recovered  t o t a l c e l l u l a r RNase a c t i v i t y i s l o c a l i z e d i n t h i s  fraction.  Moreover, the microsomal f r a c t i o n which c o n t a i n s 3 7 $ of the t o t a l c e l l u l a r RNA^ t o t a l expressed  11  c o n t a i n s comparatively  c e l l u l a r RNase a c t i v i t y .  little  These  of the  observations  imply t h a t t h e f u n c t i o n a l l e v e l s o f a c t i v i t y o f l a r g e amounts of the c e l l u l a r RNA-depolymerizing enzymes i s c o n t r o l l e d by r e s t r i c t i o n s upon enzyme-substrate i n t e r a c t i o n due t o the s e g r e g a t i o n o f enzyme and s u b s t r a t e molecules i n t r a c e l l u l a r membrane compartments. modulations o f the steady  into  different  I t also follows that  s t a t e r a t e o f RNA c a t a b o l i s m i n  the i n t a c t c e l l may be a f f e c t e d by f a c t o r s a l t e r i n g o r g a n e l l e membrane p e r m e a b i l i t y or s h i f t i n g the r a t i o o f bound t o f r e e enzyme.  149 -  S e v e r a l workers y  ? t J  -> have r e p o r t e d  t h a t a c i d RNase  i n r a t l i v e r i s a s s o c i a t e d w i t h lysosomes and e x h i b i t s the c h a r a c t e r i s t i c s t r u c t u r e - l i n k e d l a t e n c y o f other acid hydrolases.  lysosomal  A s i g n i f i c a n t f r a c t i o n o f the t o t a l  cellular  pH 7.8 RNase and pH 9«5 RNase a c t i v i t i e s have a l s o been found i n lysosomes i n l i v e r .  I n view o f the s i m i l a r i t i e s o f the  three RNase a c t i v i t i e s found i n the present  study t o those o f  r a t l i v e r , i t seems l i k e l y t h a t a l a r g e p o r t i o n o f the t o t a l a c t i v i t y which i s recovered  i n the crude m i t o c h o n d r i a l  frac-  t i o n of r a t b r a i n may a l s o be a s s o c i a t e d with the lysosomal components o f t h i s The  fraction.  compartmentalization of a c i d h y d r o l a s e s  lysosomes may serve  within  t o r e g u l a t e t h e i r a c t i v i t y and prevent  the i n d i s c r i m i n a t e a c t i o n o f these p o t e n t i a l l y d e s t r u c t i v e enzymes.  I n view o f the p r o t e c t i v e f u n c t i o n o f the lysosomal  membrane any i n j u r y t o the membrane r e s u l t i n g i n i n c r e a s e d p e r m e a b i l i t y would be expected t o enhance the r a t e o f c a t a bolism  o f cytoplasmic  constituents.  That s e g r e g a t i o n o f  a c i d RNase w i t h i n lysosomes i s an important mode o f regul a t i n g the a c t i v i t y o f t h i s enzyme i n the i n t a c t c e l l i s i n agreement with the high l e v e l o f a c i d RNase i n the c e l l , the absence o f any known s p e c i f i c i n h i b i t o r s o f a c i d RNase, and the i n a c t i v i t y toward t h i s enzyme o f pH 7*8 RNase i n h i b i t o r . Hence, a d i s c u s s i o n o f the r e g u l a t i o n o f the i n v i v o  activity  of a c i d RNase must take i n t o c o n s i d e r a t i o n the mode o f a c t i o n  - 15© -  of  lysosomal enzymes and the r e g u l a t i o n of lysosomal  function. Although the presence  of acid hydrolases w i t h i n  lysosomes i s w e l l e s t a b l i s h e d , t h e i r s i t e o f f u n c t i o n i s still to  i n dispute.  Two hypotheses  have been formulated as  how lysosomes perform t h e i r d i g e s t i v e f u n c t i o n s i n the  intact c e l l .  A c c o r d i n g to one, m a t e r i a l i s encapsulated  by membrane t o form e n d o c y t o t i c v a c u o l e s which then f u s e with lysosomal membranes and empty t h e i r contents i n t o the lysosome where they are degraded and the d e g r a d a t i o n products r e c y c l e d t o t h e s c y t o s o l . A c c o r d i n g t o t h i s model lysosomal contents n o r m a l l y remain c o n t i n u o u s l y d e l i m i t e d by an u n i n t e r r u p t e d membrane which s h i e l d s the r e s t o f the cytoplasm a g a i n s t a t t a c k by lysosomal enzymes.  T h i s hypo-  t h e s i s i s supported by o b s e r v a t i o n s t h a t ( l ) p h a g o c y t i z e d and p i n o c y t i z e d m a t e r i a l i s found i n lysosomes{ (2) primary lysosomes fuse r e a d i l y w i t h e n d o c y t o t i c  vacuoles-^ 'as 2  w e l l as with other lysosomes (but not with m i t o c h o n d r i a o r nuclei) 314,315 f  a  n  d  ^  a  n  intralysosomal s i t e of a c t i o n f o r  lysosomal enzymes, most o f which have a c i d pH optima, i s compatible with normal i n t r a l y s o s o m a l pH which has been r e p o r t e d t o be 6.34  for rat liver  lysosomes.  A c c o r d i n g t o the second h y p o t h e s i s , the enzymes comp a r t m e n t a l i z e d w i t h i n lysosomes a r e n o r m a l l y i n a c t i v e and p a r t i c i p a t e i n the c a t a b o l i s m o f c e l l  c o n s t i t u e n t s o n l y upon  -  151  -  317 r e l e a s e i n t o the s o l u b l e c e l l sap.-'  Thus, only  f  lysosomal  enzymes which occur f r e e i n the c y t o s o l r e p r e s e n t l i c a l l y a v a i l a b l e enzyme.  Support f o r t h i s h y p o t h e s i s  comes from s e v e r a l l i n e s of i n v e s t i g a t i o n . observed "*" "" 3  8  321  that increased  fragility  I t has  a c i d hydrolases  been  of lysosomes i s  c o r r e l a t e d w i t h enhanced l e v e l s of v a r i o u s a c i d i n the c y t o s o l .  metabo-  hydrolases  T h i s i m p l i e s r e l e a s e or leakage o f lysosomal i n t o the c y t o s o l and  t h i s p r o c e s s appears to  be more pronounced under c o n d i t i o n s of c e l l s t r e s s than d u r i n g normal c e l l f u n c t i o n i n g .  Some forms of s t r e s s t h a t  have r e s u l t e d i n an i n c r e a s e i n a c i d RNase a c t i v i t y of  the  322  c y t o s o l i n l i v e r c e l l s i n c l u d e hypophysectomy,-^ lectomy, and  3 2 2  and  carcinogenesis. ^  reported  to precede and  during v i r a l  Furthermore, the  1 9  a c t i v a t i o n of a c i d h y d r o l a s e s  3 2 9  An  release  i n t o the c y t o s o l has  contribute to c e l l  infection.  adrena-  been  degeneration  i n c r e a s e i n the r a t i o of f r e e  to bound lysosomal enzyme a c t i v i t y d u r i n g t i s s u e d e g e n e r a t i o n has  a l s o been r e p o r t e d . 5 6 - 2 5 8 , 2  323-325  F i n a l l y , several r e p o r t s ^ " " 3 2  3 2 8  indicate that  a c t i v i t y of lysosomal enzymes i n c r e a s e with c e l l Lysosomal membrane l y s i s and/or a c i d hydrolase under c o n d i t i o n s of extreme c e l l u l a r t i o n a l ) s t r e s s may  thus be  p r o c e s s of c e l l aging  and  (metabolic  the  age.  induction and  func-  important mechanisms i n the autolysis.  The  r o l e of lysosomal  -  152  -  RNases and other a c i d h y d r o l a s e s i n tumor r e g r e s s i o n and c e l l degeneration may,  however, be secondary  to a general  enhancement i n the r a t e of c e l l or t i s s u e c a t a b o l i s m . Although  a c i d h y d r o l a s e s would be expected  to have  much reduced a c t i v i t y a t the normal p h y s i o l o g i c a l pH  of  the c y t o s o l , some c o n d i t i o n s of c e l l u l a r s t r e s s under which fragmentation o f lysosomes may concomitant  occur are accompanied by a  a c i d s h i f t i n the normal pH of the c y t o s o l  hypoxia, eschemia,-^  0  (e.g.,  a c i d o s i s , neoplasm-^**) thus r e s u l t i n g  i n more f a v o r a b l e c o n d i t i o n s f o r the d e g r a d a t i o n of c y t o p l a s m i c c o n s t i t u e n t s by lysosomal enzymes. A method t h a t has been used to g a i n i n f o r m a t i o n r e g a r d i n g the f u n c t i o n and  s i t e of^ a c t i o n of lysosomal enzymes  has been the p h y s i o l o g i c a l p e r t u r b a t i o n of i n t a c t Thus, Pontremoli et a l . - ^  1  animals.  have r e p o r t e d the occurrence  i n c r e a s e d numbers o f lysosomes,  changes i n t h e i r morphology  i n c l u d i n g membrane breaks, as w e l l as r e l e a s e i n t o the of  s e v e r a l lysosomal enzymes i n the hepatocytes  cold-stressed rabbits.  of  cytosol  of s t a r v e d or  I f such a phenomenon r e p r e s e n t e d a  g e n e r a l mechanism of m e t a b o l i c response  to c e l l u l a r  stress,  i t would be expected t h a t c y t o s o l l e v e l s of a c i d RNase would a l s o be i n c r e a s e d i n s t a r v a t i o n or c o l d - s t r e s s e d c e l l s . However, such r e s u l t s have not been r e p o r t e d .  De Lamirande  202  and A l l a r d  have, i n f a c t , r e p o r t e d t h a t f a s t i n g  slightly  -  153  -  lowers the l e v e l s o f both a c i d RNase and pH ?.8 RNase a c t i v i t y without  a l t e r i n g the i n t r a c e l l u l a r  distribution 332  of these  enzymes i n r a t l i v e r .  Deckers-Passau e t a l . ^  have r e p o r t e d d i f f e r e n t i a l r e a c t i o n s o f s e v e r a l  lysosomal  enzymes t o changes i n p h y s i o l o g i c a l s t a t e o f the a n i m a l . Dynamic changes i n membrane p e r m e a b i l i t y may be o f c r i t i c a l importance i n the r e g u l a t i o n o f i n v i v o RNase a c t i v i t y s i n c e such changes w i l l determine the a c c e s s i b i l i t y of these enzymes t o t h e i r s u b s t r a t e s . s t a n d i n g the r o l e o f lysosomal  The key t o under-  enzyme f u n c t i o n i n c e l l u l a r  metabolism may thus l i e i n i d e n t i f y i n g those f a c t o r s r e s ponsible f o r maintaining i n t e g r i t y o f lysosomal  the s t r u c t u r a l and f u n c t i o n a l  membranes o r a f f e c t i n g  membrane p e r m e a b i l i t y thereby r e l e a s e o f lysosomal Demin and N e c h a e v a  3 3 3  lysosomal  f a c i l i t a t i n g or i n h i b i t i n g the  enzymes i n t o the c y t o s o l .  In this  regard,  have shown t h a t a d r e n a l i n a t 3 . 3 x 1 0 " M 3  causes a 27% a c t i v a t i o n o f a c i d RNase a c t i v i t y i n c e r e b r a l c o r t e x crude m i t o c h o n d r i a l f r a c t i o n incubated  i n isotonic  sucrose, whereas a c e t y l c h o l i n e a t 6 x 10""^ M i n h i b i t s the normal leakage o f l a t e n t a c i d RNase from t h i s s u b c e l l u l a r fraction.  I n l e u c o c y t e lysosomes, however, catecholamines  have been shown-^  t o i n h i b i t lysosomal  enzyme r e l e a s e and  t h i s e f f e c t has been thought to be mediated through enhanced l e v e l s o f i n t r a c e l l u l a r cAMP.  C y c l i c AMP has a l s o been  r e p o r t e d to i n c r e a s e the p e r m e a b i l i t y o f lysosomal  membranes  - 15  k  -  with r e s p e c t t o a c i d phosphatase and 3-glucuronidase i n r a t liver^  0  and to a c i d RNase se d\ d u r i n g degeneration  o f hormone-  256 dependent mammary tumors. Some o f the d i s c r e p a n c i e s between these f i n d i n g s may be e x p l a i n e d  i n terms o f t i s s u e - s p e c i f i c d i f f e r e n c e s i n the  response o f lysosomes t o these r e a g e n t s .  Also, since l y s o -  201 341 342 somes have been shown  •  s i z e and enzyme content,  %  J  t o be heterogeneous i n t h e i r  i t i s possible that v a r i a b l e a c t i v a -  t i o n or r e l e a s e o f lysosomal  enzymes may be due t o s e l e c t i v e  membrane p e r m e a b i l i t y changes i n s p e c i f i c c l a s s e s o f lysosomes or t o d i f f e r e n c e s i n p e r m e a b i l i t y of the lysosomal specific acid  membrane t o  hydrolases.  The i m p l i c a t i o n o f cAMP i n the r e g u l a t i o n o f lysosomal membrane p e r m e a b i l i t y combined with r e p o r t s o f n e u r o t r a n s m i t t e r - s t i m u l a t e d adenyl c y c l a s e i n b r a i n suggests a p l a u s i b l e mechanism by which the a c t i v a t i o n o f post s y n a p t i c c e l l face neurotransmitter metabolism.  sur-  r e c e p t o r s may modulate n e u r o n a l RNA  O s c i l l a t o r y changes i n neuronal RNA  content  accompanying e l e c t r o p h y s i o l o g i c a l a c t i v i t y - ^ may thus be 1  a t t r i b u t a b l e t o the i n t r a c e l l u l a r r e l e a s e of a r e c e p t o r transduced  metabolic  of RNA degradation substrate.  demand s i g n a l which modulates the r a t e  by r e g u l a t i n g RNase a c c e s s i b i l i t y t o  155  -  There have been c o n s i s t e n t f i n d i n g s o f a p o s i t i v e c o r r e l a t i o n between e l e v a t e d RNase a c t i v i t y o r decreased RNase i n h i b i t o r t o pH 7.8 RNase a c t i v i t y r a t i o and e i t h e r peduced r a t e s o f p r o t e i n s y n t h e s i s or a g e n e r a l catabolic a c t i v i t y .  increase i n  T h i s i s i n agreement with the view t h a t  f u n c t i o n a l l e v e l s o f these enzymes may l i m i t the a v a i l a b i l i t y and  frequency o f t r a n s l a t i o n o f mRNA molecules.  Furthermore,  the f i n d i n g t h a t reduced l e v e l s o f f r e e RNase i n h i b i t o r o r enhanced l e v e l s o f f r e e pH 7*8 RNase a c t i v i t y i s c o r r e l a t e d with  enhanced polysome breakdown, decreased c a p a c i t y o f  ribosomes f o r a c c e p t i n g o r t r a n s l a t i n g mRNA, decrease i n f u n c t i o n a l mRNA, and g e n e r a l  impairment i n the f u n c t i o n i n g  of the t r a n s l a t i o n apparatus suggests t h a t RNase i n h i b i t o r may p l a y a s i g n i f i c a n t r o l e i n r e g u l a t i n g p r o t e i n s y n t h e s i s by p r e s e r v i n g the s t r u c t u r a l and f u n c t i o n a l i n t e g r i t y o f polysomes. S e v e r a l workers have i n v e s t i g a t e d the d i s a g g r e g a t i o n of i s o l a t e d polysomes. the d i s a g g r e g a t i o n  Eker and P i h l ^ 3  3  have r e p o r t e d  that  o f polysomes i n v i t r o and the time-  dependent a c t i v a t i o n o f a l a t e n t RNase a c t i v i t y w i t h polysome p r e p a r a t i o n s  associated  i s prevented by 1 . 0 mM d i t h i o -  t h r e i t o l o r 1 . 0 mM g l u t a t h i o n e .  The e f f e c t o f these compounds  was not due t o a d i r e c t i n h i b i t i n g e f f e c t on the enzyme s i n c e these compounds do not s i g n i f i c a n t l y i n h i b i t the a c t i v i t y o f  - 156 -  p a r t i a l l y p u r i f i e d RNase p r e p a r a t i o n s which e x h i b i t s t i m u l a t i o n by pCMB n o r i n h i b i t i o n o f bovine RNase A.  I t has a l s o been r e p o r t e d  s t a b i l i z e s polysomes,344-346 in vitro.  m  RNAf  pancreatic  that r a t l i v e r  345,347  neither  cytosol  HnRNA ^ 3  a  n  d  8  RNase i n h i b i t o r has been demonstrated by s e v e r a l  workers »345*357 ^ 222  disaggregation.  0  s t a b i l i z e polysomes and prevent t h e i r  The presence o f excess f r e e RNase i n h i b i t o r  i n the c y t o s o l has thus been a s s i g n e d the p h y s i o l o g i c a l r o l e of s t a b i l i z i n g the t r a n s l a t i o n apparatus by p r e v e n t i n g the d e g r a d a t i o n o f polysomal RNA. The r e l e a s e RNase could  and a c t i v a t i o n o f i n h i b i t o r - b o u n d  pH 7.8  be e f f e c t e d by any endogenous f a c t o r capable o f  d e s t a b i l i z i n g the i n h i b i t o r - R N a s e c i a t i n g the i n h i b i t o r - R N a s e  i n t e r a c t i o n and d i s s o -  complex.  Factors  such as reduced  s u l f h y d r y l compounds capable o f modulating the i n t e r a c t i o n between f r e e RNase i n h i b i t o r and f r e e pH 7«8 RNase s h i f t the dynamic e q u i l i b r i u m between  could  RNase-inhibitor  complexes, f r e e pH 7*8 RNase, and f r e e RNase i n h i b i t o r , thereby r e g u l a t i n g t h e r a t e o f RNA d e g r a d a t i o n . brium between these molecular s p e c i e s  The e q u i l i -  c o u l d a l s o be con-  t r o l l e d by c o n s t r a i n t s t o i n t e r a c t i o n between any two components.  Thus, the i n t r a c e l l u l a r d i s t r i b u t i o n data  i n d i c a t e t h a t the compartmentalization o f f r e e RNase i n h i b i t o r may be of importance i n determining t h e d i f f e r e n t i a l  - 157 -  rates The  o f RNA d e g r a d a t i o n i n the c e l l nucleus and cytoplasm.  absence o f f r e e RNase i n h i b i t o r a c t i v i t y i n the n u c l e u s  i n d i c a t e s t h a t the n u c l e o - c y t o p l a s m i c envelope f u n c t i o n s a permeability  as  b a r r i e r which prevents the l a r g e excess o f  f r e e RNase i n h i b i t o r i n the c y t o s o l from e q u i l i b r a t i n g w i t h the  nucleoplasm. It  must be emphasized t h a t the l e v e l s o f RNase a c t i v i t y  measured under o p t i m i z e d c o n d i t i o n s  i n v i t r o cannot be taken  as a r e l i a b l e index o f enzyme f u n c t i o n The  i n the i n t a c t  cell.  a c t i v i t y o f these enzymes under normal p h y s i o l o g i c a l  conditions  The  contributions  o f each o f t h e  i s not known.A Shree RNases i d e n t i f i e d i n t h i s  study t o c e l l u l a r RNA d e g r a d a t i o n under normal p h y s i o l o g i c a l conditions  i s a l s o not apparent.  f e r e n t i a l substrate with r e s p e c t  I n f o r m a t i o n as t o the p r e -  s p e c i f i c i t i e s o f each of the three enzymes  to d i f f e r e n t classes  (e.g., rRNA, tRNA, HnRNA)  of b r a i n RNA may p r o v i d e a b e t t e r understanding o f t h e i r function  i n vivo.  I n view o f the f a c t s t h a t f r e e pH 7.8  ( l ) t h e r e i s no  RNase a c t i v i t y i n the c y t o s o l ;  e x h i b i t s n e g l i g i b l e a c t i v i t y a t pH 7A  detectable  (2) pH 6.7  and i s markedly  b i t e d by p h y s i o l o g i c a l t o n i c i t y above pH 6, and (3) pH  RNase inhi9*5  RNase e x h i b i t s o n l y 68% of i t s o p t i m a l a c t i v i t y when assayed a t pH 7«8 and i s a l s o markedly i n h i b i t e d by p h y s i o l o g i c a l i o n i c strength,  i t would seem t h a t i n t r a c e l l u l a r RNA would  -  be p r o t e c t e d  158  -  from the a c t i o n o f these enzymes under normal  physiological conditions.  The measured l e v e l s o f RNase  a c t i v i t i e s hence probably r e p r e s e n t  total cellular  capacity  f o r RNA d e g r a d a t i o n and do not r e f l e c t f u n c t i o n a l l e v e l s o f RNase a c t i v i t i e s p a r t i c i p a t i n g i n t h e breakdown o f RNA molecules under normal p h y s i o l o g i c a l c o n d i t i o n s i n v i v o . It i s , nevertheless, steady-state  c l e a r that c e r t a i n perturbations  i n the  p h y s i o l o g i c a l c o n d i t i o n s c o u l d r e s u l t i n an  a c t i v a t i o n o f the l a t e n t RNA-degrading c a p a c i t y o f b r a i n c e l l s and  hence i n an enhanced r a t e o f RNA  4.1  C o r r e l a t i o n o f Developmental Changes i n the Content, S y n t h e s i s and Degradation of RNA i n b r a i n Despite  degradation.  p r e v i o u s l y mentioned r e s e r v a t i o n s , i t seems  worthwhile t o make some t e n t a t i v e i n f e r r e n c e s as t o the p o s s i b l e f u n c t i o n a l s i g n i f i c a n c e o f the developmental changes i n RNase a c t i v i t i e s observed under t h e c o n d i t i o n s of the present  study.  How are these changes c o r r e l a t e d with develop-  mental changes i n RNA s y n t h e s i s and c e l l u l a r RNA content? F i g u r e 1 9 i s a composite developmental p r o f i l e o f the t o t a l RNA degradative  c a p a c i t y o f whole b r a i n c o n s t r u c t e d by  summing the e x p e r i m e n t a l l y  measured a c t i v i t i e s o f each  i n d i v i d u a l RNase (pH 6 . 7 RNase, pH 9 . 5 RNase, and f r e e p i 7 . 8 RNase) a t a g i v e n age. The  i n c r e a s e i n t o t a l c e l l u l a r RNA h y d r o l y t i c c a p a c i t y  per gram wet weight of whole b r a i n between 1 0 and 2 0 days  -  5  10  15  20  25  30  159  -  35 days  Post-natal  5 months  Age  FIGURE 19. T o t a l RNA d e g r a d a t i v e c a p a c i t y o f whole b r a i n as a f u n c t i o n o f the p o s t n a t a l age o f r a t . T h i s i s a t h e o r e t i c a l curve c o n s t r u c t e d from d a t a shown i n F i g u r e s 1 5 • 16 and 17 by summing the a c t i v i t y o f each i n d i v i d u a l RNase assayed a t pH 6.7. 9.5 and 7.8 ( f r e e pH 7.8 RNase a c t i v i t y ) a t each age.  -  would be  1$9  -  expected to correspond to an i n c r e a s e  r a t e of RNA  degradation, unless  p o r t i o n a l increase  i t i s accompanied by a p r o -  i n regulatory  a c t i v i t y of RNases.  mechanisms r e s t r i c t i n g  This increase  capacity d i r e c t l y p a r a l l e l s  i n the i n v i v o  i n b r a i n RNA  degradative  the concurrent i n c r e a s e  and  c e l l u l a r RNA  SNA  content of both the c e r e b r a l c o r t e x and  i n organ  content f o r i t has been shown t h a t the  continues to i n c r e a s e up accumulation ceases.  to age  total  whole b r a i n  20 days at which time  Hence, the r a t e of t o t a l RNA  exceeds the r a t e of t o t a l RNA  the  d e g r a d a t i o n by a  this  synthesis  decreasing  increment which approaches zero by age  20 days when a steady  s t a t e balance between the r a t e s of RNA  s y n t h e s i s and  t i o n i s a t t a i n e d and  degrada-  a constant organ content of RNA  is  maintained. P o s s i b l y the most r e l i a b l e RNA  synthesis  i s t h a t obtained  data on the r a t e of  with t i s s u e s l i c e s .  tunately,  developmental data f o r the r a t e of t o t a l  synthesis  i n whole b r a i n s l i c e s i s not a v a i l a b l e .  developmental s t u d i e s of the r a t e of RNA 46 cerebral cortex  slices  synthesis declines age  10 days and  20 days.  synthesis  total  UnforRNA However, i n rat  349 *  J  7  i n d i c a t e t h a t the r a t e  (undergoing a 3 to 4 - f o l d  a t t a i n s adult  of  decrease) a f t e r  steady s t a t e l e v e l s by  about  - 161  -  S i n c e the r a t e of t o t a l RNA the time when organ RNA RNA  degradative  capacity  synthesis declines  content i s accumulating and (per gram wet  i n c r e a s i n g , i t f o l l o w s t h a t any  during  the  total  weight whole b r a i n ) i s  concomitant i n c r e a s e i n the  r a t e of RNA  d e g r a d a t i o n i s l i m i t e d by the f a c t  r a t e of RNA  d e g r a d a t i o n does not exceed the d e c l i n i n g r a t e of  RNA  t h a t the  total  synthesis. S i n c e the steady s t a t e a d u l t r a t e of RNA  the f i n a l organ and 20 days and  c e l l u l a r RNA  content i s a t t a i n e d by  subsequently s u s t a i n e d ,  r a t e of t o t a l RNA  synthesis  and  age  i t also follows that  the  d e g r a d a t i o n and hence the f u n c t i o n a l l e v e l  o f RNase a c t i v i t y must a l s o reach steady s t a t e c o n d i t i o n s t h i s age.  by  However, a f t e r a t t a i n i n g peak l e v e l s at 20 days,  the  RNA-degradative c a p a c i t y of whole b r a i n subsequently d e c l i n e s up t o age  30 days.  Since between 20 and  net accumulation i n organ RNA, s y n t h e s i s remains constant,  and  3° days there i s no  s i n c e the r a t e of  RNA  i t must be t e n t a t i v e l y concluded  t h a t the d e c l i n e i n maximal RNA-degradative c a p a c i t y maximal RNase a c t i v i t y i s not accompanied by a  on  parallel  d e c l i n e i n the f u n c t i o n a l c o n t r i b u t i o n of these enzymes to the r a t e of t o t a l RNA  degradation i n v i v o .  Thus, although  the t o t a l RNA-degradative c a p a c i t y d e c l i n e s between age and  20  30 days, c o m p a t i b i l i t y w i t h the other events observed  d u r i n g t h i s time r e q u i r e s t h a t the amount of t h i s which i s f u n c t i o n a l i n v i v o must remain constant  capacity a f t e r 20  - 162  days.  -  Control factors must regulate and maintain a constant  l e v e l of RNase a c t i v i t y a f t e r age 20 days despite the i n t o t a l RNase capacity.  decline  Hence, an increasingly greater  f r a c t i o n of the t o t a l RNA-degradative capacity or maximal RNase a c t i v i t y i s functional i n vivo.  For the functional or  expressed l e v e l of RNase a c t i v i t y to remain constant while the maximal RNase a c t i v i t y measured i n v i t r o decreases, there must occur either a p r e f e r e n t i a l dropout i n the amount of nonfunctional, latent, RNase a c t i v i t y and/or a  proportional  decline of both functional and non-functional RNase  (activity)  concomitant with a progressive relaxation of control factors normally r e s t r i c t i n g the expression of RNase a c t i v i t y .  This  might be thought of as increasing the i n vivo s p e c i f i c  activity  of the enzymes. Elucidation of the precise r e l a t i o n s h i p of the developmental changes i n the measured l e v e l s of RNase a c t i v i t y to the changes i n the p h y s i o l o g i c a l l y functional l e v e l s of a c t i v i t y of these enzymes w i l l require a developmental study of the rate of turnover of t o t a l RNA  i n whole brain.  Such data would  provide a more r e l i a b l e index of the i n vivo rate of t o t a l RNA  degradation.  The difference curve between the develop-  mental p r o f i l e s f o r t o t a l RNA  turnover and f o r t o t a l  RNA-  degradative capacity would then represent developmental changes i n control factors regulating the expression of maximal RNase a c t i v i t y .  § 163 -  4.2 Regional Differences i n the Metabolism of RNA and i n the Functional Roles of RNases i n brain The developmental pattern of RNA depolymerase a c t i v i t i e s can be correlated with the occurrence of both quantitative and q u a l i t a t i v e changes i n the pattern of RNA synthesis during brain development.  On both a per gram  wet weight brain and per milligram protein basis neonatal rat brain contains a higher amount of pH 6.7 RNase a c t i v i t y than adult r a t b r a i n .  Older r a t s have higher l e v e l s of both  alkaline RNase a c t i v i t i e s .  The higher t i t e r of acid RNase  during the c e l l p r o l i f e r a t i o n stage of brain development suggests a possibly s p e c i f i c r o l e f o r t h i s enzyme i n the catabolism of RNA during c e l l d i v i s i o n .  Presumably, RNA  metabolism during c e l l d i f f e r e n t i a t i o n e n t a i l s a rapid degradation of RNA and the synthesis of new species of RNA molecules.  The r i s e i n a l k a l i n e RNase a c t i v i t i e s between day 7  and day 22 may be associated with the replacement of the mRNA molecules coding f o r mitosis and c e l l d i f f e r e n t i a t i o n by RNA molecules required f o r the maintenance functions of nondividing brain c e l l s .  Thus, these enzymes may p a r t i c i p a t e  i n the turnover of the RNA content of f u l l y - d i f f e r e n t i a t e d cells. This l i n e of reasoning gives r i s e to the expectation that s i g n i f i c a n t differences i n the developmental p r o f i l e s  - 164 -  of these RNase a c t i v i t i e s may occur i n d i f f e r e n t b r a i n regions.  Unlike  the c e r e b r a l c o r t e x  i n which c e l l d i v i s i o n  i s almost complete a t b i r t h , the c e r e b e l l u m i n the r a t e x h i b i t s r a p i d c e l l p r o l i f e r a t i o n up t o 1 6 t o 2 0 days a f t e r birth. ?»308,309 k  Hence, s i n c e a c i d RNase a c t i v i t y i s h i g h e s t  d u r i n g t h e c e l l p r o l i f e r a t i o n stage o f b r a i n development, the d e c l i n e i n s p e c i f i c a c t i v i t y o f a c i d RNase may occur a t a l a t e r p o s t n a t a l age i n the cerebellum than i n the c e r e b r a l cortex.  A l s o , the r i s e i n a l k a l i n e RNase a c t i v i t i e s might  be expected t o begin a t a l a t e r age i n the c e r e b e l l u m and/or to be more prolonged than i n the c e r e b r a l  cortex.  Such r e g i o n a l d i f f e r e n c e s i n the l e v e l s o f the v a r i o u s components of the RNase enzyme system d u r i n g m a t u r a t i o n may account f o r t h e f i n d i n g t h a t developmental changes i n RNA metabolism i n the c e r e b r a l c o r t e x whole b r a i n .  are not r e p r e s e n t a t i v e o f  The a v a i l a b l e i n f o r m a t i o n  indicates  t h a t the t o t a l RNA content o f the c e r e b r a l c o r t e x ,  u n l i k e the  c e r e b e l l u m and whole b r a i n , a c t u a l l y d e c l i n e s a f t e r a t t a i n i n g peak values a t 18 to 2 0 days o f age, and t h i s d e c l i n e appears 24 t o be due t o a p r e f e r e n t i a l d e p l e t i o n o f rRNA.  Since i t  46 349 has  been shown  c e r e b r a l cortex  7  t h a t the r a t e o f RNA s y n t h e s i s  in rat  s l i c e s d e c l i n e s up t o 2 0 days and subsequently  remains c o n s t a n t , the d e c l i n e i n RNA content o f t h i s b r a i n r e g i o n a f t e r age 2 0 days must be due to an i n c r e a s e r a t e o f RNA d e g r a d a t i o n .  i n the  - 165 This conclusion  i s i n agreement with the d i f f e r e n c e  i n the developmental p r o f i l e s o f f r e e RNase i n h i b i t o r s p e c i f i c a c t i v i t y found here f o r whole b r a i n and t h a t reported  by Takahashi and Suzuki  D  f o r cerebral  cortex.  The lower f r e e RNase i n h i b i t o r s p e c i f i c a c t i v i t y i n the c e r e b r a l cortex  of a d u l t r a t s as compared to 10 day o l d  animals i s c o n s i s t e n t w i t h an enhancement i n the r a t e of RNA  degradation i n t h i s b r a i n  region.  In the a d u l t r a t the c e r e b r a l c o r t e x c o n t a i n s RNA  per average c e l l  J  and has a 2-6  more  f o l d g r e a t e r r a t e of  if6 RNA  synthesis  age RNA  than the c e r e b e l l u m .  turnover and the t o t a l RNA  be p r o p o r t i o n a l l y g r e a t e r to the c e r e b e l l u m .  I t follows that at t h i s  degradative a c t i v i t y  will  i n the c e r e b r a l c o r t e x as compared  The d i f f e r e n c e i n the r a t e o f RNA  d a t i o n between these two areas w i l l be g r e a t e r d i f f e r e n c e i n the r a t e of RNA  synthesis  degra-  than the  such as to a l l o w  a  net d e p l e t i o n o f RNA content i n the c e r e b r a l c o r t e x a f t e r age 20  days. D i r e c t t e s t i n g of these c o n c l u s i o n s  w i l l require a  comparative study of the developmental changes i n RNA  turn-  71 over i n these b r a i n r e g i o n s .  Dawson'  s t u d i e d the r a t e o f  rRNA turnover i n 35 day o l d r a t b r a i n a f t e r an i n t r a c e r e b r a l i n j e c t i o n of l a b e l l e d u r i d i n e and found t h a t the l a b e l i n c o r porated i n t o rRNA decayed with a h a l f - l i f e o f 6 days.  Studies  -  166 -  u s i n g other r o u t e s of p r e c u r s o r  administration  have shown  t h a t i n a d u l t r a t , whole b r a i n rRNA turns over with a h a l f life  o f 12 d a y s . ^ ' - ^  i n j e c t i o n of precursor  I t seems l i k e l y t h a t i n t r a c e r e b r a l  1  i n Dawson's study may have r e s u l t e d  i n the p r e f e r e n t i a l l a b e l l i n g o f rRNA i n the c e r e b r a l cortex,  and hence, the h i g h e r t u r n o v e r r a t e o f rRNA  reported  by Dawson may r e f l e c t a h i g h e r r a t e o f rRNA s y n t h e s i s and d e g r a d a t i o n i n t h e c e r e b r a l c o r t e x as compared t o other regions  of t h e a d u l t r a t b r a i n .  The f a c t t h a t f o r whole  b r a i n the r a t e o f rRNA turnover i n the newborn r a t i s twice t h a t o f the a d u l t ^  0  i s c o n s i s t e n t w i t h the developmental  p a t t e r n o f f r e e RNase i n h i b i t o r a c t i v i t y observed f o r whole b r a i n i n the p r e s e n t study.  I f f r e e pH 7,8 RNase p a r t i c i -  pates i n the d e g r a d a t i o n o f rRNA, the h i g h e r l e v e l of f r e e RNase i n h i b i t o r i n the b r a i n o f the a d u l t would l i m i t the a c t i v i t y o f t h i s enzyme thus a f f o r d i n g a l o n g e r  h a l f - l i f e of  RNA. Although i t was d e s i r a b l e t o o b t a i n an o v e r a l l p i c t u r e of the RNase a c t i v i t i e s o f the whole b r a i n , i t must be s t r e s s e d that the data thus obtained may mask s i g n i f i c a n t region-specific differences.  I n view o f the o r g a n i z a t i o n a l  complexity o f the b r a i n and the d i f f e r e n t i a l r a t e s of development o f d i f f e r e n t b r a i n r e g i o n s , w i l l be r e q u i r e d  a r e g i o n a l d i s t r i b u t i o n study  t o t e s t some o f the s p e c u l a t i o n s  emerged i n t h e f o r e g o i n g  discussion.  which have  Moreover, the data  - 167 -  obtained  i n the present  study o f r a t whole b r a i n  represents  composite r e s u l t s f o r a complex mixture o f c e l l s ( p r i m a r i l y neurons and g l i a ) each o f which may have q u i t e d i f f e r e n t <co  degradation  c a p a c i t i e s with r e s p e c t t o RNA.  F o r example,  ?  90 Watson  has r e p o r t e d t h a t RNA turnover  proceeds more  r a p i d l y i n neurons than i n g l i a and neuronal RNA  turnover  i s markedly s u b j e c t t o v a r i a t i o n depending on the e n v i r o n mental s t a t u s o f the a n i m a l .  I t i s hence d e s i r a b l e t h a t  measurements o f the f u n c t i o n a l l e v e l s o f the v a r i o u s components o f the RNase system be extended t o l o c a l i z e d b r a i n r e g i o n s and homogeneous c e l l p o p u l a t i o n s  of d i s c r e t e  c e l l types. 4,3 T i s s u e - s p e c i f i c D i f f e r e n c e s i n RNA Turnover and RNase A c t i v i t i e s i n t h e a d u l t animal  ,  I n the a d u l t r a t rRNA o f whole b r a i n t u r n s over a t h a l f the r a t e o f l i v e r rRNA, observation  T h i s i s c o n s i s t e n t with t h e  t h a t when expressed on a p e r gram wet weight b a s i s  the l i v e r content  o f RNase a c t i v i t y i s 1 0 - f o l d h i g h e r  than  b r a i n and i t s i n h i b i t o r - b o u n d pH 7,8 RNase i s 2 - f o l d lower than b r a i n . Although the l e v e l o f f r e e RNase i n h i b i t o r i n r a t l i v e r was not measured i n the present has r e p o r t e d  activity  study,  Roth  1 8 7  t h a t i n a d u l t r a t s the s p e c i f i c a c t i v i t y o f f r e e  - 168 -  RNase i n h i b i t o r o f the c y t o s o l f r a c t i o n i s 15$ h i g h e r i n whole b r a i n than i n l i v e r s  Roth a l s o found t h a t the  s p e c i f i c a c t i v i t y of f r e e RNase i n h i b i t o r i n the c y t o s o l of l i v e r was 2 - f o l d g r e a t e r  than t h a t of kidney and i n  accordance with t h i s the s p e c i f i c a c t i v i t y r a t i o of f r e e pH 7.8 RNase i n the c y t o s o l o f kidney t o l i v e r was 0 » l . k  As would be expected, the s p e c i f i c a c t i v i t y r a t i o o f t o t a l pH 7*8 RNase (assayed i n the presence of pCMB) was con2 02  s i d e r a b l y lower ( i l ) than t h i s . k  i n a comparative  De Lamirande  and A l l a r d  study o f f r e e pH 7«8 RNase and a c i d RNase  a c t i v i t i e s i n d i f f e r e n t t i s s u e s of the a d u l t r a t found lowest l e v e l s o f both a c t i v i t i e s i n the c e r e b r a l c o r t e x (3f o l d lower than i n l i v e r ) . G r e e n s t e i n e t al.^52 d R -t and M i l s t e i n ^ have a l s o r e p o r t e d t h a t the a c i d RNase a  n  0  n  3  a c t i v i t y of whole b r a i n i s amongst the lowest of a l l r a t 215  tissues studied.  E l l e m and C o l t e r  J  s t u d i e d the l e v e l s of  a c i d RNase and f r e e pH 7«8 RNase a c t i v i t i e s i n v a r i o u s mouse tissues.  The a c t i v i t y o f a c i d RNase per gram wet weight  t i s s u e was 3-^old lower f o r b r a i n than f o r l i v e r and the a c t i v i t y of f r e e pH 7«8 RNase per gram wet weight t i s s u e was 4 - f o l d lower f o r b r a i n as compared t o l i v e r .  The l e v e l o f  both enzyme a c t i v i t i e s on a per gram wet weight t i s s u e was h i g h e r i n kidney than l i v e r and lower i n muscle brain.  basis  than  - 169 -  The  s i g n i f i c a n c e o f these t i s s u e - s p e c i f i c d i f f e r e n c e s  i n RNase a c t i v i t i e s i s n o t known.  However, the lower l e v e l  of t o t a l RNase a c t i v i t y i n whole b r a i n , as compared t o l i v e r of a d u l t r a t s , may be r e l e v a n t t o the f a c t t h a t i n the a d u l t r a t rRNA o f whole b r a i n t u r n s over a t h a l f the r a t e o f l i v e r rRNA.  A b e t t e r understanding o f the f u n c t i o n a l s i g n i f i c a n c e  of the h i g h e r l e v e l o f i n h i b i t o r - b o u n d  pH 7»8 RNase and  p o s s i b l y a l s o o f f r e e RNase i n h i b i t o r a c t i v i t y i n b r a i n , as compared t o l i v e r , must await f u r t h e r  information.  4.4 Concluding Remarks The features gradation  p r e s e n t study has e l u c i d a t e d some of the gross  o f the enzyme system which p a r t i c i p a t e s i n the deo f RNA i n b r a i n .  The RNase enzyme system i n b r a i n  i s s i m i l a r i n i t s broad and g e n e r a l  outline to that  studied  i n other mammalian t i s s u e s and may d i f f e r from other organs only i n the s o p h i s t i c a t i o n and v e r s a t i l i t y o f the r e g u l a t o r y mechanisms by which i t s a c t i v i t y i s c o n t r o l l e d and coordinated  w i t h other enzyme systems.  t h i s study was to explore  The i n i t i a l i n t e n t o f  the RNase system i n b r a i n f o r any  d i s t i n g u i s h i n g f e a t u r e s which might be s p e c i f i c a l l y r e l a t e d to the s p e c i a l i z e d f u n c t i o n s o f t h i s t i s s u e . preliminary  However, a  c h a r a c t e r i z a t i o n o f the p r o p e r t i e s o f t h i s  enzyme system i n whole b r a i n was r e q u i r e d due t o the l a c k of i n f o r m a t i o n  as to i t s RNase content and c o m p o s i t i o n .  A  - 170 -  deeper l e v e l of a n a l y s i s w i l l be r e q u i r e d physiological function r o l e s i n the i n t e g r a t e d  to evaluate the  o f these enzymes and t h e i r metabolism o f RNA  i n brain.  The e f f e c t of a number o f v a r i a b l e s d e t e r m i n a t i o n o f the component  respective  i n f l u e n c i n g the  a c t i v i t i e s of the RNase system  has been c l a r i f i e d .  This  necessary groundwork  f o r the i n v e s t i g a t i o n of v a r i a t i o n s i n  the  i n f o r m a t i o n thus p r o v i d e s the  f u n c t i o n a l l e v e l s of the v a r i o u s components  enzyme system f o l l o w i n g  of t h i s  a l t e r a t i o n s i n the m i l i e u o f the  animal or a l t e r a t i o n s i n s p e c i f i c b r a i n f u n c t i o n s .  RNase  a c t i v i t i e s and RNase i n h i b i t o r l e v e l s can now be measured i n brain following  various p h y s i o l o g i c a l  stresses  l e a r n i n g experience, sensory d e p r i v a t i o n , malnutrition,  such as  electroschock,  as w e l l as d u r i n g c e l l d e g e n e r a t i o n  axonal s e c t i o n or e l e c t r o l y t i c l e s i o n s .  Such  following  measurements  of the f u n c t i o n a l l e v e l s and s u b c e l l u l a r d i s t r i b u t i o n o f the v a r i o u s components  o f the RNase system under c o n d i t i o n s  to be accompanied by changes i n c e l l u l a r RNA  content  p r o v i d e a c l e a r e r understanding of the c o n t r i b u t i o n of the component  known  may o f each  a c t i v i t i e s to the s p e c i f i c changes i n RNA  metabolism and c e l l u l a r RNA to occur i n b r a i n .  content which have been r e p o r t e d  T h i s would r e s u l t i n a c l e a r d e f i n i t i o n  of the complex r e l a t i o n s h i p between the a c t i v i t y o f each of  - 171  the  -  i d e n t i f i e d components of t h i s enzyme system to n e u r o n a l  functional  states.  Further information i s required the  (1)  s i g n i f i c a n c e of the h i g h l e v e l of RNase i n h i b i t o r i n  b r a i n and how  to determine  how  i t i s geared to s p e c i f i c t i s s u e f u n c t i o n ;  regulatory  coupled to the (3) how  mechanisms c o n t r o l l i n g RNA  catabolism  are  e l e c t r o p h y s i o l o g i c a l a c t i v i t y of neurons,  these mechanisms respond to changes i n the  f u n c t i o n a l demand f o r  and  metabolism i n the  RNA  d e g r a d a t i o n must a l s o be  r a t e of de novo RNA  neuron's  intact cell,  RNA  function  changes i n the r a t e  synthesis,  nucleocytoplasmic transport.  of  c o r r e l a t e d with changes i n  the  c o n v e r s i o n of p r e c u r s o r  t r a n s c r i p t i o n products i n t o f u n c t i o n a l l y mature RNA, The  detection  these parameters i n response to enhanced be  and  RNA.  To achieve a c l e a r p i c t u r e of i n t e g r a t e d  a c t i v i t y may  (2)  c r i t i c a l to the  molecular events which u n d e r l i e  and  of changes i n  electrophysiological  i d e n t i f i c a t i o n of those the m o d i f i c a t i o n  of a  neuron's f u n c t i o n a l r e l a t i o n s h i p with other neurons. 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