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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 W i l l i a m P h i l i p Popow B.A.., U n i v e r s i t y of B r i t i s h Columbia 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of B i o c h e m i s t r y We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1975 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of / / o g ^ g ^ - ' ^ ^ y The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date S /S>-T ABSTRACT A study was made of the enzyme system responsible for the catabolism of ribonucleic acid in rat brain. I n i t i a l work with whole brain homogenates and extracts revealed the presence of three ribonucleases (RNases) distinguishable by the pH at which they exhibit optimal activity. The identified RNases are referred to according to their 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 inhibitor of the pH 7*8 RNase. The components of this multi-enzyme-inhibitor system were separated and partially purified by ammonium sulphate fractionation of whole brain extracts followed by DEAE-cellulose column chromatography of the ammonium sulphate precipitable fractions. The DEAE-cellulose eluate RNases were characterized with regard to the effect of various reagents upon their activity. The intracellular distribution and developmental profiles of the three RNase activ i t i e s and the pH 7«8 RNase inhibitor activity were also determined. ***-»»#« - i i -TABLE OF CONTENTS PAGE List of Tables List 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 in brain RNA content and composition during postnatal maturation 5 1.12 Changes in brain RNA content and composition during aging . . . . . . 19 1.2 Turnover of Brain RNA 22 1.3 Changes in Brain RNA Content and Composition Accompanying Sensory Stimulation, Physiological Challenges and Learning Experience 25 1.4 The Role of the RNase Enzyme System in RNA Metabolism 33 1.5 Regulation of #Nase Activity and RNA Degradation • • 36 1.6 Intracellular RNases of Non-Neural Mammalian Tissues . . . . . . . . 41 1.61 Acid RNase 41 1.62 pH 9.5 RNase 43 1.63 pH 7.8 RNase . 44 1164 RNase Inhibitor 46 1.65 Ribosomal RNase . 4 8 1.66 Nuclear RNases . 49 - i i i -PAGE 1.7 Changes in RNase Activities under Various Physiological and Pathological Conditions • 51 1.8 The RNase Enzyme System of Brain 53 II. MATERIALS AND METHODS 2.0 Materials . . . . . . . . . . . . . 57 2.01 Experimental animals 57 2.02 Chemicals 57 2.1 Methods 58 2.11 Preparation of tissue homogenates and extracts • 58 2.12 Fractionation of isotonic sucrose homogenates by differential centrifugation 59 2 . 1 3 Purification procedure . 60 2.131 Ammonium sulphate fractionation . . . 6 0 2.132 DEAE cellulose, column chromato-graphy of ammonium sulphate fractions 61 2.14 Enzyme assays 61 2.141 Determination of RNase activity . . . 61 2.142 Assay for deoxribonuclease activity • 6 3 2.143 Assay for phosphodiesterase activity • • 6 3 2.144 Assay for pH 7.8 RNase inhibitor activity . 64 III. EXPERIMENTAL RESULTS 3.0 Characteristics of the RNase Activities of Adult Rat Whole Brain Homogenates and Extracts 66 3.01 Effects of pH and buffer system on the RNase activ i t i e s assayed in isotonic sucrose homogenates 66 - i v -PAGE 3.02 Solubilization of latent RNase acti v i t i e s by horaogenization i n Q,lfo Triton X100 68 3 . 0 3 Effects of pH, buffer system, ionic strength, and NaCl on RNase activ i t i e s assayed in 0,1% Triton X100 extracts . . . 71 3.04 Evidence indicating the presence of a protein inhibitor of pH 7.8 RNase activity in brain ?$8 3.041 Time-dependent activation of pH 7.8 RNase activity in stored enzyme preparations 7 8 3.042 Inhibition of bovine pancreatic RNase A activity by brain extracts • 7 8 3 . 0 4 3 Activation by pCMB of RNase activity assayed at pH 7 * 8 . . . . • 7 9 3.044 Effect of EDTA on RNase activity assayed at pH 7 * 8 . . . . . . . . . . 80 3 . 0 4 5 Comparison of the effects of pCMB on RNase activ i t i e s in li v e r and brain • 8 2 3 . 0 5 Summary comment on the variables influencing the determination of RNase activities i n crude extracts . . . . 8 6 3.1 Separation and Partial Purification of the Components of the Multi-Enzyme-Inhibitor System of Adult Rat Whole Brain 6.1$ Triton X100 Extracts 8 7 3.11 Ammoniu# sulphate fractionation of G.1% Triton X100 extracts of adult rat whole brain 8 8 3 . 1 2 DEAE-cellulose column chromatography of the 25-858* and the 75-100$ saturated ammonium sulphate fractions . . . . . . . 94 3,2 Properties of the Three Separated RNase Activities . 100 - V -PAGE 3.21 Effect of pH 101 3.22 Effect of NaCl 105 3.23 Effect of MgCl 2 105 3.24 Effect of EDTA 10? 3.25 Effect of pCMB. 109 3.26 Effect of 3-mercaptoethanol and dithiothreitol 109 3.27 Effect of detergents 110 3.28 Effect of storage 112 3.3 DNase and Phosphodiesterase Activities of DEAE-cellulose eluate enzyme fractions . . . . 113 3.4 Intracellular Distribution of RNase Activities and RNase Inhibitor Activity . . . . 116 3»5 Characterization of RNase Activities in Separated Subcellular Fractions 129 3,6 Developmental Changes in RNase Activities and in RNase Inhibitor Activity in Rat Whole Brain 139 IV. DISCUSSION 4.0 Regulation of the In Vivo Function of RNases in brain 148 4.1 Correlation of Developmental Changes in the Content, Synthesis and Degradation of RNA i n brain • . . . . . . . . • 158 4.2 Regional Differences in the Metabolism of RNA and in the Functional Roles of RNases i n brain , . . . « • . . . • 163 4.3 Tissue-specific Differences in RNA Turnover and RNase Activities in the Adult Animal • 167 B„ BIBLIOGRAPHY . ., 173 *«*«*«* s f l -LIST OF TABLES TABLE PAGE I. Effect of buffer concentration on RNase activity i n 0.1% Triton X100 extracts . . . . . . . 74 II. Effect of pCMB and EDTA on pH 7.8 RNase activity in 0.1% Triton X100 extracts 77 III. pCMB reversal of EDTA-inhibited pH 7.8 RNase activity • 81 IV. Enhancement of RNase inhibitor activity i n the presence of 1 mM EDTA 82 V. Recovery of RNase ac t i v i t i e s in ammonium sulphate precipitated fractions . . . . 89 VI, Effect of various reagents upon DEAE-cellulose eluate RNase Activities . . . . . . . . . 106 VII. Effect of various detergents upon DEAE-cellulose eluate RNase act i v i t i e s • 111 VIII. DNase activity of DEAE-cellulose eluate enzyme fractions . . . . . . . . 114 IX. Phosphodiesterase activity of DEAE-cellulose eluate enzyme fractions . . . . . . . . . 115 X, Intracellular distribution of pH 6.7 RNase activity • • • • • . . . . . 118 XI. Intracellular distribution of pH 9.5 RNase activity . . . . . . . 120 XII. Intracellular distribution of free pH 7,8 RNase activity . . . . . . . . 122 XIII. Intracellular distribution of total pH 7.8 RNase activity 124 XIV. Calculated intracellular distribution of latent, inhibitor-bound pH 7.8 RNase . 125 XV. Intracellular distribution of free RNase inhibitor activity . • 126 *««##*« - v i i -LIST GF FIGURES FIGURE PAGE 1. pH profiles of RNase activity i n isotonic sucrose homogenates of adult rat whole brain . . . 67 2. pH profiles of RNase activity in 0.1% Triton X100 extracts of adult rat whole brain« effect of various buffers . . . . . . . . . . 72 3. Effect of NaCl on the activity and pH optimum of acid RNase activity i n 0.1% Triton X100 extracts of adult rat whole brain 75 4. pH dependence of 0.1 mM pCMB-stimulated RNase activity i n 0.1$ Triton X100 extracts of adult rat whole brain 83 5. pH-activity profiles and effect of 0,1 mM pCMB on RNase activity i n 0.1% Triton X100 homogenates of adult rat l i v e r . . . . . . . . . . 83a 6a pH-activity profile of 75-100$ saturated ammonium sulphate fraction . . . . 92 16b pH-activity profile of 25-55% saturated ammonium sulphate fraction • 93 7. Elution profile of 75-100% saturated ammonium sulphate fraction chromatographed on DEAE-cellulose 95 8. Elution profile of 25-55$ saturated ammonium Sulphate fraction chromatographed on DEAE-cellulose 97 9a pH-activity profile of DEAE-cellulose 8 eluate acid RNase activity . 102 19b pH-activity profile of DEAE-cellulose eluate pH 9 .5 RNase activity 103 9c pH-activity profile of DEAE-cellulose eluate pH 7.8 RNase activity 104 10. Reactivation of EDTA-inhibited pH 9 .5 RNase activity by Mg++ 108 11a pH-activity profile of nuclear fraction in presence of 2.0 M urea or 0.2 mM pCMB . . . . . 132 - v i i i -FIGURE PAGE l i b pH-activity profile of nuclear fraction in presence of 1 mM EDTA 132 12a pH-activity profile of crude mitochondrial fraction in the presence of G.2 mM pCMB . . . . 133 12b pH-activity profile of crude mitochondrial fraction in the presence of 1 mM EDTA or 0.5 mM MgCl2 133 13. pH-activity profile of microsomal fraction i n the presence of 2.0 M urea 135 14. pH-activity profile of cytosol fraction . . . . 138 15. Developmental profile of pH 6.7 RNase v activity - _•- 139a 16. Developmental profile of pH 9*5 RNase activity • 139b 17. Developmental profile of pH 7.8 RNase activity . 139c 18. Developmental profile RNase inhibitor activity • 143 : 19. Total RNA-degradative capacity of whole brain as a function of developmental age . . . . 159 - ix -ACKNOWLEDGEMENTS I would like to express my immeasurable gratitude to Shan-Ching Sung for his limitless patience, trust, under-standing and encouragement without which this work could not have been sustained or completed, I would like to thank my laboratory colleagues Jen-Fu Chiu, Vijendra Singh, Raja Rosenbluth and Jim Nagy for their 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, feeling and thought, for the privilege of knowing them and through knowing them expanding the limits of my understanding and appreciation of the inneruworkings of the human brain. Finally, I thank the many others who intersected my l i f e in the course of this project for the inspiration they i n s t i l l e d i n me and for a l l they have taught me. It i s only i n collaboration with a l l of the abovemen-tioned people that the ongoing i n vivo processes of my own brain have been sustained and regulated throughout the course of this work. ««#«««« I. INTRODUCTION 1.0 General Frame of Reference 1—7 It i s generally regarded ' that tissue-specific differences in macromolecular composition are a consequence of the differential expression of genome information common to a l l somatic c e l l s . The problem of formulating neuro-biological phenomena in molecular terms i s hence essentially that of understanding the specific nature, expression and Regulation of the genetic capabilities 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 arising from the organizational complexity and morphological heterogeneity of the mammalian central nervous system have hindered progress toward this goal and, consequently, the molecular under-standing 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 in molecular com-position which produce or parallel morphological changes as cell s differentiate toward their mature structural and functional states. Information as to the molecules and molecular events occurring in the course of development w i l l permit a more detailed understanding of how they participate - 2 -to yield the specific morphology, metabolism, and functional capabilities of the f u l l y differentiated c e l l types of the adult brain. Secondly, considerable effort has been directed to-ward achieving an understanding of the molecular composition and metabolism of normal adult brain and correlating brain-specific molecular constituents with the unique morphology and functional characteristics of neuronal tissue. Thirdly, attempts have also been made to detect alterations in molecular composition and molecular function accompanying normal adult changes in neuronal function. Morphological changes in fully-differentiated neurons have Q been demonstrated to occur i n response to changes in sensory input and behavior-modifying challenges from the external environment• An understanding of the molecular events correlated with or responsible for such morphological alterations w i l l help to elucidate the nature and limits of interaction between sensory experience and intracellular metabolism; that i s , how perturbations of intraneuronal metabolism are effected, the capacity of the molecular mechanisms regulating metabolic adaptation and hence the limits of response a b i l i t y of metabolic processes within fully-differentiated neuronal c e l l types. It i s hoped that such work w i l l eventually - 3 -result in the identification of those brain-specific mole-cules and molecular events which may mediate the processing and storage of sensory information or which may underlie cognitive and affective processes and other psychobiological phenomena peculiar to the central nervous system. Finally, there have been studies of how alterations in the genome can affect defined functions of the nervous system in neurological and behavioral mutants; such studies can provide clues concerning the contributions made by single, identified gene products to complex and integrated brain function and behavior. It has become clear from such studies that both the comparatively gross molecular changes which occur throughout 1 3 development t J and the more subtle neurochemical correlates of experientally- and behaviorally-related neural activity 9-l6 in the adult brain 7 entail quantitative and qualitative changes in the protein composition of c e l l s . These changes in cellular protein composition may occur within a particular c e l l , in a population of cells linked to one another structurally and functionally, and in even more complex systems. Proteins by their intervention as enzymes and membrane constituents influence a l l other molecular constituents of the c e l l . Hence, such changes 17 in protein composition could be expected ' to correspond - 4 -to the establishment of new functional connections between neurons, alterations i n the properties of certain neurons, families of neurons, and in their steady-state mode of 1ft .? 1 synaptic function " * Because ribonucleic acid i s the primary gene product and any specific information manifested at this level of genome expression i s subsequently transferred to proteins through the mechanism®©.? translation (protein synthesis) to yield the tissue- and c e l l type-specific metabolism, mo»-phology and functional capabilities of an organ, i t seems l i k e l y that alterations in cellular protein composition may be secondary to changes i n de novo RNA synthesis and RNA turnover. Changes in cellular protein patterns thus arise as a consequence of modulations in gene expression--either directly through (1) changes in the rate of transcription of constitutively expressed genetic information, or through (2) qualitative shifts in the read-out of the reversibly expressible pool of genetic information (i.e., through induc-tion or repression) i or more indirectly by (3) post-transcrip-tional mechanisms regulating gene expression (modulation of RNA turnover, RNA translation and protein turnover). It i s this third general category of regulation of cellular protein - 5 -composition which i s the domain of concern specifically dealt with in the present thesis. Prior to discussing the role of the enzyme system chiefly responsible for the post-transcriptional metabolism of RNA and the post-transcriptional control of cellular RNA c o n t e n t , i t w i l l be useful to review the information avail-able to date on changes in content, composition and turnover of RNA i n the infant and adult central nervous system. 1.1 Changes in Brain RNA During Ontogenesis 1.11 Changes in brain RNA content and composition during post-natal maturation In the rat, total RNA content of whole brain increases up to the 15th to 18th day postnatally. 2 2• 23» 2^ Subsequently, 24 2 5 the nuclear and transfer RNA content ' J of whole brain re-mains relatively constant throughout adulthood whereas the 24 26 microsomal and ribosomal RNA content declines. ' There also occurs a change in 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 rat, the increase in a l l classes of RNA ceases at 18 days and i s followed by a 40?S decrease in polysomal RNA by the time the animals reach - 6 -adulthood (200-250 g. body weight)/** This decline in polysomal RNA content per cerebrum i s accompanied by an increase in the pyri^aidine and a decrease in the purine content of this RNA fraction, whereas the base composi-tions of nuclear RNA of cerebral cortex of both young and adult animals remains the same. Such developmental changes in the base composition of RNA isolated from the microsomal and polysomal fractions of brain are most lik e l y accounted for in terms of (1) shifts in the base sequence and base compositions of specific mRNA molecules associated with ribosomes at different stages of development, and/or (2) an age-dependent increase in the mRNA/rRNA ratio in these fractions. The a b i l i t y of RNA molecules homologous in base sequence to compete in hybridization to complementary base sequences of DNA has been utili z e d to demonstrate alterations in the kind of genome information transcribed at different •' 27 31 32 stages of ontogenesis. * % J -j:c@Eoasei;elblal< have studied the ab i l i t y of total RNA isolated from different regions of the adult mouse and human brain to compete with total RNA from the same brain regions of feta l or infant mouse and human brain hybridized to unique sequence DNA. They found that in both mouse and human brain there occurs with development - 7 -a net increase in the variety of transcribed RNA. Moreover, the greater transcriptional diversity evident in adult as compared to fetal brain i s attributable to diversification of RNA in the cerebral cortex since the number of different kinds of RNA molecules present in other regions of the adult brain was similar to that of whole fet a l brain. Twenty per cent of the maximal unique sequence genome information was expressed in whole adult brain compared to 6$ for other organs in the human, and 12$ as compared to 6% in the mouse. 28 Similar competition hybridization studies have indicated that in mouse l i v e r the variety of mRNA molecules synthesized declines during development. Bondy and Roberts 3 3 demonstrated that during maturation chromatin prepared from whole rat brain as well as specific brain regions exhibits a decline i n i t s capacity to function as a template for E. c o l i RNA polymerase-catalyzed RNA synthesis in vitro, indicating that during development there occurs a progressive template-limiting decline in the rate of transcription. Chromatin from brain was found to support the incorporation of more label into RNA than hepatic chromatin. The decline in template activity of chromatin was correlated with a concurrent decline in the non-histone protein content of chromatin. Such a develop-mental drop-out in non-histone chromosomal proteins asso-ciated with DNA has also been observed by Kurtz and Sinex 3^ - 8 -in mouse brain and may be relevant to the explanation of the results 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 for transcription. Bondy and Roberts 3 3 found that whereas the total mRNA transcribed from whole brain chro-matin was lower in the adult than in the newborn rats, the mRNA fraction of the total RNA transcribed was proportionally higher in the adult than in the infant. Thus, although the total amount of mRNA-like RNA in rat brain also decreases during development, J the decrease in total rRNA i s s t i l l greater and results in a net increase in the mRNA/rRNA ratio. Bondy and Roberts3** also found that nuclear IfM* from both whole brain and l i v e r of adult rats hybridized to rat total 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 in the adult a larger proportion of RNA synthesis i s directed towards mRNA produc-tion in brain as compared to l i v e r . Such an inferrence i s not unequivocally supported by the data since in this case the hybridization of RNA transcribed from single copy genes i s not clearly distinguished from that of RNA which is the product of repetitive genes. Also, nuclei contain rapidly synthesized RNA molecules which are not transferred to the - 9 -cytoplasm 3^'^ and this type of RNA presumably does not function as mRNA. This conclusion i s , nevertheless, supported by a considerable body of other evidence. Comparison of the base composition of RNA isolated from various subcellular fractions of brain and l i v e r indicate that the adult brain contains more RNA which i s DNA-like in i t s base composition than li v e r . 3 ' ' By this criterion brain has a greater nuclear heterodisperse RNA and mRNA content than l i v e r . Zomzely et al. 3** have also demonstrated that in the adult a larger proportion of RNA synthesis in vivo results i n mRNA in brain as compared to other organs in the rat. Whereas in brain the number of polysomes i s relatively high at birth and declines during development,3^ in l i v e r they are relatively few in number at birth and increase with de-velopment. Concurrent with the decrease in polysomal RNA and in the total number of polysomes, as the brain matures there occurs a decrease i n the number of membrane-bound poly-k i 2 5 4l somes and a decrease in the size J and st a b i l i t y of isolated polysomes. It remains uncertain to what extent the smaller polysome size class distribution in the adult may represent (1) a de-velopmental shift in the size of mRNA molecules associated with ribosomes 3^ , i + 2 or (2) breakdown of polysomes during their 10 -isolation"* 3 which may reflect age-related differences between the RNase activity levels of the post-mitochon-dr i a l supernatant from which polysomes are prepared. The proportion of polysomes to free ribosomes in 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 acid incor-poration in vivo into nuclear RNA i s the same for both the adult and 4-day old rat cerebral cortex. However, there i s a lag period of about 6 0 minutes in the young animals which i s not present i n adults, during which labelling of the micro-somal and ribosomal fractions of the cytoplasm proceeds only slowly. * J V Sharma and Singh observed a similar lag i n the rate of transfer of label from the nucleus to RNA in the cytoplasm in newborn rat whole brain slices incubated in vitro. Mandel*1'-' in studying developing chick brain observed that newly synthesized RNA remains within the nucleus of chick embryo neuroblasts and spongioblasts unti l about 15 . days of embryonic l i f e . The nature of the nucleocytoplasmic barrier responsible for this phenomenon i s unknown. Murthy3^ has shown that the entry of polyribosomal RNA into the cyto-plasm follows the temporal sequence) mRNA, 18S RNA, 28S RNA. - 11 It i s not known to what extent the rate-limiting step con-tributing to this difference i s due to the rate of precursor RNA conversion to functionally mature RNA, the rate of RNA assembly into ribosomal subunits and informosomes or the rate of transport across the nuclear envelope* Comparison of in vivo rates of RNA synthesis in the brain or specific brain regions of animals of different ages is complicated by the fact that intracranial routes of admini-stering the labelled precursor have been demonstrated to result in uneven distribution of the label in different brain regions. Also, such studies have not controlled for age-dependent changes in the rate of uptake of labial into the c e l l s nor for differences i n intracellular precursor pool size. Guroff et a l . ^ reported that the in vitro rate of 3H-uridine incorporation/mg DNA was 3-fold greater i n cerebral cortical slices than in cerebellar slices of the IB-day old rat. Between age 10 days and 4 months, the rate of RNA synthesis/mg DNA decreases 6-fold in the cortex and 10-fold in the cerebellum, so that the rate of RNA synthe-sis/mg DNA i s about 6-fold higher in the adult cortex as 46 compared to the adult cerebellum. Guroff also reported that the rate of RNA synthesis/mg DNA was 2-fold higher i n slices of l i v e r than in slices of cerebral cortex of the adult rat. - 12 -Sung**' found that the in vitro rate of ^C-uridine incorporation/mg DNA in tissue slices incubated for one hour was slightly higher in the cerebellum than in the cortex of 2-day old rats. By age 10 days, the rate of RNA synthesis/mg DNA had decreased 2.2-fold in the cerebellum, whereas the cortex exhibited a 1.3-fold increase relative to the rate of RNA synthesis at age 2 days. 48 32 Johnson studied the i n vitro incorporation of J P-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 in the rate of RNA synthesis up to age 7 to 9 days. Several workers have measured the rate of RNA synthesis in isolated nuclei incubated in vitro under conditions which preferentially detect either RNA polymerase A- or RNA poly-merase B- catalyzed RNA synthesis. Bbndy and Waelsch^'^ 0 studied the incorporation ofi ^C-UTP and "^C-ATP into RNA of liv e r and brain c e l l nuclei ionic strength—conditions determined to be optimal for 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 liv e r of rats at a l l ages between 8 days and 4 months. Moreover, the capacity incubated for 3 minutes - 13 -of nuclei for RNA polymerase B- catalyzed RNA synthesis increased almost proportionally in both organs during this time period resulting in a net increase of 20% for brain and 3 0 % for l i v e r . These workers also studied the regional dis-tribution of RNA polymerase B- catalyzed RNA synthesis in the adult brains of the rabbit and squirrel monkey. Nuclei prepared from the cerebral cortex, caudate and hippocampus were found to exhibit 2-fold higher RNA synthesizing activity per mg DNA than those from the thalamus-hypothalamus, corpus callosum, pons-medulla and cerebellum. Gfiugfrida et a l . ^ 1 studied the RNA synthesizing activity of c e l l nuclei prepared from the cerebral cortex,, cerebellum and brainstem of 5 » 10, and 18-day old rats. U t i l i z i n g assay conditions which preferentially discriminate between the rates of RNA synthesis catalyzed by RNA polymerase A and RNA polymerase B they found that under both conditions, nuclei isolated from a l l three brain regions of older rats exhibited substantially lower rates of %-UTP and 3H-GTP incorporation than those derived from the 5-day old rats. In 5-day old animals, nuclei from the cerebellum had a 1 .4-fold higher rate of RNA polymerase B- catalyzed RNA synthesis than nuclei from both the cerebral cortex and brainstem, and underwent a 3 . 3 - f o l d decrease to become 2-fold lower than the activity in the cerebral cortex and brainstem by 18 days. By - 14 -18 days the RNA polymerase B activity/rag DNA had declined in the cerebral cortex and brainstem to 75% and 86% respec-tively of their 5-day old l e v e l . RNA polymerase A- catalyzed RNA synthesis/mg DNA, assayed in the presence of Mg + + and low ionic strength, was slightly higher in the cortex and brain-stem than in the cerebellum of 5-day old animals and under-went a 2 . 5 - f o l d decrease in a l l three brain regions between 5 and 18 days. Thus, there i s a predominant amount of evidence indi-cating that on a per unit DNA and hence per nucleus basis, there occurs a decline in the rate of total cellular RNA synthesis as well as RNA polymerase A- and B- catalyzed RNA synthesis during development in the whole brain as well as i n specific brain regions. On the basis of the consistent body of evidence i t can be inferred that the demand for mRNA and rRNA, as indicated by the rate of RNA synthesis, i s highest during c e l l division, and c e l l differentiation and, subsequently, declines to lower levels as the brain matures and attains i t s adult number of ce l l s by age 18 to 2 0 days. However, the apparently anomalous results of Bondy and Waelsh*^'-*0 are not to be disregarded. These workers have reported finding an increase in the rate of RNA polymerase B-catalyzed RNA synthesis during maturation in a l l regions of the rabbit brain, in the whole brain of the rat and in the l i v e r of both rabbit and rat. - 15 -The discrepancy between the r e s u l t s of d i f f e r e n t workers may be related to differences i n the assay condi-tions f o r RNA polymerase B a c t i v i t y . Whereas Bondy and WaelsJbh's data were obtained with an incubation period of 3 minutes, G i u f f r i d a et a l . ^ 1 have found that the Mn++/ (NH_y)2S O k - stimulated reaction i s l i n e a r f o r K 5 minutes after an i n i t i a l l ag period of at le a s t 5 minutes. This, however, does not o f f e r an obvious explanation of the apparent discrepancy, since Barondes-'-' i n a study of Mn** /(NHk) 2S0k - stimulated RNA syntheis by "aggregate enzyme" during a s i m i l a r l y short incubation period of 5 minutes found a 2-fold lower rate of C-CTP incorporation into RNA (per unit DNA) i n the whore brain of 9-month old as compared to 12-day old r a t s . •Also,in 9-month old rats, t h i s enzyme preparation from brain was only 37$ as active as that from l i v e r . The explanation for why the rate of RNA synthesis has been reported to be lower i n brain as compared to l i v e r during long incubation periods i s not l i k e l y to be due to a more rapid degradation of newly synthesized RNA-^  i n brain as compared with l i v e r i n view of the data reported by Dutton and Mahler. J These workers found that i n £dult rats the rate of RNA polymerase A- catalyzed RNA synthesis (Mg++ and low i o n i c strength) of nuclei prepared from the cerebral - 16 -cortex was only 2% of that of l i v e r nuclei. They also found that the incorporation of %-CTP into RNA was enhanced 2 5 to 50% by preincubation of brain nuclei at 3 7 ° (see similar 46 observation by Guroff for brain slices) whereas preincuba-tion of l i v e r nuclei for 3° minutes completely inhibited their capacity to synthesize RNA.-^  These workers concluded that the RNA synthetic capacity was more stable and the nuclear RNA more protected from nucleases in brain nuclei than in l i v e r nuclei of adult animals. Since results following brief incubation periods are probably more reliable measures of the i n i t i a l rate of 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 in vivo rate of RNA synthesis than results obtained with linger incubation periods where the rate of degradation of the newly .synthesized RNA becomes an important consideration, particularly if:the rate of nuclear RNA turnover varies with age or shows tissue specific differences. However, the differential sensitivity of brain and l i v e r nuclei to pre-incubation might also explain the discrepancy in results. If the duration between preparation of nuclei and assay of nuclear RNA synthesizing activity was long in Bondy's case, then the l i v e r nuclei should be preferentially inhibited resulting in lower activities than brain nuclei; but this - 17 -presupposes that the observations b y Dutton and Mahler of the differential stability of the polymerase A activity of brain and l i v e r nuclei i s also valid for their polymerase B a c t i -vity—and this would not account for an increase i n poly-merase B act i v i t y in brain during maturation. In any case, the resolution of this apparent discrepancy must await more comprehensive data on the enzyme systems responsible for RNA i metabolism, particularly the rate of degradation of nuclear RNA in brain as a function of age. A l l the aforementioned data consistently agrees that by age 18 days when net increase in organ content of DNA and 22 2? 24 cellular content of RNA has ceased"" • cells of the cerebral cortex have at least a 2-fold higher rate of RNA synthesis (6-fold higher with tissue slices) than ce l l s of the cerebellum and most other regions of the brain (Bondy and CQ Waelsch's data-' suggest that caudate and hippocampus have levels of RNA polymerase B- catalyzed RNA synthesis activity comparable to that of cortex), and hence presumably a propor-tionally higher rate of RNA turnover. The functional signi-ficance of this regional difference i n RNA metabolism measured in vitro i s not known. However, the data of Giuffrida et al.-* indicate that the difference i s predominantly due to a 2-fold higher rate of RNA polymerase B- catalyzed RNA synthesis in the cerebral cortex since the level of RNA polymerase A-- 18 -catalyzed RNA synthesis i s nearly equal in 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, this suggests a higher rate of synthesis and ut i l i z a t i o n of mRNA in the cells of the adult cerebral cortex (and possibly caudate and hippocampus) in comparison to other brain regions. It i s not known to what extent the high RNA polymerase B activity of adult cerebral cortex i s due to the higher pro-portion of neuronal nuclei (25%) in this region of the brain or whther i t i s due to a high RNA synthesis activity specific to the neurons and/or non-neuronal c e l l types of the neo-cortex. Guiffrida et al."* 1 have demonstrated that within a given region of the brain different classes of nuclei are found having differences of at least 2-fold i n their RNA polymerase a c t i v i t i e s . Neuronal nuclei were found to be more active than oligodendroglial or astrocytic nuclei.51»59.60 Yamagami et a l . - ^ have studied the composition of rat brain nuclear RNA at various stages of development and found that as the brain matures the high molecular weight compo-nents decrease. Their results indicate that during the maturation of the brain, both mRNA and rRNA are decreased - 19 -significantly in the nuclei after the thirtieth day and, subsequently, this decrease continues u n t i l the nuclear RNA is predominantly composed of small molecular weight RNA 74 molecules in the adult brain.' No evidence has yet appeared indicating to what extent these results represent a preferential decrease in the synthesis of high molecular weight RNA species and/or to increased nuclear RNA degra-dation^ 1 i n the adult. These developmental changes in cellular RNA correspond 4 l to a decline in the rate of protein synthesis and would be expected to be accompanied by a qualitative shift in the mRNA species as the genetic program synthesizing mRNA coding for proteins required for c e l l division switches sequentially to the synthesis of mRNA species coding for the synthesis of proteins required for c e l l differentiation, c e l l growth and f i n a l l y , maintenance functions of the mature c e l l . Thus at early stages of development brain RNA characteristics are compatible with the rapid rate of protein synthesis observed. There i s a higher requirement for rRNA in the rapidly dividing c e l l s . 1.12 Changes in 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 in neuronal perikarya, neuropil, g l i a l elements and - 20 -blood vessels which are believed to be responsible for the 6 2 concurrent deterioration of function. It i s not known to what extent the changes in morphology of aging brain re-present genetically coded, time-dependent changes in the c e l l or result from exposure to harmful environment. Such factors as errors introduced into protein synthesis, chronic ischemia, slow virus infection, nutritional deficiencies, intoxications, and failure of auto-oxidation are among those to be seriously considered. L i t t l e i s known about the molecular processes underlying "normal" c e l l senescence. It 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 yield insight into the molecular events responsible for or contributing to the com-plex processes of c e l l degeneration and c e l l death. According to Hollander and Barrows^3 whole brain RNA/ DNA ratio in the C57 BL/6J mouse and Wistar rat does not 64 change during aging. Chaconas and Finch found a slight decrease in the RNA/DNA ratio of the striatum, but l i t t l e or no decline in other regions of the C57 BL/6J mouse brain. Hyden^-* studying individual anterior horn spinal neurons of humans found a progressive decline in the total RNA content of these cells after sixty years. Ringborg^ has also reported a similar decline in the total RNA content of single pyramidal cells of rat hippocampus. In the latter, the amount of RNA per c e l l increases from 24 pg in newborn rats to 110 pg - 21 -f o r mature rats and, subsequently, drops to 53 pg i n very old r a t s . In the very old rats, the G+C/A+U r a t i o of the RNA of these c e l l s r i s e s from 1.66 i n mature rats to 1.95. Thus, i n d i v i d u a l neurons i n the CA^ layer of the hippocampus of 36-month old rats contain about h a l f the RNA found i n these c e l l s i n 2-month old r a t s . Wulff and Freshman^ using a microspectrophotometric technique f o r quantitating the t o t a l RNA content of single c e l l s found a s l i g h t RNA loss i n spinal motoneurons and cerebellar Purkinje c e l l s of aged ra t s , but found no change i n the RNA content of neurons of the supraoptic nucleus of the hypothalmus. • " It 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 reported to occur with aging. The r e l a t i o n s h i p of those decreases i n c e l l u l a r RNA which have been demonstrated to the concomitant development of d e f i c i t s i n integrated brain function and behavior remains to be elucidated. I t must be noted that upon such basal l i f e - c y c l e changes i n c e l l u l a r RNA content are superimposed short-l a s t i n g , r e v e r s i b l e fluctuations i n RNA content that r e s u l t from increases or decreases i n functional demand. - 22 1.2 Turnover of Brain RNA In adult rat, the h a l f - l i f e of both the RNA and proteins in brain ribosoraes was found to be 12 to 18 days.^*^ 0 In 35-day old r a t P 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 in the turnover of ribosomes appears to be peculiar to the brain, since in a comparative study of other rat tissues Menzies et al.'' 3 found no age-dependent differences in the turnover of rRNA in l i v e r , spleen, lung or intestinal mucosa. 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 significant tissue-specific difference in 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 half the rate of l i v e r rRNA. Since rRNA constitutes nearly 90% of the total cellular BNA^ i t s turnover rate would be expected to have a decisive bearing on such processes as chromatolysis following axonal se c t i o n . ^ A half-ikife of at least 6 days is compatible with estimates of histological methods of the rate of chromatolysis.'''' However, the disappearance of Nissl staining after severe anoxia and possibly also after intense chronic stimulation and s t r e s s , 1 0 * * " 1 0 ^ i s too rapid to be accounted for by failure of RNA synthesis in conjunction - 2 3 -with a normal rate of rRNA turnover. Rapid disappearance of N i s s l substance may hence involve a c t i v a t i o n of ribonu-cleases present i n the cytosol or associated with ribosomes. No age-associated difference has been observed i n the turnover of tRNA. This class of RNA exhibits a double expo-n e n t i a l turnover pattern; one component has a h a l f - l i f e of 5 to 8 days, similar to that of l i v e r tRNA 7^' 7^ and the second component 1 3 to 1 6 d a y s . 7 0 , 7 2 * 7 ® I t i s not known to what extent these re s u l t s r e f l e c t d i f f e r e n t i a l turnover of di f f e r e n t components of tRNA population within assingle c e l l type or to d i f f e r e n t i a l turnover of the t o t a l tRNA population within d i f f e r e n t c e l l types such as neurons and g l i a . Mitochondrial RNA of rat brain has been shown to have a h a l f - l i f e of 1 1 . 6 and 1 0 days i n young and adult animals respectively. Data on the turnover rates of mRNA i n rat brain i n d i -cates the presence of at least two populations of mRNA. A more l a b i l e f r a c t i o n representing most of the mRNA turns over with a h a l f - l i f e of less than k hours,7 9 - 8 3 a n (_ another smaller population of mRNA molecules turns over with a mean Qh. h a l f - l i f e of 1 0 to 12 hours. Reports that polysomes from rat cerebral cortex J and whole brain decrease i n s t a b i l i t y during maturation presumably due to an enhanced s u s c e p t i b i l i t y to degradation of the mRNA i n the polysomes would suggest a higher rate of mRNA turnover i n the adult; however, d i r e c t - 24 -data on developmental changes in the turnover rates of mRNA i s lacking. There i s l i t t l e available data on RNA turnover i n the nuclei of brain c e l l s . Bondy^® studied the turnover of i n vivo labelled nuclear RNA in whole brain of adult rats and found that a l l of the acid-insoluble radioactive label lost from the nucleus in the f i r s t 18 h after intracranial injec-tion of C-cytidine could be accounted for by the increased labelling of cytoplasmic RNA. That i s , the decrease in specific radioactivity of newly synthesized nuclear RNA was due to i t s rapid conversion to cytoplasmic RNA and transport out of the nucleus. Within the nucleus of adult brain cells there would thus appear to be l i t t l e degradation of RNA to acid-soluble products of the type reported for HeLa c e l l nuclei by Harris. - >' 0 0 Nuclear RNA exhibited a heterogeneous breakdown pattern with half-lives of from 26 h to 11.5 days. The specific radioactivity of nuclear RNA did not f a l l below that of the cytoplasmic RNA fractions in the manner which one would expect i f a simple precursor-product relation existed between nuclear and cytoplasmic RNA. This suggests that a large amount of the RNA synthesized in 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 re-utilized in RNA synthesis i s not known. Watts^1 has reported that the turnover rate of nuclear RNA i s more rapid in the adult than in the infant brain. The combined evidence suggests that in the adult, newly synthesized RNA i s processed and degraded in the nucleus as well as trans-ported to the cytoplasm at an enhanced rate. It should be emphasized that the available informa-tion on RNA turnover in brain has been derived from a complex mixture of cell-types which may differ considerably in their RNA content and metabolism.59 .60 ,86 -89 Watson^0 n a s reported that RNA turns over more rapidly i n neurons than in g l i a and neuronal RNA turnover i s markedly subject to variation de-pending on the functional activity of neurons, the physio-logical condition of the organism and i t s environmental status. Information as to the turnover rates of different classes of RNA (and of particular molecular species within particular class) within a single cell^type under various physiological conditions and states of neural activity i s not yet available. 1.3 Changes i n Brain RNA Content and Composition Accompanying Sensory Stimulation, Physiological Challenges and Learning Experience Consistent with the brain's specialization for de-tecting and responding to changes i n i t s ambient stimulus 6*5 91-97 f i e l d there have been numerous reports J* 7 J- 7 1 of - 26 -perturbations in 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, specific brain regions, subregions, or the activity of specific neurons. When adult rats were kept f i r s t in the dark for 3 days and then exposed to the light and sounds of a laboratory for 15 minutes, the polysome to monosome ratio increased by 8 3 % in the cerebral cortex but was not affected in the l i v e r . Evidence has also been reported indicating that the total RNA of neurons in 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 levels within 24 00 hours. 7 On the other hand, convulsions produced by methionine sulphoximine or electroshock caused a disaggregation of polysomes. 1 0 0' 1 0^ A more detailed study 1 0 2 of the time-course of electroshock showed a decrease in number of poly-ribosomes during the f i r s t 15 minutes, and an increase over the following 15 to 30 minutes. By 60 minutes after electro-shack no significant difference from control was observed. 1 0 3 Other workers 1 0 i +" 1 0^ have reported a rapid turnover of ribonucleoprotein (Nissl substance) and a decrease in total cellular RNA content under conditions of chronic - 27 -stimulation and stress. On the basis of such observations. Hyden has proposed that moderate neuronal excitation pro-duces increases in RNA as an adaptation to increased func-tional demand on the neurons whereas intense chronic stimu-lation produces a decrease in neuronal RNA content through (1) slowing down RNA synthesis, possibly by diverstion 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 result from a non-specific increase of a l l classes of cellular RNA.^2 That i s , the increased RNA content i s not due to a preferential or dispro-portional increase in the quantity of a particular class of RNA molecules, Hyden 1 1 0 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 activity and found that the new RNA has the same base-ratio characteristics as the bulk of RNA present in control c e l l s . Whether the RNA which remains after intense chronic stimula-tion exhibits a base composition different from that of resting c e l l s has not been determined. Shashoua has reported that KCl-induced convulsions and generally stressful physio-logical conditions resulted i n no detectable base composition changes in the RNA of whole goldfish b r a i n . H o w e v e r , the occurrence of local changes in specific brain regions would not be detected in such an analysis of whole brain RNA. - 28 -Although changes in RNA content serve as a reliable indicator of alterations in the metabolic and functional state of neurons, a deeper understanding of the significance of these changes w i l l require direct electrophysiological monitoring of the bioelectric activity of neurons as well as a more careful examination of the specific contribution of such parameters as stimulus frequency, intensity and duration to the net effects on cellular RNA content, composition and metabolism* Since m-, t-, and r-RNA subserve protein synthesis i t has been anticipated that under those conditions in which neuronal RNA content (or rate of synthesis) i s increased, there might be a comparable, even greater concomitant effect on protein content and synthesis. Sufficient comparative data has not accumulated to test this hypothesis. ' J Numerous reports have appeared demonstrating increases 114-117 in the total content f as well as changes in the base 1J4 118—124 composition »-"--»-« -»•<- 0 f neuronal RNA during the acquisition of a new behavior pattern. 1 2~*~ 1 3 1 Trained mice, compared to quiet and yoked mice, showed greater incorporation of radio-active uridine into b r a i n 1 1 ' ' * 1 3 2 nuclear and ribosomal RNA, greater incorporation of uridine into polysomes isolated from b r a i n , 1 1 3 and a higher ratio of brain polysomes to free ribosomes. 1 3 2 These results have been confirmed by autoradio-graphic studies with mice13** and rats given similar - 29 -training. 135,136 These changes were not observed i n l i v e r and kidney. 117,132-133. Reports 117,125 of increased uridine incorporation into RNA during training experience cannot always be re-lia b l y interpreted as indicating an actual increase i n the rate of RNA synthesis since increased labelling could also result from a reduction in the size of the endogenous un-labelled (most l i k e l y intranuclear) ribonucleotide precursor p o o l . 1 3 0 * 1 3 ' ' Changes in ribonucleotide precursor pool sizes have, in fact, been observed under such conditions.^ In-creased incorporation of label into RNA might also be observed i f a decreased rate of RNA degradation were combined with an unchanged rate of RNA synthesis. However, results obtained following brief pulse times are measures of the i n i t i a l rate of synthesis and are not significantly influenced by the rate of RNA degradation.^ Measurements of the turnover rate of neuronal RNA under these conditions have not been reported. The increases in neuronal RNA content which have been detected i n learning experiments may be an unspecific sign of increased activity, not related to a specific learning or information consolidation process. In view of the large i n -fluence of the organism's behavioral history upon these 112 139-l40 measures, '-^ i t would seem to be necessary to use as controls animals who have been previously adapted to some of the simple stimulus components of the training situation in - 30 -order to i d e n t i f y changes i n molecular species s p e c i f i c to the occurrence of instrumental learning during t r a i n i n g experience. I t has been r e p o r t e d 1 1 ^ * 1 1 ® " 1 2 1 that i n contrast to conditions of increased physiological sensory and/or motor a c t i v i t y i n non-learning situations, the RNA content i n -creases which accompany learning exhibit base-ratio changes i n the d i r e c t i o n of the base composition c h a r a c t e r i s t i c s of mRNA ( i . e . , higher adenine and u r a c i l content). This would indicate that i n d i v i d u a l neurons are capable of discriminating between sensory information processing contingent upon the a c q u i s i t i o n of a new behavior and a c t i v a t i o n which does not e n t a i l the a c q u i s i t i o n of new information. Such s p e c i f i c i t y of neurochemical response indicates that the regulation of neuronal RNA metabolism and possibly gene expression by sensory information i s mediated v i a the receptive pole of the neuron at which the e l e c t r i c a l l y - c o d e d information i s trans-duced into molecular messengers. RNA metabolism i s not d i r e c t l y dependent upon or coupled to the b i o e l e c t r i c response output or electrophysiological a c t i v i t y of neurons, 1 3 ® » l k 3 » 1 5 ^ but would seem rather to be regulated by neurohormones pre-sumably acting upon receptors of the post-synaptic membrane. The nature of the i n t r a c e l l u l a r molecular messages into which sensory information i s transduced and the i n t r a c e l l u l a r causal sequence of events by which the modulation of RNA metabolism - 31 -i s effected remains to be elucidated. Also>, i t i s not yet possible to i n f e r from the experimental evidence available that new molecular species of RNA are synthesized through the release of genome information i n order to account f o r the observed base-composition changes i n neuronal RNA. Although hormonal induction of genome information has been well 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 of the reported a l t e r a t i o n s i n RNA base-composition i n neurons have not been adequately tested. Such changes might be due to ( l ) a change i n the quantity of one or more species of mRNA or a d i f f e r e n t i a l quantitative s h i f t i n the rate of synthesis or degradation of two classes of RNA r e s u l t i n g , f o r example, i n a net increase i n mRNA/rRNA r a t i o , (2) enhanced a c t i v i t y of terminal addition enzymes; f o r example, of the kind catalyzing the po s t - t r a n s c r i p t i o n a l adeny-l a t i o n of the 3 '-terminal end of mRNA, or (3) i n t e r c e l l u l a r transfer of s p e c i f i c RNA molecules between g l i a and 91 110 i i ' neurons. ' Direct evidence of the occurrence of q u a l i -t a t i v e l y new molecular species of RNA or protein during learning (which might be obtained by competitive hybridiza-t i o n 1 ^ 1 ' 1 ^ 2 or electrophoretic p r o f i l e studies) i s not yet available to supplement the base-composition data and f a c i -l i t a t e the int e r p r e t a t i o n of t h e i r s i gnificance to the molecular mechanisms underlying learning and memory formation. - 32 -The questions of whether the changes in neuronal RNA base-composition which are correlated with the acquisition and consolidation of sensory information represent the syn-thesis of qualitatively new species of mRNA and whether 12 114 144 eventually new species of proteins • * also are formed during the establishment of a new behavioral response have not been definitively answered. However, sufficient data have, nevertheless, accumu-lated to implicate at least an indirect involvement of RNA i n the molecular processes which underlie changes in neuronal function brought about by changes in sensory stimulation and learning experience. The determination of how transient per-turbations of the steady-state neuronal levels of RNA are effected w i l l require a more detailed understanding of the enzyme systems responsible for 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 li k e l y coupled to neuronal membrane receptors. The particular concern of the present study i s the RNA degradation system of the c e l l , i t s participation i n cellular RNA metabolism and i t s contribution to the changes in cellular RNA which have been observed to occur i n the course of brain development, aging and during alterations in neuronal function i n the adult brain. - 33 -1.4 The Role of the RNase Enzyme System in RNA Metabolism It i s probable that the determination of the lifetime of various RNA molecules by RNases operates as a key factor in the control of cellular biosyntheses. Considerable effort has, therefore, been invested in 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 in RNA. The participation of enzymes cleaving the diester linkages between ribonucleotides has been implicated i n diverse aspects of celiular metabolism. Biological functions for which some evidence i s available include (l) the maturational processing of precursor transcription products, and (2) the degradation of endogenous ribosomal, messenger, transfer and nuclear RNA. Other possible functions for which experimental evidence i s more tenuous include (3) defense against foreign RNA , and (4) provision of ribonucleotides for reutilization by the RNA synthesizing apparatus. There is sufficient evidence now to conclude that in both procaryotic and eucaryotic cells mRNA1^"1^0, ^ i-RNA 1 5 1" 1 5 3 , 1 3 5 t l 5 6 ind tRNA 1 5 7' 1 5 8 are a l l transcribed as precursor RNA molecules which are subsequently trimmed of their extra nucleotide sequences by maturation endoribonu-cleases to form functionally mature RNA molecules. The processing of the large nuclear pool of rapidly turning over - 34 -HnRNA appears l i k e l y to involve ( l ) s i t e - s p e c i f i c endori-bonucleases which produce s e l e c t i v e cleavage(s) of the primary t r a n s c r i p t i o n products, and (2) RNases of more general s p e c i f i c i t y which degrade the non-functional cleavage products to monoribonucleotides. Considerable research i s currently being directed toward elucidating t h i s c r i t i c a l r o l e of RNases i n nuclear RNA metabolism and i n the provision of fu n c t i o n a l l y mature RNA molecules to the protein-synt&esizing apparatus of the cytoplasm. The RNase enzyme system also regulates the functional l e v e l s of both nuclear and cytoplasmic RNA content by i t s p a r t i c i p a t i o n i n RNA degradation. The determination of the li f e t i m e of various RNA molecules by RNases i s of obvious importance i n regulating the a v a i l a b i l i t y and l i m i t i n g the extent of use of fu n c t i o n a l l y mature RNA molecules i n protein synthesis. A t h i r d possible function of RNases suggested by H e r r i o t 1 - ^ i s the degradation of inf e c t i o u s v i r a l RNA. That a b a r r i e r exists to i n f e c t i o n of plant and animal c e l l s by naked v i r a l RNA i s well e s t a b l i s h e d , 1 6 0 ' 1 6 1 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 f u n c t i o n . 1 6 2 ' 1 6 3 ISwfvirJ iK f l a n t l 4EI Svlrlii content of RNase i n leaves i s not correlated with s u s c e p t i b i l i t y to 164 i n f e c t i o n by in t a c t v i r u s . Direct evidence l i n k i n g RNases with protection against infection by RNA virions i n plant, animal or bacterial cells has not yet been reported. Finally, a possible function of RNA depolymerizing enzymes which has received l i t t l e consideration i s the pro-vision of ribonucleotide precursors to the RNA synthesizing apparatus of the nucleus. Some question e x i s t s 1 6 ^ as to the sufficiency of nuclear de novo nucleotide synthesis and nucleotide influx from the c^jfoplasm as mechanisms for the replenishment of ribonucleoside triphosphates within nuclei during RNA synthesis. It has been alternatively hypo-t h e s i z e d 1 6 ^ ' 1 6 6 that rate-limiting control of RNA synthesis may be achieved through the regulation of the free ribonu-cleotide 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 syn-thesis and RNA degradation are tightly coupled or co-ordinated to achieve steady-state turnover rates, (2) how RNA synthesis can proceed at rapid rates without rapid depletion of the small pools of free ribonucleotides i n the nucleus, 1 6^ and (3) a possible function of the rapidly turning over homopolyribonucleotides 1 6 7" 1 7 0 and other nuclear RNA which does not reach the cytoplasm and whose - 36 -function in the nucleus i s to date undetermined. 1 7 1" 1 7^ 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 identified in guinea pig l i v e r , 1 7 6 * 1 7 7 rat l i v e r 1 7 8 and Ehrlich ascite carcinoma 1 7^ c e l l nuclei. The ribonuclease enzyme system thus modulates the levels of functional RNA molecules i n the c e l l by p a r t i c i -pating in the biosynthesis and degradation of functionally mature RNA. Much remains to be learned as to the specific contribution of this group of enzymes and i t s individual components to the specific changes i n cellular metabolism accompanying each stage of the c e l l cycle ( c e l l division, c e l l differentiation, c e l l growth, c e l l maintenance and c e l l degeneration). 1,5 Regulation of RNase Activity and RNA Degradation It must be postulated out of logical necessity that a l l tissues and cells possess an enzyme system for degrading RNA molecules. If this enzymatic apparatus were allowed to function uncontrolled i t would completely hydrolyze the cellular RNA. Since this does not occur, i t follows that Regulatory mechanisms must exist which control the functional levels of RNase activity and prevent the indiscriminate breakdown of RNA within the c e l l . - 3 7 -The molecular mechanisms by which intracellular RNase activity and RNA degradation are regulated are l i t t l e under-stood. The question as to which or whether a particular type of RNase in a c e l l specializes i n attacking only one class of RNA has not been definitively answered. While a l l classes of RNA are li k e l y to be competent substrates for any one RNase in variously manipulated i n vitro conditions, the intracellular situation of the RNA and of the enzyme may impose considerable restriction in vivo. Thus, apart from the degree of specificity inherent i n the enzyme-substrate interaction which may entail recognition of primary, secon-dary and tertiary structural features of the substrate, additional constraints on substrate-enzyme interation may depend on the functional level of various RNases as well as their accessibility to substrate. Several means by which such regulation or restriction of enzyme-substrate inter-action may be achieved in vivo include» (l) spatial segrega-tion of substrate and enzyme by differential intracellular compartmentalizatiom ( 2 ) complexing of RNA with protein to form ribonucleoprotein structures resistant to the action of RNaSes? ( 3 ) complexing of enzymes with inhibitor molecules; ( 4 ) allosteric modulation of the substrate-binding or cata-lytic^of VR^ K s e s by small effector molecules, and ( 5 ) altera-tions in the rate of de novo synthesis or turnover of RNases. - 38 -S p a t i a l segregation of the enzyme i s c l e a r l y involved i n the prevention of indiscriminate degradation of RNA by i fin the large amounts of RNase present i n lysosomes. Lyso-somal RNase i s u n l i k e l y to be involved i n the turnover of a single s p e c i f i c class of RNA. The a c t i v i t y of lysosomal RNase depends on the fusion of vacuolized RNA p a r t i c l e s with the lysosomes or the rlease of lysosomal RNases into the cytosol following a change i n lysosomal membrane permea-b i l i t y . Such lysosomal membrane changes have been reported T fin i fti to occur under conditions of stress • and are followed by a considerable breakdown of messenger and r&bosomal RNA. Upon being synthesized most RNA becomes associated with s p e c i f i c proteins. The protein-associated state of the RNA provides an obvious means of regulating i t s accessi-b i l i t y and s u s c e p t i b i l i t y to degradative enzymes. That RNA-associated proteins r e s t r i c t the a c t i v i t y of RNases i n vivo i s indicated by the fact that i n i n t a c t polysomes, rRNA i s unaffected by l e v e l s of RNase which r e a d i l y attack the inter-ribosomal segments of mRNA and the rRNA of free ribosomal ftfi 1 O p subunits. * Ribonucleoprotein p a r t i c l e s may even bind 183 184 RNase without destruction of th e i r RNA. Bovine pan-cr e a t i c RNase A-catalyzed degradation of ribosomes has been demonstrated to depend on t h e i r functional state, and J354 Yanofsky has suggested that mRNA while associated with r i b o -somes i s afforded some protection from the action of RNases. - 39 -Associated proteins thus confer resistance upon the RNA of ribonucleoprotein complexes by imposing constraints 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 sequences i n the RNA molecules. Maturational processing and turnover of RNA must, therefore, proceed i n a regulated sequence of steps with exposed cleavage s i t e s being attacked f i r s t possibly by s i t e - s p e c i f i c endoribonucleases. I n i t i a l c l e a -vages would be expected to induce conformational changes i n the ribonucleoprotein structure possibly accompanied by the d i s s o c i a t i o n or loosening of bound protein and hence the exposure of new cleavage s i t e s which could then be attacked by RNases of more general s p e c i f i c i t y . Thus, the degradation of a single RNA molecule might e n t a i l the p a r t i c i p a t i o n of several d i f f e r e n t RNases at d i f f e r e n t stages of i t s degrada-t i o n . Also, the concerted action of RNases and proteases i n the degradation of ribonucleoprotein i s suggested by the synchronous turnover of the RNA and protein components of r i b o s o m e s . 7 0 Any impairment i n the f i d e l i t y of trans-c r i p t i o n would r e s u l t i n defective RNA molecules which could not p a r t i c i p a t e i n ribosome and informosome assembly. This defective RNA unprotected by function would thus be more susceptible to attack by RNases and, therefore, would be degraded rapidly, whereas the functional pool of RNA would undergo normal turnover. Such a mechanism of d i f f e r e n t i a l degradation of functional versus non-functional RNA molecules - 40 -would minimize the manifestation of t r a n s c r i p t i o n a l errors at the l e v e l of protein and c e l l function. Thus, the control of RNA turnover could depend greatly on changes i n l o c a l i o n concentrations and other factors which dissociate ribonucled-protein complexes as well as upon more d i r e c t regulation of the functional l e v e l s and a c t i v i t i e s of s p e c i f i c RNases. The complexing of enzymes with s p e c i f i c protein i n h i -b i t o r s has 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 a c t i v i t y of such catabolic enzymes as pro-t e a s e s , 1 8 5 deoxyribonuclease 1 8 6 and pH 7.8 RNase. 1 8 7 The functional l e v e l of the l a t t e r enzyme thus depends on the p a r t i t i o n i n g between active and inhibitor-bound forms of the enzyme and could be regulated by any factors a f f e c t i n g t h i s -l DO balance. The reported absence of detectable RNase i n h i -b i t o r protein i n nuclei may p a r t i a l l y account f o r the rapid turnover of RNA i n the nucleus, whereas RNA transported out of the nucleus into the cytosol which contains a large excess of RNase i n h i b i t o r , escapes rapid degradation. 189 Some evidence does exi s t 7 f o r the control of functional l e v e l s of i n t r a c e l l u l a r RNases through the regula-t i o n of the rate of synthesis and t r a n s l a t i o n of the mRNA molecules coding f o r these enzymes. The factors responsible f o r e f f e c t i n g t h i s control are unknown. The factors regu-l a t i n g the l i f e t i m e and turnover of s p e c i f i c RNases are also unknown. - 41 -Much remains to he understood as to the mechanisms by which RNA degradation i s regulated and coordinated with RNA synthesis. 1.6 Intracellular RNases of Non-Neural Mammalian Tissues Intracellular RNases of mammalian tissues have not been purified to homogeneity and knowledge of their structure and function does not approach in completeness that for bovine pancreatic RNase A or RNase T^. Extensive study of this enzyme system in animal tissues has, nevertheless, revealed i t to be complex, consisting of many distinguishable components widely divergent i n their structural and functional ppoperties. The intracellular RNase system of rat l i v e r has been studied more extensively than that of any other tissue, and the following discussion w i l l hence deal primarily with those components of the RNase system which have been iden-t i f i e d and more or less thoroughly characterized in this tissue. 1.61 Acid RNase An RNase with an acid pH optimum has been charac-terized and partially purified by a number of investi-gators. 190-193 _«his enzyme i s highly unstable and i s rapidly inactivated by heat and dilute acid. It has a molecular weight of about 24,000 to 28,000 Daltons, 1^ a pH optimum - 42 -of 5*8, and no metal ion requirement. Acid RNase has been reported to degrade p a r t i a l l y hydrolyzed RNA more rapidl y 192 than high molecular weight RNA, 7 to hydrolyze a l l purine and pyrimidine phosphodiester bonds (although i t has a pyrimidine bond preference) to 3'-ribonucleotides with the intermediate formation of 2•, 3'-cyclic ribonucleoside 191-195 phosphates 7 J and hence to leave no undialyzable RNA 196 197 ' core. 7 ' 7 1 The single-stranded form of RNA i s s p e c i f i -c a l l y or p r e f e r e n t i a l l y attacked. Although a high p u r i f i -cation of t h i s enzyme from r at mammary carcinomas has 189 recently been reported, ' d e f i n i t i v e substrate s p e c i f i c i t y studies with highly p u r i f i e d enzyme preparations have not yet been done. I n i t i a l studies 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 of t h i s enzyme found i t to be associated primarily with the 198 199 200 crude mitochondrial f r a c t i o n . • 7 7 De Duve et a l . subsequently demonstrated that t h i s enzyme along with other acid hydrolases i s associated not with mitochondria per se ibuti With a separate class of organelles which they named 196 "lysosomes." Reid and Nodes 7 obtained s i m i l a r r e s u l t s and concluded that acid RNase was present i n c e l l p a r t i c u l a t e s intermediate i n size between mitochondria and that class of 201 lysosomes containing acid phosphatase. Rahman also studied 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 acid RNase i n comparison with that of several other acid hydrolases and - 43 -found i t to be present i n a class of lysosomes s i m i l a r to that containing cathepsin D but distinguishable from those lysosomes containing acid phosphatase and cathepsin C. De 202 20? Lamirande and A l l a r d • J also found that acid RNase i s not contained i n the same class of c e l l p a r t i c u l a t e s as acid phosphatase, but concluded on the basis of the s i m i l a r i t y of i t s d i s t r i b u t i o n with glutamate dehydrogenase that i t must be present predominantly i n mitochondria. Considerable amount of the t o t a l c e l l u l a r acid RNase a c t i v i t y has also been recovered i n the high speed supernatant f r a c t i o n of 194 202 c e l l horaogenates. 7 * I t i s not clear whether t h i s f r a c t i o n of the enzyme a c t i v i t y a c t u a l l y exists i n s i t u i n the soluble phase of the cytoplasm or whether i t represents enzyme released from lysosomes during the tissue homogeniza-t i o n and subcellular f r a c t i o n a t i n g procedures. Some evidence 194 has been obtained 7 suggesting that the acid RNase a c t i v i t y of the cytosol may be due to an enzyme d i f f e r e n t from that found i n the crude mitochondrial f r a c t i o n . 1.62 pH 9.5 RNase An al k a l i n e RNase with a pH optimum of 9$5 was f i r s t 204 205 detected by Rahman ' J i n rat l i v e r . I t i s strongly i n h i -bited by most monovalent and divalent cations and t h i s i n h i b i t o r y e f f e c t i s not due to i o n i c strength. However, at a concentration of 0.5 to 1.0 x 10 J M Mg , unlike other divalent cations, produces a 30 to 40$ a c t i v a t i o n of t h i s enzyme. Inhibition m EDTA can also be overcome by Mg++ at a suitable concentration. This enzyme i s heat and acid labile and exhibits substrate inhibition with an RNA concen-tration of more than 2.0 mg/ml. The above characteristics 204 have been reported for the unpurified enzyme and no puri-fication procedure or substrate-specificity data have yet been published. In a study of i t s intracellular distribution Rahman reported the pH 9 . 5 RNase to be associated prima-r i l y with mitochondria and microsomes. L i t t l e of the total cellular content of this enzyme was recovered in the lyso-somal and supernatant fractions, although lysosomes possessed the highest specific pH 9 » 5 RNase activity. 1 . 6 3 pH 7 . 8 RNase An alkaline RNase with a pH optimum of 7 . 8 has been extensivley studied and partially 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 activity maxima between pH 6 . 7 and 8 . 5 , i t has no divalent cation requirement, and i s stable at 7 0 ° for 5 minutes. It hydrolyzes RNA more rapidly than poly U and poly C and shows no activity toward poly A and poly G. It appears to s p l i t principally pyrimidine phosphodiester bonds to yield ribonucleoside-3'-phosphates via 2 * , 3 ' - c y c l i c pyrimidine nucleotide intermediates, and leaves a resistant undialyzable oligoribonucleotide core. y ' This enzyme i s thus similar in i t s functional properties to bovine pancreatic RNase A. pH 7 * 8 RNase has been reported - 4 5 -to have a molecular weight of between 11 ,500 to 12,000 \qL 208 Daltons. Beard and Razzell have achieved a 3,000-fold purification of this enzyme from hog liver and Gordon 2°^ has purified rat l i v e r pH 7.8 RNase 6,000-fold. Results obtained by various workers concerning the intracellular distribution of this enzyme are not entirely 207 in agreement. Roth ' separated the rat l i v e r homogenate into only three fractions—nuclei, crude mitochondria, and supernatant—and reported that the total pH 7.8 RNase activity was recovered in the mitochondrial and supernatant fractions with a small amount of enzyme in the nuclei. Reid 196 and Nodes 7 achieved a further fractionation of the crude mitochondrial fraction into mitochondria and lysosomes and reported that the highest specific activity of pH 7.8 RNase occurred not i n mitochondria but in a class of lysosomes "less readily sedimented than those particles containing acid phosphatase." However, these investigators did not present their data in terms of the per cent distribution of the total activity. Rahman subsequently confirmed that the lysosomal fraction possessed the highest specific pH 7.8 RNase activity (3-fold that of any other fraction) and found that the per cent recovery of total activity was lowest in the lysosomal and highest for the mitochondrial fraction. F i f t y per cent of the total recovered pH 7.8 RNase activity was also approximately equally distributed between the - 46 -microsomal and supernatant fractions. De Lamirande and 199 Allard 7 7 under their subcellular fractionation and assay-conditions found pH 7.8 RNase to be evenly distributed among mitochondria, lysosomes and microsomes. Discrepancies in the intracellular distribution data probably reflect d i f f e -rences in assay procedures employed in different labora-tories. 1.64 RNase Inhibitor 210 Pirotte and Desreux f i r s t described an RNase inhibitor protein in the cytosol of guinea pig l i v e r . The properties of the inhibitor from rat li v e r have been studied,187,211,212,213,155.158 a n d i t h a g b e e n p a r t i a l l y 214 215 purified. It has also been studied in mouse tissues, J ? 16 ? 1 7 ? i ft mouse ascites tumours, rat adipose tissue ' and blood. The rat l i v e r RNase inhibitor i s a labile protein readily inactivated by acid, heat, heavy metal ions such as Hg++ and Pb++ and by sulfhydryl-blocking reagents such as p-chloromer-curibenzene sulfonic acid. '* The free inhibitor i s present in large excess in the rat l i v e r supernatant fraction as indicated by the inhibition by this fraction 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 inhibitor present which i s complexed to the endogenous pH 7«8 - 47 -RNase."1""''•tJ"L On treatment of the supernatant f r a c t i o n with sulfhydryl-blocking reagents, acid, or heat the i n h i b i t o r i s inactivated and the inhibitor-bound latent pH 7*8 RNase i s 211 released i n active form. Some c h a r a c t e r i s t i c s of the i n t e r a c t i o n between RNase i n h i b i t o r and modified derivatives of bovine pancreatic 219 RNase A have been studied. * I t was determined that the reaction between the two proteins i s not dependent on the active s i t e of the RNase, nor on the free amino groups of the enzyme. Interaction i s , however, strongly dependent on a r e l a t i v e l y native configuration of the RNase and on hydrogen bonding. There i s considerable evidence that free sulfhydryl 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 . Roth has suggested that since pancreatic RNase A has no free sulfhydryl groups, the formation of a disulphide bond between RNase and i n h i b i t o r i s u n l i k e l y . Rather, the s u l f h y d r y l group(s) of the i n h i b i t o r may p a r t i c i p a t e i n the formation of hydrogen bond(s) with the enzyme, or may be required to maintain the active conformation of the i n h i b i t o r . RNase i n h i b i t o r i s active p r i n c i p a l l y i n the pH range between 7 and 9 and has been reported to produce l i t t l e or no i n h i b i t i o n of acid R N a s e 1 9 5 * 2 1 3 or pH 9.5 RNase. 2 0^ The s p e c i f i c i t y of the i n h i b i t o r action i s not wholly accountable i n terms of the pH dependence of the inhibitor-enzyme i n t e r -action since the 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 3 whereas RNase Tj which i s similar in several respects to pancreatic RNase A was not inhibited when assayed at pH 7 . 8 . 2 1 3 RNase inhibitors isolated from different mammalian sources are not specific and do cross-react in varying 212 degrees with heterologous pH 7 . 8 RNases. There are considerable tissue- and species-specific differences in the relative amounts of free and RNase-bound i n h i b i t o r . 1 8 7 RNase inhibitor has recently been purified to near 2 2 0 2 2 1 homogeneity • and i t has been found to be an acidic protein with a molecular weight of 5 0 , 0 0 0 to 6 0 , 0 0 0 Daltons. 2 2 2 1 , 6 5 Ribosomal RNase Another alkaline RNase has been detected in ribosome and polysome preparations from rat l i v e r . 2 2 3 " 2 2 5 * This ribosomal RNase has a pH optimum of 8 . 5 . i s relatively stable in acid, but rapidly inactivated above 5 5 ° . It i s stimulated by an optimal divalent cation (Ca.++ or Mg++) concentration of 1 0 mM and a monovalent cation (K+ or Na+) concentration of 5 0 mM. Its molecular weight has been estimated to be 3 7 , 0 0 0 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 in a latent form bound to RNase inhibitor. Although - 49 -direct inhibition of the purified enzyme by the cytosol or purified inhibitor has not yet been demonstrated, several workers have reported thatppolysomes are stabilized by RNase inhibitor preparations. The fact that, despite considerable effort, i t has not been possible to isolate polysomes free of RNase activity suggests that the bound RNase may be an integral part of polysome structure and may be involved in their normal O O f\ function. Roth has made the interesting observation that the activity of the alkaline RNase present in rat liver micro-somes must be functionally intact for the incorporation of 14 C-l-leucine into protein. This suggests that this 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 for the rapid turnover of RNA in the nucleus since the activity of these enzymes may regulate the kind and amount of RNA that does find i t s way into the cyto-plasm. op o op Q Heppel et a l . '* have detected an alkaline RNase with a pH optimum between 8 . 5 and 9 . 5 in nuclei of guinea pig l i v e r . This enzyme i s inactivated below pH 6 and above 5 0 ° . It hydrolyzes poly A and poly U to ribonucleoside-5'-phosphates - 5 0 -and oligoribonucleotides of two to s i x units i n length t e r -purj 229 minated by 5 '-phosphates. 1 7 6 An 80-fold i f i c a t i o n of t h i s enzyme has been achieved by Razzell.' 230 Cunningham and Steiner J have reported further properties of what appears to be the same enzyme. A polyribonucleotide phosphorylase has also been reported to be present i n guinea p i g l i v e r n u c l e i 1 7 7 and HeLa c e l l n u c l e i . 2 3 1 This enzyme i s supposedly associated with chromosomes and may p a r t i c i p a t e i n the breakdown of newly synthesized heterogeneous nuclear RNA (HnRNA) to ribonucleoside - 5'-diphosphates. An RNase a c t i v i t y has also been detected i n the nucleolar f r a c t i o n obtained from normal, 23 2* 233 neoflsiiifei© 233 234 235 p l a s t i c J J * J and regenerating J J rat l i v e r as well as from H e L a 2 3 6 * 2 3 7 and L cells. 2 3® This enzyme i s an endori-bonuclease and has been implicated i n the processing of 4 5 S precursor ribosomal RNA into mature 18 S and 28 S tRNA. However, t h i s putative function remains to be d e f i n i t i v e l y demonstrated. Other endoribonucleases analogous and possibly homo-logous to those functioning i n the maturational processing of precursor t R N A 2 3 9 , 2 2 + 0 and precursor r R N A 2 k l " 2 k 3 i n bacteria have not yet been demonstrated i n mammalian c e l l s . 244 245 Stein and Hausen ' J have reported an i n t e r e s t i n g enzyme (RNase H or hybridase) from c a l f thymus which - 51 -s p e c i f i c a l l y degrades the RNA moiety of DNA-RNA hybrids. The recent f i n d i n g that RNase H a c t i v i t y appears to be a 246 247 universal property of reverse transcriptases ' ' from RNA tumor viruses has led to the suggestion that the bio-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 o l i g o r i -bonucleotide which normally functions as a primer i n DNA 248 249 2 50 2 51 r e p l i c a t i o n ' 7 and which becomes covalently linked J »<•-'•»• to the newly synthesized DNA chain. Stimulation of i n v i t r o t r a n s c r i p t i o n by rat l i v e r RNA polymerase B of native rat l i v e r DNA and chromatin has also been r e p o r t e d 2 ! 2 to occur i n the presence of rat l i v e r RNase H. This stimulation was attributed 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 release of newly synthesized RNA from the t e m p l a t e . 2 5 2 In general, studies of nuclear RNases have not achieved a high degree of r e s o l u t i o n . Those enzymes which have been detected have not been extensively p u r i f i e d or characterized. Further work i s required to define the role of these enzymes i n the turnover of RNA i n the nucleus &n_d i n the nuclear maturation of cytoplasmic RNA. 1.7 Changes i n RNase A c t i v i t i e s under Various Physiological and Pathological Conditions A number of workers have monitored variations 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 various components of the RNase enzyme system i n several tissues under various - 52 -p h y s i o l o g i c a l and pathological conditions with the expecta-t i o n that such information might y i e l d a better understanding of the functional role of these enzymes i n the regulation of c e l l u l a r RNA content and turnover. C e l l u l a r RNase a c t i v i t y has been found to be elevated under several circumstances i n which the RNA/DNA r a t i o i s r e d u c e d 2 5 3 ' 2 5 5 and protein s y n t h e s i s 1 8 9 , 2 5 3 - 2 6 7 ^ g r e ( } U c e ( i t Thus, decreases i n RNase i n h i b i t o r or increases i n free pH 7 . 8 RNase a c t i v i t y l e v e l s have been observed i n tissues exhibiting a low rate of protein synthesis or an increase i n t h e i r catabolic a c t i v i t i e s . 2 5 7 " 2 6 7 In some c a s e s 1 8 9 , 2 5 7 , 2 5 8 , 2 6 5 increased acid RNase a c t i v i t y has also been demonstrated under s i m i l a r conditions. These observations suggest that the de-creased protein synthesis under these conditions i s due to elevated functional l e v e l s of various c e l l u l a r RNase a c t i -v i t i e s and hence enhanced degradation of c e l l u l a r RNA. Conversely, increased RNA/DNA r a t i o s and increased 268—? 7 5 protein synthesis have been demonstrated " ( J to be p o s i t i v e l y correlated with decreased functional l e v e l s of several c e l l u l a r RNase a c t i v i t i e s . Thus, increases i n the r a t i o of 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 tissues characterized by a high rate of RNA OAft oot synthesis and/or c e l l d i v i s i o n • f D such as i n regenerating r a t l i v e r 2 7 6 ' 2 7 7 and l i v e r of hypophysectomized r a t s , 2 5 5 * 2 7 8 2 7 0 2 fiO nephrotic k i d n e y C ( y * and during compensatory renal - 53 -hypertrophy following u n i l a t e r a l nephrectomy.'',J"L In the case of nephrotic kidney decreases i n pH 9.5 RNase and acid RNase were also found to be p o s i t i v e l y correlated with the elevated rate of protein synthesis. These observations have suggested that the increased protein synthesis i n these con-di t i o n s may r e s u l t from the preservation of mRNA and possibly also tRNA and rRNA. p Q p p Q 0 RNA turnover studies ' J have indicated that i n these situations the changes i n c e l l u l a r RNA content are due predominantly to changes i n the rate of RNA catabolism rather than i n the rate of RNA synthesis. On the basis of these 2 72 findings, Shortman ' has hypothesized that 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 physiological conditions are effected through RNases by influencing the rate of RNA degradation. This work thus points to a need f o r a thorough in v e s t i g a t i o n of the contribution of the RNase enzyme system to the changes i n c e l l u l a r RNA of brain which have been reported to occur i n response to changes i n physiological stimulation and during learning and memory formation. 1.8 The RNase Enzyme System of Brain Whereas some information exists concerning the enzymes involved i n the synthesis of RNA i n brain, comparatively l i t t l e i s known of the molecular processes by which c e l l u l a r - 54 -RNA content i s post-transcriptionally controlled. Infor-mation as to the enzymes participating in the maturational processing of precursor transcription products into functionally mature transfer, ribosomal, and messenger RNA as well as in the degradation of these molecules i s not available in the same degree of detail for brainnas for many other mammalian organs. R o t h 1 8 7 in a comparative study of various rat tissues found that RNase inhibitor activity was highest in brain and hence presumably has a particularly important role in regu-lating the metabolism of RNA in this tissue. Ellem and 215 Colter, J however, in a similar study of mouse tissues found that mouse brain contained very low levels of both pH 7.8 284 RNase and RNase inhibitor activity. Guroff in a develop-mental study of rat brain RNase activity assayed at pH 5 found that the level of RNase activity expressed at this pH was lower i n older animals than i n young animals. Suzuki and Takahashi 2 8 5 studied the regional distribution of RNase inhibitor in rabbit brain and found that those areas (cere-bral cortex, cerebellum, hippocampus) rich in neurons corres-ponded to the areas having highest RNase inhibitor activity. The maximal variation in the level of RNase inhibitor activity between the different brain regions studied was not more than 30$. These investigators have also reported a developmental profile for RNase inhibitor activity in rat - 55 -cerebral cortex. This component of the RNase system exhibits a sharp peak between the 5th and 10th day after birth, f a l l s to near neonatal levels by the 13th day, and subsequently 221 remains relatively constant. Takahashi, Mase and Suzuki have also recently reported a high purification of RNase inhibitor from pig cerebral cortex. Datta and co-workers have detected two RNase activ i t i e s with pH optima of 5»4 and 7.9 in ribosomes isolated from goat cerebral cortex. Detec-tion of significant activity required incubation periods of 14 to 18 hours. Both activities were tightly bound to the ribosomes and could not be solubilized. These workers have suggested that these ribosomal RNase activ i t i e s may be res-ponsible for the chromatolytic changes which ribosomes undergo during neuronal stress. An RNase activity with a pH optimum of 7.6 has been detected ' in the cerebrospinal f l u i d of humans. 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 infarction, neoplasm, and demyelination due to multiple sclerosis) exhibited elevated levels of this RNase activity. In general, the available information on the RNase enzyme system of brain i s fragmentary and unconfirmed. - 56 -1.9 The Present Investigation Because the RNase enzyme system plays such an important r o l e i n regulating i n t r a c e l l u l a r RNA content and hence i n regulating 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 to the protein-synthesizing apparatus of the c e l l , i t i s possible that any d i s t i n c t i v e charac-t e r i s t i c s of t h i s enzyme system i n brain may have a d i r e c t r e l a t i o n s h i p to the specialized function of t h i s t i s s u e . A M Q C S inasmuch as changes i n brain c e l l u l a r RNA content have been demonstrated to occur i n response to learning and sensory stimulation, the role of RNases may achieve special s i g n i -ficance i n brain i n comparison with other tissues such as l i v e r which have been more extensively studied. Thus, f o r example, the enhanced l e v e l s of neuronal RNA r e s u l t i n g from sensory experience may be predominantly due to decreased RNA degradation which, i n turn, may be due to decreased 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 inve s t i g a t i o n reported upon i n t h i s thesis was hence aimed at obtaining more detailed and comprehensive information about the c h a r a c t e r i s t i c s of the RNase enzyme system i n rat brain and at achieving some understanding of the contribution of the i n d i v i d u a l components of t h i s enzyme system to the regulation of RNA metabolism i n t h i s t i s s u e . I I . MATERIALS AND METHODS 2.0 Materials 2.01 Experimental animals Rats of the Wistar s t r a i n were obtained from the Vivarium of the Department of Zoology, University of B r i t i s h Columbia. 2.02 Chemicals T r i t o n X100 was a product of Rohm and Haas. U l t r a -pure sucrose and ammonium sulphate were obtained from Swartz/Mann. Para-chloro-mercuribenzoicacid was a product of N u t r i t i o n a l Biochemicals Corporation. This reagent was dissolved i n .02 M Tris-HCl buffer, pH 8.9. D i t h i o t h r e i t o l (Cleland's reagent) was 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 water immediately p r i o r to use. Gelatin was a product of Eastman Organic Chemicals and was p u r i f i e d by the method of Short-man. DEAE c e l l u l o s e with an exchange capacity of 0.84 mi l l i e q u i v a l e n t monovalent anion per gram was obtained from BioRad Laboratories and was p u r i f i e d by the method of 288 Peterson and Sober. A l l other common laboratory chemicals were "reagent grade" and were used without further p u r i f i -cation. - 57 -- 58 -Fleischmann's yeast s-RNA prepared by the method of 289 Holley 7 was the product of Calbiochem. Solutions of t h i s substrate prepared i n d i s t i l l e d water could be stored at 4° without exhibiting s i g n i f i c a n t increase i n acid-soluble A260. Highly polymerized double-stranded calf-thymus DNA was a product of Calbiochem, and was dissolved i n .01 M NaCl. Biif-(p-nitrophenyl) phosphate (Na) was a product of Sigma. C r y s t a l l i n e bovine pancreatic RNase A (ca. 50 Kunitz units/mg) was a product of N u t r i t i o n a l Biochemicals Corpora-t i o n . Bovine serum albumin was a product of Calbiochem. 2.1 Methods 2.11 Preparation of tissue homogenates and extracts Wistar s t r a i n white rats of body weight 200-400 g were decapitated and the whole brain quickly removed from the cranium and placed i n a glass p e t r i dish kept on i c e . The tissue was weighed, suspended i n i c e - c o l d 0.1$ (v/v) T r i t o n X100 or 0.32 M sucrose i n the r a t i o of one gram wet weight tissues 9 ml of homogenizing medium, and homogenized at 0° with nine to ten strokes i n a glass homogenizer f i t t e d ,with a t e f l o n pestle (Arthur H. Thomas Co.; clearance, 0.13-0.18 mm). - 5 9 -Tissue used fo r p u r i f i c a t i o n work was processed as above with the exception of being homogenized at 0° i n a s t a i n l e s s s t e e l OmniMixer (Ivan S o r v a l l Inc.) f o r 3 0 seconds at speed control 5 t 3 0 seconds pause,and 3 0 seconds at speed control 6 . The 10$ (w/v) tissue homogenates were centrifuged at 3 k , 800 x g for 6 0 minutes at 0-4° and the supernatant decanted and referred to as the "extract." 2.12 Fractionation of isotonic sucrose homogenates by d i f f e r e n t i a l centrifugation For subcellular d i s t r i b u t i o n studies isotonic sucrose homogenates were fractionated by d i f f e r e n t i a l centrifugation at 0° i n a type S S - 3 k rotar of a S o r v a l l RC2 centrifuge and a Spineo rotar # 5 0 of a Beckman Model L u l t r a c e n t r i f u g e . Isotonic sucrose homogenates prepared as described i n section 2.11 were centrifuged at 800 x g for 10 minutes, the crude 800 x g p e l l e t was resuspended i n a volume of i c e - c o l d 0 . 3 2 M sucrose equal to l / 5 the volume of the i n i t i a l homogenate (resuspension was carried out i n a glass homogenizer, the pestle being gently rotated by hand), and centrifuged again at 800 x g for 10 minutes to y i e l d the f i n a l 800 x g nuclear p e l l e t . Supernatants from the preceding two centrifugations were pooled and centrifuged at 8,000 x g f o r 20 minutes to y i e l d the 8,000 x g mitochondrial p e l l e t . The 8,000 x g - 60 -supernatant was then centrifuged at 105,000 x g for 60 minutes (Spinco rotar #50) to y i e l d the 105,000 x g microsomal p e l l e t and the 105,000 x g supernatant or 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 to a f i n a l volume with i c e -cold 0.32 M sucrose equal to one-half the s t a r t i n g volume of the homogenate, and rehomogenized at 0° with f i v e strokes i n a glass 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 ultrapure ammonium sulphate was sprinkled onto the surface of a magnetically s t i r r e d crude enzyme extract maintained at 0° i n an ice-water bath. The rate of addition of ammonium sulphate was controlled so as not to exceed the rate at which the s a l t dissolved. The amount of ammonium sulphate added to give a desired saturation was 290 calculated according to the formula of Noda and Kuby. 7 After 30 minutes of additional s t i r r i n g , the pre c i p i t a t e s were co l l e c t e d by centrifugation at 3k,800 x g f o r 15 minutes, suspended i n ic e - c o l d .02 M Tris-HCl,pH ?. k and dialyzed at k : ! ° i n the cold room against the same buffer. Nessler's reagent was used to test f o r complete removal of (NHu^SOk from the dialysates according to Umbreit, Brunis and S t a u f f e r . 2 3 6 * - 6 1 -2 . 1 3 2 DEAE cellulose column chromatography of ammonium sulphate fractions Diethylaminoethylcellulose was treated according to p OQ the procedure of Peterson and Sober. The washed DEAE cellulose, suspended in . 0 2 M Tris-HCl, pH 7»4 eq u i l i -brating buffer, was poured into columns at 4° i n the cold room and allowed to settle under gravity. The packed columns were washed with 5 0 0 ml equilibrating buffer prior to being loaded with the dialyzed ammonium sulphate frac-tions. After the protein solution had completely entered the column matrix i t was washed in with ten or more bed volumes of equilibrating buffer and then eluted with a linear gradient of NaCl in equilibrating 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 fractions was sampled with a Beckman Model DU spectrophotometer. 2.14 Enzyme assays 2.141 Determination of RNase activity RNase activity was determined by measuring the increase in 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 . 0 5 M buffer, . 0 2 5 $ (w/v) ( . 2 5 mg/ml) Fleischmann's yeast s-RNA (in d i s t i l l e d H2O), 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. The reaction was terminated by placing 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 centri-fuge. The supernatant was carefully decanted and its absorbance at 260 nm measured immediately in s i l ica 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 non-enzymatic 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° • - 6 3 -A unit of RNase activity 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 3 7 ° • Specific activity was ex-pressed as units per mg protein. 2.142 Assay for deoxyribonuclease activity 291 The assay procedure for measuring DNase activity 7 was the same as that used for measuring RNase activity except highly polymerized double-stranded calf thymus DNA at a f i n a l concentration of . 0 2 5 $ (w/v) was used as sub-strate. Incubation was for 6 0 minutes at 3 7 ° . 2.143 Assay for phosphodiesterase activity Phosphodiesterase activity was measured by the liberation 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. 2 9 3 The reaction mixture contained 3 3 mM buffer, 0 .4 mM bis-(p-nitrophenyl) phosphate (Na), and enzyme sample in a total volume of 3 * 0 ml. Incubation was for 60 minutes at 3 7 ° • The reaction was terminated by transferring the incubation tubes to an ice bath and adding 3 * 0 ml of 0.04 N NaOH to each tube. The A400 was measured immediately. - 64 -2.144 Assay for pH 7 . 8 RNase inhibitor activity RNase inhibitor activity was determined by measuring the reduction in the activity of a standard amount of bovine pancreatic RNase A in the presence of various amounts of inhibitor sample. 1 8 7 Crystalline bovine pancreatic RNase A (ca 5 0 Kunitz units per mg) was dissolved in 0 . 1 $ (w/v) purified gelatin solution to a concentration of 0 , 0 1 ;ug/ml. The standard RNase inhibitor 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 dilutions of tissue sample to give a level of inhibition between 3 0 $ and 7 0 $ , and . 0 2 5 $ (w/v) sRNA i n a total volume of 2 . 0 ml. The substrate was added last after mixing the previous com-ponents. Controls for the activity of 0 . 5 ng crystalline bovine pancreatic RNase A alone, and for the RNase activity of the tissue samples alone, as well as appropriate reagent blanks were incubated and assayed concurrently. Corrections for any RNase activity in the inhibitor samples were made i n calculating the per cent inhibition of control pancreatic RNase A activity. Incubation was for 3 ° minutes at 3 7 ° . A l l other aspects of the assay procedure were the same as that previously described for the assay of RNase activity. The level of RNase inhibitor activity in the tissue samples was interpolated from experimentally constructed standard - 6 5 -curves r e l a t i n g per cent of control bovine pancreatic RNase A a c t i v i t y to protein concentration of the i n h i b i t o r sample. A unit of RNase i n h i b i t o r a c t i v i t y was defined as that 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 con-t r o l a c t i v i t y of 0 . 5 ng bovine pancreatic RNase A under these conditions and i s hence numerically equivalent to the weight of bovine pancreatic RNase A i n h i b i t e d . 2 . 1 5 Protein determination Protein was measured according to the method of Lowry 2 9 k et a l . using 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 of the RNase A c t i v i t i e s of Adult Rat Whole Brain Homogenates and Extracts I n i t i a l experiments were aimed at detecting RNase a c t i v i t i e s i n adult rat whole brain homogenates and extracts, and determining the effect on these a c t i v i t i e s of such variables as the medium used f o r homogenization of the ti s s u e , and the pH, buffer, and io n i c strength of the incubation medium. These experiments yielded information as to some general c h a r a c t e r i s t i c s of the RNase enzyme system i n brain and permitted s e l e c t i o n of optimal conditions of assay f o r the RNase a c t i v i t i e s which were detected. 3 . 0 1 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 Figure 1 shows three representative curves of the RNA-depolymerizing a c t i v i t y assayed at d i f f e r e n t pH values i n f r e s h l y prepared isotonic sucrose homogenates of adult rat whole brain. RNase a c t i v i t y i s detectable throughout the entire range of hydrogen ion concentrations tested with Tris-HCl buffer. Poorly defined a c t i v i t y maximae occur betwen pH 6 to 7 and between pH 8 to 9 » Relative to the a c t i v i t y expressed i n .05 M Tris-HCl, equimolar concentra-tions of imidazole-HCl buffer y i e l d 6 5 $ higher a c t i v i t y - 6 6 -- 67 -O CD ° "5 .y a M— a c v> \ CO ~ .5 .4 .3 .2 .1 ~A V J L _ i L. J__J 1_ 7 8 PH 10 FIGURE 1. The e f f e c t of pH on the a c t i v i t y of RNase i n i s o t o n i c sucrose homogenates of adult rat whole brain. Aliquots of 0.1 ml were assayed from a homogenate containing 11.3 mg-protein/ml. Incubation was f o r 60 minutes. The buffer systems used were Tris-HCl —A— , NHty-acetate —<5r-imidazole-HCl --+-- , and the f i n a l buffer concentration was 50 mM i n a l l experiments unless stated otherwise. - 6 8 -at pH 6 . 6 . A c t i v i t y l e v e l s are about 3 0 % lower with equi-molar concentrations of NHif-acetate buffer and the a c t i v i t y maxima i s s h i f t e d to more acid pH values. Since homogenization i n isotonic sucrose i s most l i k e l y to preserve the 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 organelles i t may be 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 conditions 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 added substrate when the subcellular p a r t i c l e s are s t i l l r e l a t i v e l y i n t a c t . The amount of RNase a c t i v i t y expressed i n isotonic sucrose homogenates i s about 5 0 , ? 6 , and 6 8 per cent at pH 6 . 7 » 7 * 8 and 9 . 5 respectively of the t o t a l extractable RNase a c t i v i t y assayed i n 0.1% T r i t o n X100 homogenates. Hence, i t may be t e n t a t i v e l y concluded that under conditions which most c l o s e l y approximate the i n s i t u state of these enzymes, more of the t o t a l detergent-extractable a l k a l i n e RNase a c t i v i t y (as compared to acid RNase) of brain i s accessible to added substrate. 3.02 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 adult r a t whole brain i n 0.1% (v/v) of the non-ionic detergent T r i t o n X100, which i s known to disrupt,and s o l u b i l i z e l i p o p r o t e i n membranes, resulted i n l e v e l s of RNase a c t i v i t y which were 5 0 , 24, and 3 2 per cent higher at pH 6 . 7 , 7 . 8 , and 9 * 5 respectively than that expressed i n isotonic sucrose homogenates. The 0,1% T r i t o n - 69 -homogenate and the 0.1$ T r i t o n extract prepared from i t according to the procedure described i n section 2.11 of Methods exhibited nearly congruent pH versus RNase a c t i v i t y curves. 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 recovered i n the extracts. This observation indicates that homogenization i n 0.1$ T r i t o n XI00 was e f f e c t i v e i n producing complete c e l l breakage and i n releasing i n soluble form any active RNase bound to or sequestered within 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 to acid RNase a c t i v i t y r a t i o i s con-siderably lower f o r the 0.1$ T r i t o n X100 extract (see F i g . 2 on page 72) compared to that 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 that homogenization of the tissue i n detergent as compared with isoto n i c sucrose resulted i n the extraction of r e l a t i v e l y more acid 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 . The large difference between the l e v e l s of measurable RNase a c t i v i t y under these two homogenization conditions suggests that a large f r a c t i o n of the t o t a l RNA-degrading capacity of brain c e l l s may be present i n s i t u i n an inactive or latent state 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 to the r e l a t i v e contribution of these two possible i n s i t u - 70 -enzyme states to the observed latency i n enzyme a c t i v i t y . Also, no study was made of whether the r a t i o of RNase a c t i v i t y i n sucrose homogenates varies with developmental age. T r i t o n most l i k e l y acts to release and activate those enzymes present i n a latent, non-functional state 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 structures. The p o s s i b i l i t y that Triton-enhancement of expressed l e v e l s of RNase a c t i v i t y may be p a r t i a l l y due to a more d i r e c t action on the enzyme molecules per se or to i n a c t i v a t i o n of 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 of the e f f e c t of 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 indicated 50$ stimulation of acid 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 al k a l i n e RNases assayed at t h e i r respective pH optima (see Table VII, page i l l . Since the e f f e c t of added T r i t o n on the assay of RNase a c t i v i t y i n more crude but soluble enzyme preparations (such as hypotonic buffer extracts prepared without detergent) was not determined, i t must be considered that T r i t o n may act at l e a s t i n part by d i s s o c i a t i n g or preventing the formation of non-specific molecular aggre-gates involving RNase enzymes, and by minimizing the i n t e r -action of added substrate with non-specific proteins. In summary, the preceding r e s u l t s c l e a r l y demonstrate the importance of ensuring complete disruption of i n t r a -c e l l u l a r membrane compartment i f the f u l l RNase capacity - 71 -of brain tissue i s to be measured. Investigators of the components of 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 that maximal RNase a c t i v i t y could only be detected upon disruption of i n t r a -c e l l u l a r organelle membranes by hypotonic shock5 sonica-296 196 214 t i o n , 7 repeated freeze-thaw cycles, 7 * or non-ionic 204 295 20*4 detergent treatment. • y j Rahman, f o r instance, found that acid RNase i n r a t l i v e r i s o t o n i c sucrose homogenates exhibited only 5 to 10% of the t o t a l acid RNase a c t i v i t y extractable with 0.1% T r i t o n X100. He also reported that RNase a c t i v i t i e s assayed at both pH 7»8 and pH 9*5 were equally activated about 60% by 0.1% T r i t o n X100. 1 3.03 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 Buffer-dependent differences i n the a c t i v i t y and pH optimum of RNA-depolymerizing enzymes assayed i n a f r e s h l y prepared 0.1% T r i t o n X100 extract of adult r a t whole brain are shown i n Figure 2. The pH optimum of the acid RNase a c t i v i t y varies with the buffer system used, A d i s t i n c t pyramidal a c t i v i t y maxima i s observed at pH 6 .7 with both imidazole-HCl and Tris-HCl buffers, and at pH 6 ,4 and 5.9 with NH4-acetate and Na-phosphate buffers 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 ^ adult r a t whole brain. AJiqbotsfof 0 . 1 mi of a frfeshly prepared extract containing 5»2 mg.protein/ml were assayed i n various buffer systems as a function of the hydrogen ion concen-t r a t i o n of the incubation .mixture. Incubation was f o r 60 minutes. The buffer systems used were Tris-H61 — • — , NH4-acetate— A — , imidazole-HCl , and Na-phosphate --o-- . - 7 3 -Of the various buffers tested, maximal acid RNase a c t i v i t y was obtained with imidazole-HCl. Tris-HCl and N H 4 -acetate buffers yielded a c t i v i t i e s which were about 8 7 % and 6 9 % respectively of that obtained with imidazole-HCl, The l e v e l of a c t i v i t y at t h e i r respective pH optimae i s nearly i d e n t i c a l i n equimolar concentrations of Tris-HCl and Na-phosphate buffers. However, i n Na-phosphate buffer there occurs with decreasing hydrogen ion concentration a rapid 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 concentration of the buffer was found to have a considerable e f f e c t on both acid RNase a c t i v i t y and a l k a l i n e RNase a c t i v i t y assayed at pH 7 . 9 (Table I ) . The a c t i v i t y of acid RNase declines and a s h i f t to more acid pH optima occurs with increasing io n i c strength ( F i g . 3 ) » The higher i o n i c strength of an equimolar concentration of Na-phosphate buffer, as compared with the monovalent buffer systems tested, may thus account f o r the more acid pH optimum obtained with t h i s buffer. In view of the dependence of acid RNase a c t i v i t y and;. pH optimum upon i o n i c strength, i t seems l i k e l y that apparent discrepancies i n the maximal a c t i v i t y and pH optimum reported by several workers for t h i s enzyme i n other mammalian tissues i s due to differences i n the i o n i c strength under which 204 a c t i v i t y was assayed. Thus, f o r example, Rahman reported a pH optimum of 5*5 f o r acid RNase from rat l i v e r homogenate assayed i n 200 mM acetate buffer. - 7 k -TABLE I. 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 buffer concentration (mM) Increase i n A 2 6 0 per 6 0 minute incubation 6 . 4 Na-phosphate 2 0 . 5 4 9 6 . 4 N.a-phosphate 5 0 . 3 6 9 7 . 9 Na-phosphate 2 0 . 1 6 7 7 . 9 Na-phosphate 5 0 . 0 5 6 . 7 . 8 Tris-HCl 5 0 . 2 6 6 A s i m i l a r interdependence of io n i c strength and pH 2 9 7 optimum has been observed with bovine pancreatic RNase A ' 298 and bovine brain acid DNase. 7 Low i o n i c strength and low pH favor the denaturation of polynucleotide substrates. Increasing the s a l t concentration or i o n i c strength, on the other hand, by shielding the mutually repelled charged phosphate groups of the polynucleotide chain, s t a b i l i z e s the hydrogen-bonded secondary structure and favors a more t i g h t l y c o i l e d double-stranded configuration. Thus, the i n h i b i t o r y e f f e c t on enzyme a c t i v i t y of increasing i o n i c strength may be p a r t i a l l y , though not completely, counteracted by con-currently decreasing the pH, thereby maintaining a s i n g l e -stranded form of the polynucleotide substrate. Assuming t h i s to be the primary mechanism of action of s a l t , i t can . be inferred that acid RNase, l i k e bovine pancreatic RNase A, p r e f e r e n t i a l l y attacks single-stranded sequences of RNA. - 75 -M o l a r i t y of N a C l FIGURE 3. The e f f e c t of NaCl on acid RNase a c t i v i t y , Aliquots of 0 . 1 ml of 0 . 1 % T r i t o n X 1 0 0 extracts of adult r a t whole brain were assayed (30 minute incubation) at the following pHi NH-4-acetate, pH4 — o — ; NH4-acetate, pH5 —w— ; NH4-acetate, pH.6—v— ; Tris-HCl, p H 7 "+*- . The a c t i v i t y assayed i n Tris-HCl at pH 6 . 4 i n the absence of added NaCl i s also shown , RNase a c t i v i t y i s expressed as the increase (corrected f o r substrata and.enzyme blanks) i n A 2 6 0 per 3° minute incubation. - 7 6 -In a single experiment, i t was indeed found that 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 acid RNase preparation was 3 0 % higher with heat-denatured rat brain nuclear RNA than with native nuclear RNA. However, u n t i l t h i s putative "explanation i s more conclusively tested, the d i r e c t e f f e c t of ion i c strength on enzyme conformation, s t a b i l i t y and s o l u b i l i t y must also be taken into consideration i n attempting to account f o r the observed ionie-strength-dependent changes i n the a c t i v i t y and pH optimum of acid RNase. In the alkaline region, the pH-RNase a c t i v i t y p r o f i l e obtained with Tris-HCl buffer ( F i g . 2) has a composite pattern with the appearance of several superimposed curves between pH 7 . 5 and 9 « 5 » This p r o f i l e i s suggestive of the presence of at leas t two a l k a l i n e RNase a c t i v i t i e s with overlapping pH curves. Phosphate and imidazole buffers 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 within which they r e t a i n e f f e c t i v e 213 buffering capacity. This i s possibly due to the known J chelating a c t i v i t y of these buffers which may s t a b i l i z e the pH 7,8 RNase-inhibitor complex or prevent the i n a c t i v a t i o n of RNase i n h i b i t o r by metal ions. Such an i n t e r p r e t a t i o n i s suggested by the observations that (1) EDTA does not appre-c i a b l y suppress RNase a c t i v i t y assayed at pH 7 . 8 i n Na-phosphate buffer, and (2) the l e v e l of RNase a c t i v i t y assayed at pH 7 . 8 i n the presence of 0.2 mM pCMB i s the same i n both Tris-HCl - 7 7 -and Na-phosphate "buffers (Table I I ) . A l t e r n a t i v e l y phosphate and imidazole ions may intera c t with 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 Tris-HCl buffer, to produce a l e s s active confor-mation of the enzyme molecule. I t may be worth noting for the purpose of comparison that bovine pancreatic RNase A, a 299 basic protein with p l = 7 . 8 , i s known 7 to have a strong a f f i n i t y f o r multivalent 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 Reagent present at Increase i n A260 per pH Buffer system* ffnailconceniftfration 6 0 minute incubation 7 . 8 Tris-HCl . 2 6 6 7 . 9 Na-phosphate .049 7 . 9 Na-phosphate EDTA (ltO mM) . 0 2 9 7 . 9 Na-phosphate EDTA ( 2 . 0 mM) .044 7 . 8 Tris-HCl pCMB ( 0 . 2 mM) . 6 8 7 7 . 9 Na-phosphate pCMB ( 0 . 2 mM) . 6 9 2 • F i n a l buffer concentration i n a l l cases was 5 0 mM. Several other buffer systems (Tes-H61, glycine-NaOH, and Na2C03-NaHC03) with e f f e c t i v e buffering capacities i n the alk a l i n e pH range were found to y i e l d very low RNase a c t i v i t i e s compared with Tri s - H C l . - 78 -3.04 Evidence indicating the presence of a protein inhibitor of pH 7»8 RNase activity in brain 3.041 Time-dependent activation of pH 7.8 RNase activity i n stored enzyme preparations A time-dependent activation of RNase activity assayed at pH 7»8 was observed upon storage of freshly prepared enzyme preparations at 0°. RNase activity assayed at pH 7«8 reached a maximum after about 6 days storage of 0.1% Triton X100 extracts at 0°, and subsequently remained relatively constant up to 14 days. Activity after 6 days storage at 0° was 86% greater than the activity assayed in freshly prepared ex-tracts. Figure 14 on page 138 shows the pH versus RNase activity profiles of a cytosol fraction assayed immediately upon preparation and after two weeks storage at 0°. Maximal activation of RNase activity i s observed at about pH 8,3 and the activity at this pH i s 476% above that of freshly pre-pared cytosol. A 10% increase in RNase activity assayed at pH6.7 also occurred i n the l4 day old cytosol. 3.042 Inhibition of bovine pancreatic RNase Acactivity by brain extracts Freshly prepared extracts inhibit bovine pancreatic RNase A activity and the degree of inhibition i s propor-tional to the amount of extract added. Parachloro-mercuribenzoate prevented the inhibition of bovine pancreatic RNase A activity by freshly prepared extracts. - 79 -3.043 Ac 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 The sulfhydryl blocking reagent, parachloromercuri-benzoate (pCMB), activates RNase a c t i v i t y i n fr e s h l y prepared extracts assayed at pH 7*8 and the degree of a c t i v a t i o n i s p o s i t i v e l y correlated with the extract's capacity to i n h i b i t bovine pancreatic RNase A a c t i v i t y . The time-dependent decline i n the capacity of extracts to 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 decline i n the capacity of pGMB to stimulate the endogenous pH 7.8 RNase a c t i v i t y . The e f f e c t s of 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 of a l a b i l e endogenous protein i n h i b i t o r which depends on the i n t e g r i t y of free 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 release of active pH 7«8 RNase from a RNase-inhibitor complex. The protein nature of the i n h i -b i t o r i s indicated by the facts that i t i s completely i n a c t i -vated by heating at 100°, i t does not pass through d i a l y s i s membranes, and i t i s inactivated 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 indicated by the time-dependent increase i n pH 7«8 RNase a c t i v i t y observed upon storage of extracts and the concomitant decrease i n pCMB stimulation of pH 7.8 RNase a c t i v i t y i n stored extracts. The 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 during storage of extracts could be prevented by 1.0 mM EDTA - 80 -apparently due to the s t a b i l i z a t i o n of RNase i n h i b i t o r by t h i s reagent. The i n h i b i t o r a c t i v i t y could also be s t a b i l i z e d by B-mercaptoethanol or d i t h i o t h r e i t o l . These sulfhydryl reducing agents were subsequently found to have no detectable ef f e c t on the 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, page 106). 3,04'+ Ef f e c t of EDTA on RNase a c t i v i t y assayed at pH 7 .8 The e f f e c t of removing traces of heavy metal ions was examined by adding the chelating agent, EDTA, to incubation mixtures of fr e s h l y prepared extracts. EDTA was found to produce near t o t a l i n h i b i t i o n of RNase a c t i v i t y assayed at pH 7 .8 i n 50 mM Tris-HCl buffer. To determine whether t h i s i n h i b i t i o n was due to augmentation of RNase i n h i b i t o r a c t i v i t y or to the binding of divalent cations more d i r e c t l y e s s e n t i a l f o r RNase function, pH 7 .8 RNase a c t i v i t y of extracts was measured i n the presence of both 1 .0 mM EDTA and 0.2 mM pCMB (Table I I I ) . The l e v e l of pH 7 .8 RNase a c t i v i t y obtained under these conditions was s l i g h t l y greater than that measured i n the presence of 0.2 mM pCMB alone. The fa c t that pCMB can completely restore the pH 7 .8 RNase a c t i v i t y i n h i b i t e d by EDTA, combined with the observation (Table VI) that neither pCMB nor EDTA altered the a c t i v i t y of p a r t i a l l y p u r i f i e d pH 7 .8 RNase preparations indicates that the e f f e c t of these reagents i s primarily upon the a c t i v i t y of RNase i n h i b i t o r . EDTA thus appears to act by l i b e r a t i n g RNase - 81 -i n h i b i t o r from inactive complexes with traces of metal ions, the free i n h i b i t o r thereby becoming a v a i l a b l e to combine with free pH 7 . 8 RNase. A c o r o l l a r y of t h i s explanation of the mechanism of EDTA action i s that i n h i b i t i o n of bovine pan-c r e a t i c RNase A by the i n h i b i t o r endogenous to extracts i s greater i n the presence of EDTA than i n i t s absence (Table IV). TABLE I I I . ACTIVATION OF EDTA-INHIBITED DEAE-CELLULOSE ELUATE pH 7 . 8 RNase ACTIVITY BY pCMB Enzyme preparation* Reagent present at Increase i n A 2 6 0 per added (mis) f i n a l concentration 30 minute incubation . 0 5 . 0 6 6 . 0 5 EDTA ( 0 . 5 mM) . 0 1 0 . 0 5 EDTA ( 5 . 0 mM) . 0 3 5 . 0 5 pCMB ( 0 . 2 mM) . 9 2 8 . 0 5 EDTA ( 0 . 5 mM)+ pCMB (0 .2 mM) .97** . 0 5 EDTA ( 5 . 0 mM)+ pCMB ( 0 . 2 mM) 1 . 1 1 0 •The pooled DEAE-cellulose eluate pH 7 * 8 RNase a c t i v i t y (fractions k 7 - 6 5 i n F i g . 8 ) was dialyzed against 2 0 mM Tris-HCl, pH 7 . 4 buffer and aliquots of the dialysate were assayed i n 5 0 mM Tris-HGl, pH 7 . 8 buffer. - 82 -TABLE IV. ENHANCEMENT GF RNase INHIBITOR ACTIVITY BY EDTA ~~ — Increase in A260 Addition to assay per 3 0 minute incubation 0 . 5 ng pancreatic RNase A . 7 0 3 0 . 5 ng pancreatic RNase A EDTA ( 1 . 0 mM) . 7 0 3 cytosol* . 0 4 3 cytosol* EDTA ( 1 . 0 mM) .009 0 . 5 ng pancreatic RNase A cytosol* . 4 9 3 0 . 5 ng pancreatic RNase A cytosol* EDTA ( 1 . 0 mM) . 3 5 8 •Aliquots (0.15 ml) of a 1/10 dilution (in ice-cold .02 M Tris-HCl, pH 7 . 8 buffer) of a cytosol fraction freshly prepared from adult rat whole brain were assayed. Assay conditions were as described in section 2.144 of Methods. 3.045 Comparison of the effects of pCMB on RNase activ i t i e s i n liver and brain A comparison of the level 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 activity from each organ. This date i s shown in Figures 4 and 5» A plot of the RNase activity as a function of pH showed that in the absence of pCMB the specific activity of acid RNase and alkaline RNase of liv e r was 3-fold and 5-fold greater respectively than that of brain. When total pH 7 . 8 RNase - 83 -c o u c o 200 h 180 L 160 140 120 ? 100 u ro 80 — CD ~ z CO OC 3 6 0 40 20 0 _L 7 8 P H 10 FIGURE k. The stimulation by pCMB of RNase a c t i v i t y i n 0.1% T r i t o n X100 extracts of adult rat whole brain, Aliquots of 0,5 ml of extract were incubated f o r 60 minutes i n 50 mM Tris-HCl buffer. Per cent stimula-t i o n was calculated as the difference i n A260 between samples assayed at a given pH with and without 0.1 mM pCMB divided by the central a c t i v i t y i n the absence of pCMB. - 8 3 a -FIGURE 5. The effect of pCMB on RNase activity in 0.1% Triton X100 homogenate of adult rat l iver . Aliquots of 0.05 ml of a 5% (wet weight/ l iver/f inal 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 stimula-tion and per cent inhibition by pCMB of control RNase activity. - 84 -a c t i v i t y was assayed i n the presence of 0.1 mM pCMB, the s p e c i f i c a c t i v i t y was about 3-fold greater f o r l i v e r than brain. The a c t i v i t y released by pCMB was, however, 95$ greater i n brain than l i v e r . In both organs, pCMB a c t i v a -t i o n was greatest around pH 7.5. The pH-dependent e f f e c t of pCMB may be due to changes i n the 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 to pCMB of the c r i t i c a l s u lfhydryl groups at di f f e r e n t pH. Thus, i n addition to stimulating enzyme a c t i v i t y , pCMB produces a change i n the shape of the pH-RNase a c t i v i t y curve. This may be related to the observation of Colter et a l . who mixed various d i l u t i o n s of RNase i n h i b i t o r with a standard amount of bovine pancreatic RNase A. The pH-activity curve of the uninhibited enzyme had a sharp optimum at pH 7.8. As the r a t i o of RNase i n h i b i t o r to pancreatic RNase A i n -creased, the p o s i t i o n of the optimum s h i f t e d to more a l k a l i n e pH values. Hence, the apparent a c t i v i t y optimum at about pH 8.5 observed i n isotonic sucrose homogenates (F i g . 1) and 0.1$ T r i t o n X100 extract©, (F i g . 2) of brain assayed without pCMB may be due to the pH-dependence of RNase-inhibitor complex formation—more uninhibited RNase a c t i v i t y being expressed at more a l k a l i n e pH due to d i s s o c i a t i o n of inhibitor-bound pH 7.8 RNase. Between pH 6 and pH 7, pCMB consistently stimulates RNase a c t i v i t y i n extracts by 20 to 30$. This stimulation - 8 5 -i s not due to a di r e c t e f f e c t on acid RNase, nor i s i t due to release of latent acid RNase from an inhibitor-bound form, since acid RNase a c t i v i t y assayed at a l l stages of p u r i f i -cation subsequent to i t s separation from a l k a l i n e RNase a c t i v i t y was i n h i b i t e d 3 0 % by the same concentrations of pCMB (see Figure 6 a , page 9 2 ). Rather, t h i s a c t i v a t i o n most l i k e l y represents the net outcome of d i r e c t i n h i b i t i o n of acid RNase a c t i v i t y by pCMB and the release 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 ret a i n s 6 8 % of i t s a c t i v i t y when assayed at pH 6 . 7 (see F i g . 9c, page 104). A rel a t e d 2 Oii-observation has been reported by Rahman who observed a 1 5 to 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 acid pH range upon reco n s t i t u t i n g the supernatant and sedimentable p a r t i -culate f r a c t i o n s 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 also l i k e l y to be a composite r e s u l t . The expected increase i n t r e s i d u a l pH 7 . 8 RNase a c t i v i t y normally expressed at t h i s pH upon release from i n h i b i t o r i s masked by the strong d i r e c t i n h i b i t o r y effect of the sulfhydryl blocking reagent upon some other alkaline 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 brain extracts assayed i n the pH range between 7«Q and 8 , 5 was obtained at pGMB; concentrations of 0 . 2 mM. Higher concentrations of - 8 6 -pCMB had no further e f f e c t , suggesting that pCMB has no di r e c t stimulatory or i n h i b i t o r y e f f e c t on the pH 7 » 8 RNase a c t i v i t y per se. This was subsequently confirmed by measuring the eff e c t of 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 variables influencing the determination of RNase a c t i v i t i e s i n crude extracts The l i t e r a t u r e shows a marked lack of agreement between workers 1 9 3 . 2 0 2 , 2 1 4 , 3 0 0 w h Q e s t i m a t e d the l e v e l s of acid RNase 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 . In t h i s organ, the pH 7 . 8 RNase to acid 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 homo-2 0 2 genates of 0 . 0 5 , and De Lamirande and A l l a r d obtained a r a t i o of 2 . 3 . In the present study a pH 7 « 8 RNase to acid RNase a c t i v i t y r a t i o of 0 . 3 was obtained f o r brain extracts, as compared to 0 . 7 when 0 . 1 mM pCMB was present i n the assay. I t i s apparent from the present study that a wide spectrum of r e s u l t s could be obtained ranging from no RNase i n h i b i t o r and high pH 7 « 8 RNase a c t i v i t i e s to high 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 the care taken to avoid i n h i b i t o r i n a c t i v a t i o n i n the course of homogenate prepara-t i o n and assay. Also, as previously mentioned, differences i n such parameters as buffer system, ion i c strength, and the extent of rupture of subeellular organelles do a f f e c t the - 8? -apparent l e v e l of RNase a c t i v i t i e s measured i n v i t r o . Yet another reason f o r the marked discrepancies i n the absolute 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 i n v e s t i -gators i s the lack of standardization of the RNA-precipitating 202 agent used to stop the reaction. Thus, i t i s often d i f f i -c u l t to compare the r e s u l t s reported by d i f f e r e n t i n v e s t i -gators since i n the absence of common bases of reference no r e l i a b l e basis for comparison e x i s t s . 3.1 Separation 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-enzyiae-In h i b i t o r System of Adult Rat Whole Brain 0.1% T r i t o n X100 Extracts The preceding experiments revealed the presence i n rat whole brain homogenates and extracts of at l e a s t two ribonu-cleases distinguishable by the pH at which they exhibit optimal a c t i v i t y . The a l k a l i n e RNase a c t i v i t y referred to as pH 7»8 RNase appears to be present l a r g e l y i n a l a t e n t form bound to a protein i n h i b i t o r . Although i t i s important to characterize enzyme a c t i -v i t i e s under conditions which most c l o s e l y approximate t h e i r i n s i t u state i n order to permit extrapolation of i n v i t r o experimental r e s u l t s to the i n vivo condition, the hetero-geneity of RNase a c t i v i t i e s present i n crude extracts of r a t brain complicates the i n t e r p r e t a t i o n of r e s u l t s obtained using such a complex mixture of molecular components. An attempt - 88 -was, therefore, made to separate the components of the RNase enzyme system from each other and from other proteins 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 sulphate followed by anion exchange column chromatography of the ammonium sulphate pr e c i p i t a b l e f r a c t i o n s . 3«11 Ammonium sulphate f r a c t i o n a t i o n of 0 . 1 % T r i t o n X100 extracts of adult rat whole brain T r i t o n X100 ( 0.1%) extracts of adult r a t whole brain were fractionated with powdered ammonium sulphate according to the procedure described i n section 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 Table V shows that t h i s procedure effected a clear separation of acid and a l k a l i n e RNase a c t i v i t i e s . A considerable loss of pH 6 . 7 RNase a c t i v i t y occurs during the ammonium sulphate f r a c t i o n a t i o n and subsequent removal of the s a l t by d i a l y s i s . Total per cent recovery of pH 6 . 7 RNase a c t i v i t y i n a l l the dialyzed ammonium f r a c t i o n s was consistently about 6 0 % . The protein 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 saturation contained the highest s p e c i f i c a c t i v i t y and accounted f o r nearly 7 0 % of the t o t a l recovered pH 6 . 7 RNase a c t i v i t y . TABLE V. RECOVER*OF RNase ACTIVITIES IN AMMONIUM SULPHATE PRECIPITABLE FRACTIONS* RNase activity assayed at pH 7.8 in the Calculated latent Enzyme RNase activity RNase activity RNase activity presence of RNase activity preparation assayed at pH 6 . 7 assayed at pH 9 .5 assayed at pH 7.8 0.2 mM pCMB at pH 7.8  : Total % Re- "Total % Re- Total % Re- Total % Re- Total fo Re-activity covery activity covery activity covery activity covery activity covery 0.1% Triton X100 extract 1,104.0 100 591.1 100 4 l 4 . 0 100 920.0 100 506.0 0-25% satu-rated (NHJ^)2S0i». fraction 13.2 1.2 15.4 2.6 11.7 2 .8 10.4 1.1 0.0 0 .0 25-55% satu-rated (NH/f)2S0^ fraction 157.5 12 .5 687.8 116.4 389.6 94.1 948.6 103.1 559.0 110 .5 55-75% satu-rated (NH4)2S0/| fraction 65 .4 5 .9 28.9 4 . 9 2 . 7 0.6 42.4 4 . 6 39 .7 7.8 75-100% saturated (NHif)2S04 fraction 424.2 38.4 69 .8 11.8 2.0 0.4 51 .5 5 .5 4 9 . 5 9.8 % of i n i t i a l activity re-covered in a l l (NHif)2S04 fractions , 5,8.0 1.35»7, 97.9, , 114.3 | 128.1 •An in 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 .05 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 0 0 CD - 9 0 -Total recovery of pH 9 . 5 RNase a c t i v i t y was generally higher but somewhat more variable, ranging from 7 5 to 1 3 5 $ • About 8 5 $ ' of the t o t a l recovered pH 9 . 5 RNase a c t i v i t y as well as the highest s p e c i f i c a c t i v i t y was found i n the ammonium sulphate 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 3 5 $ saturation. Most of the free pH 7 . 8 RNase a c t i v i t y (assayed without pCMB) as well as the latent, inhibitor-bound pH 7 « 8 RNase a c t i v i t y (the difference between 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 and the free pH 7 . 8 RNase a c t i v i t y assayed without pCMB) was also r e -covered i n the 2 5 to 5 5 $ saturated ammonium sulphate f r a c t i o n . Total recoveries of both free 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 capacity of d i f f e r e n t amounts of each ammonium sulphate f r a c t i o n to i n h i b i t the a c t i v i t y of a standard amount of bovine pancreatic RNase A was not measured, no quantitative statement can be made as to the amount of free RNase i n h i b i t o r a c t i v i t y recovered i n each ammonium sulphate f r a c t i o n . However, free RNase i n h i b i t o r a c t i v i t y appears to be d i f f u s e l y d i s t r i b u t e d throughout fr a c t i o n s 2 5 - 5 5 , 5 5 - 7 5 , and 7 5 - 1 0 0 $ saturation since each of these was capable of i n h i b i t i n g bovine pancreatic RNase A a c t i v i t y and t h i s i n h i b i t i o n could be prevented by 0 . 2 mM - 91 -pCMB. This observation i s consistent with Roth's report that free RNase i n h i b i t o r of r a t l i v e r i s p r e c i p i t a t e d mostly between 35-55$ ammonium sulphate saturation with 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 at greater than 60% saturation. The f a c t that the t o t a l recovery of free pH 7.8 RNase a c t i v i t y was never greater than 100% suggests that the endogenous pH 7.8 RNase-inhibitor complex was not dissociated during ammonium sulphate f r a c t i o n a t i o n . This indicates the firmness of the binding between i n h i b i t o r and RNase. Shortman has r e p o r t e d 2 1 3 that s a l t concentrations up to 0.3 M did 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 pancreatic RNase A. In summary, ammonium sulphate f r a c t i o n a t i o n does not effect any resolution of the following molecular species 1 free pH 7.8 RNase, free RNase i n h i b i t o r , pH 7.8 RNase-i n h i b i t o r complex, and pH 9«5 RNase. However, i t succeeds i n removing considerable amounts of inactive protein and i n separating the above components from the acid RNase a c t i v i t y (see Pigs. 6a and 6b). FIGURE 6 a .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 7 5 and 100 per cent saturation with ammonium sulphate. An i n i t i a l volume of 160 ml of a 0.1$ T r i t o n X100 extract of adult r a t whole brain was fractionated according to the procedure described i n Materials and Methods. The protein f r a c t i o n p r e c i p i t a t i n g between 75-100S saturation of ammonium sulphate was dissolved i n and dialyzed against 20mM Tris-HCl, pH 7 . k buffer and 20 u l aliquots of the dialysate were assayed i n 5 0 mM buffer i n the absence (NH4-acetate • { Tris-HCl — • — ) and i n the oresence (NH4-acetate • s T r i s -HCl --O- ) of 0.1 mM pCMB. - 93 -1.3 1.2 c 0 o 1.1 _o *) V c 1.0 a .9 C i .8 o CO \ .7 o •o CM .6 < .5 .4 Figure 6 b ' L I I I _ l L 7 8 p H 10 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 per cent saturation with ammonium sulphate. The procedure f o r the preparation and assay of t h i s f r a c t i o n i s the same as that described i n the legend to Figure 6a. with the exception that 50 >uls of the dialysate was used f o r assay and incubation was f o r 30 minutes. RNase a c t i v i t y was assayed i n 50 mM Tris-HCl buffer i n the absence — « — , and i n the presence --o---of 0.1 mM pCMB. - 94 -3 . 1 2 DEAE-cellulose column chromatography of the 2 5 * 5 5 $ and the 75 - 1 0 0 $ saturated ammonium sulphate f r a c t i o n s An attempt was made to achieve further separation and p u r i f i c a t i o n of the constituent molecular species of the 7 5 to 1 0 0 $ and the 2 5 to 5 5 $ ammonium sulphate f r a c -tions by chromatography of each of these f r a c t i o n s on c columns of DEAE-cellulose according to the procedure de-scribed i n section 2 . 1 3 2 of Methods. Figure 7 shows a t y p i c a l e l u t i o n p r o f i l e of protein and pH 6 . 7 RNase a c t i v i t y upon chromatography on DEAE-ce l l u l o s e of a 7 5 - 1 0 0 $ saturated ammonium sulphate f r a c -t i o n . Only one RNase a c t i v i t y peak, eluting between 0 . 1 and 0 . 2 M NaCl, was detected i n the eluate f r a c t i o n s . When the 2 5 to 5 5 $ saturated ammonium sulphate f r a c t i o n was chromatographed on a column of DEAE-cellulose two peaks of RNase a c t i v i t y were detected i n the eluate f r a c t i o n s ( F i g . 8). The f i r s t enzyme peak coincides with the unadsorbed protein washed through the column with e q u i l i b r a t i n g buffer. The second enzyme peak was eluted at a NaCl concentration between 0,15 and 0 , 2 5 M„ When the eluate f r a c t i o n s were assayed f o r t h e i r capacity to i n h i b i t bovine pancreatic RNase A a c t i v i t y , a broad peak of i n h i b i t o r a c t i v i t y was detected e l u t i n g between 0 , 1 5 and 0 . 5 M NaCl and overlapping the free pH 7 , 8 RNase a c t i v i t y O 20 40 60 80 IOO 120 F r a c t i o n N u m b e r FIGURE 7. E l u t i o n p r o f i l e of 75-100% saturated ammonium sulphate p r e c i p i t a b l e f r a c t i o n chromatographed on DEAE-cellulose. - 95 -- 9 6 -FIGURE 7 . E l u t i o n p r o f i l e of 7 5 - 1 0 0 $ saturated ammonium sulphate 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 protein 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 saturation with ammonium sulphate was dialyzed against . 0 2 M Tris-HCl, pH 6 . 9 buffer, and 4 . 0 mis of the dialysate containing 5 6 . 8 mg protein and 1 5 2 units of RNase a c t i v i t y assayed at pH 6 . 7 was loaded on a 1 cm by 1 5 cm column of DEAE-cellulose pre-equilibrated with 5 0 0 ml of . 0 2 M Tris-HCl, pH 6 . 9 buffer. The sample was eluted with a l i n e a r l y increasing s a l t gradient ( 2 5 0 ml Tri s - H C l , pH 6 . 9 to 2 5 0 ml 1 . 0 M NaCl i n . 0 2 M Tris-HCl, pH 6 . 9 ) . Fractions of 4 . 4 ml were c o l l e c t e d . Aliquots ( 0 . 2 ml) of the undialyz eluate f r a c t i o n s were assayed f o r RNase a c t i v i t y at pH 6 . 7 i n 5 0 mM Tris-HCl buffer. Incubation was f o r 6 0 minutes. o b 1 M Q G » O W 00 -a w •1 M a C o o rt H* H-•tj O t~ 3 <+ f» V cr 1 01 H* o 0 n w> Ml M -n P o o Q «+ •"•> o a H- ^ o O rt Q 3 zr 3 <D o Z *1 Ox C O 1 3 o 3 o p) vj\ cr rt n o cn CD n p V> rt _ M O CD P a. c+ n> o o. 3 P — D 3 51 o W3 1 t— O C O. S I-1 CO e c o o >d CQ ET co (a • rt • S Absorbance at 280 nm. p • Ln T A • .v. o U NaCl concentration (M) • • I L °/o inhibition of pancreatic RNase A activity —*-—J 1 l o b ro 'o o RNase activity ( units / ml. ) -16 -- 98 -FIGURE 8. E l u t i o n p r o f i l e of the 2 5 - 5 5 % saturated ammonium sulphate 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 protein 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 per cent saturation with ammonium sulphate was dialyzed against . 0 2 M T r i s - H C l . pH 6 . 9 buffer and 1 0 ml of the dialysate containing 3 9 3 mg of protein was loaded on a 1 cm by 19 ,5 cm column of DEAE-cellulose pre-equilibrated with . 0 2 M Tri s - H C l , pH 6 . 9 buffer. The sample was eluted with a l i n e a r l y increasing s a l t gradient (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 Tri s - H C l , pH 6 .9 ) . Fractions of 4,8 ml were c o l l e c t e d , Aliquots (0.2 ml) of the undialyzed eluate f r a c t i o n s were assayed immediately f o r RNase a c t i v i t y at pH 9*5 —° and f o r free RNase i n h i b i t o r a c t i v i t y . — • — , and on the following day f o r free and t o t a l (with 0 .2 mM pCMB)— - RNase a c t i v i t y at pH 7.8. Incubations 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 described f o r the standard assay condition i n Materials and Methods. - 9 9 -assayed i n the absence of pCMB as well as 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. The wash-through RNase a c t i v i t y was found to be very 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 the cold room ( 4 ° ) . This a c t i v i t y was i n h i b i t e d by pCMB when assayed at pH 7 . 8 or pH 9*5* At a f i n a l conentration of 0 . 2 mM, pCMB produced k 0$ and 7 0 $ i n h i b i t i o n at pH 7 . 8 and pH 9 . 5 respectively. The fac t that t h i s RNase a c t i v i t y i s not adsorbed on DEAE-cellulose columns equilibrated at pH 7 . 4 indicates that i t has no net negative charge at t h i s pH and i s hence a basic protein. The free pH 7 « 8 RNase a c t i v i t y recovered i n the eluate f r a c t i o n s was up to 300% greater than the amount loaded on the column. This i s inf e r r e d to be due to a p r e f e r e n t i a l i n a c t i v a t i o n of the i n h i b i t o r component of pH 7 . 8 RNase-i n h i b i t o r complexes, as well as to the d i s s o c i a t i o n of such complexes and the p a r t i a l separation of free pH 7 « 8 RNase a c t i v i t y from free i n h i b i t o r a c t i v i t y during the chroma-tography. The recovery of 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) i n the eluate f r a c t i o n s ranged between 7 5 and 1 0 0 $ of the amount loaded. The free RNase i n h i b i t o r a c t i v i t y i n the eluate f r a c t i o n s was very unstable. Upon storage i n the cold room, - 100 i t exhibited a rapid loss i n i t s capacity to i n h i b i t pancreatic RNase A a c t i v i t y . The per cent i n h i b i t i o n of bovine pancreatic RNase A a c t i v i t y was highest at pH 7*5» thus i n d i c a t i n g that the free RNase i n h i b i t o r a c t i v i t y has a pH optimum of 7»5» 3.2 Properties of the Three Separated RNase A c t i v i t i e s Three apparently distinguishable RNase a c t i v i t i e s were thus separated from 0.1$ T r i t o n X100 extracts by means of ammonium sulphate f r a c t i o n a t i o n followed by DEAE-cellulose column chromatography. In order to conclusively 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 to further characterize t h e i r properties, the e f f e c t of various reagents on the a c t i v i t y of the DEAE-cellulose eluate enzyme fracti o n s was studied. The c h a r a c t e r i s t i c s of the pH 7.8 RNase reported i n the following sections (Pig. 9c, Tables VI and VII) were determined using DEAE-cellulose eluate f r a c t i o n s which had been stored for some time and hence exhibited 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 indicated by the f a i l u r e of pCMB to stimulate 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 of these enzyme f r a c t i o n s to i n h i b i t bovine pancreatic RNase A a c t i v i t y . - 10.1 -3.21 E f f e c t of pH DEAE-cellulose eluate f r a c t i o n s with peak RNase a c t i v i t i e s were assayed at various hydrogen ion concen-trat i o n s i n 0,05 M buffer. The pH optimum of the DEAE-cellulose eluate acid RNase a c t i v i t y was found to have shifted to a more acid pH, with maximal a c t i v i t y now occurring at pH 6.4 with Tris-HCl and at pH 6.0 with NH*-. acetate buffers ( F i g . 9a). This a c t i v i t y was free of contamination by pH 7.8 RNase as indicated by the fac t that 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 at pH 7.8. The DEAE-cellulose washwthrough RNase a c t i v i t y exhibits maximal a c t i v i t y above pH 9 and retains 56% of a c t i v i t y when assayed at pH 7.8. The shoulder i n the pH curve between pH 8.0 and 8.5 suggests that t h i s enzyme f r a c t i o n may consist of a mixture of more than one d i s t i n c t enzyme species. Further p u r i f i c a t i o n i s required to test t h i s p o s s i b i l i t y . The pH curve of the DEAE-cellulose eluate pH 7.8 RNase a c t i v i t y i s shown i n Figure 9c. This enzyme exhibits a broad but symmetrical pH p r o f i l e with a pH optimum of about 7.7. fft^retains 68% of i t s a c t i v i t y when assayed at pH 6 .7 . - 102 -FIGURE 9a.The e f f e c t of pH on DEAE-cellulose eluate acid RNase a c t i v i t y . 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 Figure 7 were pooled, dialyzed against .02 M Tr i s - H C l , pH 7.4 buffer, and the dialysate was brought to 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 preparation were assayed at various pH i n .05 M NH4-acetate — , or i n .05 M Tris-HCl — — buffe r s . Incubation was for 60 minutes. - 103 -FIGURE 9b.Effect of pH on DEAE-cellulose wash-through RNase a c t i v i t y . Aliquots (0 .2 ml) from f r a c t i o n number 4 i n Figure 8 were assayed at various pH i n . 0 5 M Tris-HCl buffer. Incubation was fo r 60 minutes. - 104 -FIGURE 9 c .Effect of pH on DEAE-cellulose eluate 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 eluted between 0 . 1 and 0 . 2 M NaCl (eluate f r a c t i o n s 4 3 - 6 5 i n Figure 8) were pooled and stored undia-lyzed f o r one month at 4 ° C p r i o r to assay. Aliquots of 0 , 2 ml were assayed at various pH i n . 0 5 M Tris-HCl buffer. Incubation was fo r 6 0 minutes. - 105 3 . 2 2 Effect of NaCl The effect of NaCl and buffer concentration on the DEAE-cellulose eluate acid RNase activity was not de-termined. DEAE-cellulose eluate pH 9 . 5 RNase activity was markedly inhibited by NaCl as well as by increasing buffer concentration. The inhibitory effect of NaCl was much greater than could be accounted for in terms of an equiva-lent increase in ionic strength. DEAE-cellulose eluate pH 7.8 RNase activity was stimulated by NaCl with maximal activity occurring at the NaCl concentration of 1 3 5 mM. NaCl concentrations greater than 180 mM were inhibitory. The stimulatory effect of NaCl on pH 7.8 RNase activity can probably be completely accounted for in terms of ionic strength since the increment in enzyme activity i s nearly the same for both a 5 0 mM increase in NaCl and a 5 0 mM increase i n buffer concentration. 3 . 2 3 Effect of MgCl2 Divalent cations were found to strongly inhibit the activity of a l l three RNases. pH 6.7 and pH 9 . ^ RNase acti v i t i e s were completely inhibited and pH 7.8 RNase half inhibited at a MgCl2 concentration of 5 mM. However, within a very narrow concentration range around 1 mM, Mg++ 10& -TABLE VI. EFFECT OF VARIOUS REAGENTS UPON DEAE' CELLULOSE ELUATE RNase ACTIVITIES F i n a l Con- j° of Control RNase A c t i v i t y centration Assayed i n 50 m Tris-HCl Reagent Added (mM) buffer  pH 6.7 pH 9.5 pH 7.8 RNase RNase RNase Tris-HCl 25 124 64 5 0 100 100 100 100 - 66 1 3 5 5 68 10 45 2 5 24 118 40 1 2 9 NaCl 100 149 1 3 5 186 180 164 2 2 5 1 2 3 2 7 0 75 3 1 5 46 0.5 16 1.0 2 3 2.5 7 9 5.0 1 4 55 MgCl 2 10.0 2 40 15.0 29 2 5 . 0 1 5 0.5 1 3 1.0 1 2 3 1 0 5 EDTA 2.0 88 2.5 1 3 10.0 112 pCMB . 0 5 84 .1 72 .2 61 3 6 9 8 . 3 104 1.0 120 D i t h i o t h r e i t o l 1 . 2 5 2.5 187 9 5 5.0 120 190 10.0 181 90 15.0 87 I - 10? -»ay found to stimulate pH 9«5 RNase a c t i v i t y (see section The influence of MgGl2 on the reaction i s l i k e l y to be complex. Mg++ may act by d i r e c t l y a l t e r i n g the conforma-t i o n of the enzyme molecules or by forming a cation bridge between enzyme and other proteins, or between proteins and RNA. I t i s k n o w n 3 0 1 " 3 0 3 that divalent eations can con-siderably a l t e r the structure of RNA molecules and thus a f f e c t the a c c e s s i b i l i t y of substrate bonds to the action of RNases.30** Thus, the mechanism of Mg++ i n h i b i t i o n of RNase a c t i v i t y may be related to the fa c t that Mg + + s t a b i l i z e s the secondary structure of RNA and may thereby maintain a substrate configuration unfavorable to enzymatic attack. 3.24 E f f e c t of EDTA One mM EDTA activated acid RNase a c t i v i t y assayed at pH 6 . 7 by 2 3 % , whereas at a concentration of 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 suppression of pH 9.5 RNase a c t i v i t y . This e f f e c t could be reversed by restoring MgCl2 to the reaction mixture at a suitable concentration. MgCl2 was then found to stimulate t h i s enzyme a c t i v i t y by up to 40% (see P i g . 10). This enzyme thus appears to have a divalent cation requirement - 108 -160 0.5 1.0 1.5 2.0 MgCl2 (mM) FIGURE 10. Reactivation of EDTA-inhibited pH 9 . 5 RNase a c t i v i t y by Mg++. 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 Figure 8 ) was assayed i n 5 0 mM Tri s - H C l , pH 9 . 5 buffer. Various concentrations of MgC12 were added to incubation mixtures con-t a i n i n g 1 . 0 mM EDTA and the enzyme f r a c t i o n . Reagents were added to the incubation mixture i n the following orderi buffer, EDTA, enzyme N f r a c t i o n , MgC12, sRNA. Incubation was f o r 6 0 minutes. Control pH 9 . 5 RNase a c t i v i t y was that assayed i n the absence of both EDTA and MgC12. - 109 which can be met by Mg++. Hence, the previously observed inhibition by Mg++ of pH 9*5 RNase activ i t y assayed without EDTA was due to the fact that this requirement was already met by traces of divalent cations present in the reaction mixture. The addition of MgCl2 produced super-optimal concentrations of divalent cation which resulted i n strong inhibition. EDTA has no significant effect on free pH 7»8 RNase activity. This supports the conclusion that the inhibitory effect of EDTA on RNase activity i n crude enzyme extracts assayed at pH 7.8 i s due to the stabilizing influence of this chelating agent on RNase inhibitor activity. 3.25 Effect of pCMB Parachloromercuribenzoate inhibited acid RNase activity about 40% and pH 9»5 RNase activity about 64% at 0.2 mM concentration. Free pH 7.8 RNase activity was unaffected by up to 0.3 mM pCMB. 3.26 Effect of 3-mercaptoethanol and dithiothreitol Dithiothreitol stimulated acid RNase activity 20% at 1 mM. Higher concentrations of either of these sulfhydryl reducing agents had no additional effect. The opposite effects of pCMB and DTT on acid RNase activity are consistent - 110 -with the conclusion that t h i s enzyme contains free s u l f -hydryl group(s) which are required f o r optimal enzymatic a c t i v i t y , A s i m i l a r conclusion can be made with regard to 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 cts of equal concentrations of these reagents on the l a t e r enzyme i n d i -cates that the c r i t i c a l s u l f h y d r y l groups involved are either more reactive and/or more es s e n t i a l to the a c t i v i t y of t h i s enzyme i n comparison with acid 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 . This i s consistent with i t s i n s e n s i t i v i t y to pCMB and suggests that t h i s enzyme has no free sulfhydryl group requirements or that i t s sulfhydryl groups are un-reactive and inaccessible to these reagents at pH 7«8. The lack of influence of DTT, pCMB and EDTA on p a r t i a l l y p u r i f i e d pH 7.8 RNase indicates that the previously observed e f f e c t of these reagents on RNase a c t i v i t y assayed i n crude enzyme preparations at pH 7-8 was mediated through the action of these reagents on RNase i n h i b i t o r . 3.27 E f f e c t of detergents A l l three RNase a c t i v i t i e s were almost completely i n h i b i t e d by the in c l u s i o n of .005$ (w/v) sodium l a u r y l sulphate i n the assay mixtures (Table VII). - I l l -TABLE VII. EFFECT OF VARIOUS DETERGENTS UPON DEAE-CELLULOSE ELUATE RNase ACTIVITIES F i n a l $ of Control RNase A c t i v i t y Detergent Concentration assayed i n 50 mM Tris-HCl Added {%} buffer  pH 6.7 PH 9.5 pH 7.8 RNase RNase RNase Sodium l a u r y l . 0 0 5 15 12 0 sulphatel 18 . 0 2 5 4 0 Sodium . 0 5 133 24 1 0 7 desoxycholate 4 3 .10 11 1 1 3 T r i t o n XIOO2 . 0 2 5 171 1 0 6 108 .10 153 95 108 F i n a l concentration given i n per cent (weight/volume). ^ F i n a l concentration given i n per cent (volume/volume). Sodium deoxycholate at a f i n a l concentration of . 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 stimulated pH 6.7 RNase a c t i v i t y by 33$. Increasing the sodium deoxy-cholate concentration to 0 . 1 $ resulted i n 57$ i n h i b i t i o n of pH 6.7 RNase a c t i v i t y . At these concentrations sodium deoxy-cholate had 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 . Both pH 7.8 RNase and pH 9.5 RNase a c t i v i t y was unaffected 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 stimulated 71$ and 53$ by T r i t o n X100 at con-centrations of . 0 2 5 $ (v/v) and 0 . 1 % (v/v) respectively. - 1 1 2 -3.28 E f f e c t of storage Although crude enzyme preparations routinely stored at 0° f o r up to two weeks exhibited no noticeable loss i n acid RNase a c t i v i t y , at a l l subsequent stages of 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 a c t i v i t y . The l a b i l i t y of pH 6.7 RNase a c t i v i t y from brain i s consistent with observations of t h i s enzyme a c t i v i t y i n 190 191 202 other organs by several investigators 7 *J-^J-* Who found i t 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 than pH 7.8 RNase. In the present study, DEAE-cellulose eluate f r a c t i o n s containing peak pH 6.7 RNase a c t i v i t y l o s t kQ$ of t h e i r i n i -t i a l a c t i v i t y a f t e r storage at 0° f o r 1 1 days i n 20 mM T r i s -HCl, pH 7.k buffer containing 1 0 $ (v/v) g l y c e r o l . However, storage at 0° f o r 1 3 days i n either 20 mM NHk-acetate, pH 5 buffer or i n 20 mM Tris-HCl, pH 6.7 buffer containing 3 0 $ (v/v) g l y c e r o l resulted i n only 8$ loss i n a c t i v i t y . S t a b i l i z a t i o n of t h i s enzyme a c t i v i t y could thus be achieved by storage at acid pH or i n the presence of 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 also found to undergo rapid i n a c t i v a t i o n upon storage. This i n a c t i v a t i o n appears to be due to the oxidation of free s u l f -hydryl groups since ,3-mereaptoethanol or dithiothreitol were able to reactivate t h i s enzyme and prevent i t s loss of a c t i v i t y . DEAE-cellulose wash-through pH 9 « 5 RNase a c t i v i t y - 113 -stored at 4° for 14 days in 20 mM Tris-HCl at pH 6.4, 7.0, 8.0 and 9.5 retained 94$, 93$, 68$ and 35$ respectively of i t s i n i t i a l activity. Stored under the same conditions at pH 9.5 in the presence of 5mM B-merceptoeibhanol 66$ of the original activity was retained. The pH 7.8 RNase activity was found to be compara-tively stable and no attempt was made to determine optimal stabilization conditions for this enzyme. 3.3 DNase and Phosphodiesterase Activities of DEAE-cellulose eluate enzyme fractions The possibility that the identified RNA-depolymerizing enzyme act i v i t i e s might be due to nucleases or phosphodie-sterases capable of cleaving phosphodiester bonds in ribonu-cleic acid molecules was tested by assaying for these a c t i -vities using double-stranded calf thymus DNA or bis-p-nitro-phenyl phosphate as substrates. Table VIII shows that the DEAE-cellulose eluate enzyme fractions do not significantly degrade double-stranded DNA but exhibit preferential specificity for the polynucleotide substrate containing ribose and uracil moieties. Table IX shows that the partially purified enzyme preparations exhibit no significant amounts of either J'PQL-or 5' POk-forming phosphodiesterase activity as compared to crude enzyme preparations of whole brain. - 114 -TABLE VIII. DNase ACTIVITY OF DEAE-CELLULOSE ELUATE ENZYME FRACTIONS Increase i n A260 per 60 minute incubation Enzyme Preparation .Buffer f i n a l &•& J . j . i Substrate* 75-100% 50 mM sRNA .815 saturated Tris-HCl, (NH4)2S0^ pH 6.7 ' Wk .036 p r e c i p i t a b l e f r a c t i o n DEAE-cellulose 50 mM. sRNA .234 eluate f r a c t i o n Tris-HCl, number 32 pH 6.7 DNA .025 ( F i g . 7) DEAE-cellulose 50 mM sRNA .860 pooled eluate Tris-HCl, .010 f r a c t i o n s pH 7.8 DNA 55-70 (Fig.8) DEAE-cellulose 50 mM. sRNA .301 pooled eluate Tris-HCl, DNA .011 f r a c t i o n s pH 9.5 5-16 ( F i g . 8) *0.5 ml of a 0.1% (w/v) stock s o l u t i o n of either sRNA dissolved i n d i s t i l l e d H2O or double-stranded c a l f thymus DNA dissolved i n 10 mM NaCl was added to the incubation mixture as substrate. Other d e t a i l s of assay procedure are described i n section 2.142 of Methods. - 1 1 5 -TABLE IX. PHOSPHODIESTERASE ACTIVITY OF DEAE-CELLULOSE ELUATE ENZYME FRACTIONS* Increase i n AkOO Enzyme F i n a l buffer per 6 0 minute Preparation concentration incubation 0 . 5 ml whole 3 3 mM Tris-HCl, pH 8 . 9 1 . 3 6 3 g e n a t e h 0 m ° " 3 3 m N H 4 - a c e t a t e , pH 5 . 0 1 . 5 8 9 0 . 5 ml whole 3 3 mM-Tris-HCl, pH 8 . 9 0 . 2 7 0 brain extract . 0 5 ml 7 5 - 1 0 0 $ 3 3 mMTris-HCl, p H . 6 . 7 . 0 k 7 saturated (NH k) 2S0k f r a c t i o n 0 . 2 ml DEAE- 3 3 mM Tris-HCl, pH 6 . 7 . 0 0 0 c e l l u l o s e eluate f r a c t i o n # 3 2 ( F i g . 7 ) 0 . 2 ml DEAE- 3 3 mM Tris-HCl, pH 7 . 8 . 0 1 7 c e l l u l o s e eluate pooled f r a c t i o n s 5 5 - 7 0 ( F i g .8 ) 0 . 2 ml DEAE- 3 3 mM.Tris-HCl, pH 9 . 5 . 0 0 0 c e l l u l o s e eluate pooled f r a c t i o n s 5 - 1 6 ( F i g . 8 ) •Conditions of assay are as described i n section 2 . 1 k 3 of Methods. - 1 1 6 -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 of RNase A c t i v i t i e s and RNase In h i b i t o r A c t i v i t y Isotonic sucrose homogenates of adult r a t whole brain were prepared and fractionated by d i f f e r e n t i a l centrifugation into nuclear, crude mitochondrial, microsomal, and cytosol f r a c t i o n s according to the procedure described i n section 2 . 1 2 of Methods. Each subcellular f r a c t i o n was assayed f o r RNase a c t i v i t y i n 5 0 mM Tris-HCl buffer at pH 6 . 7 , pH 9 . 5 , and at pH 7 . 8 with and without 0 . 2 mM pCMB. The r e s u l t s recorded i n Table X, , XI, and XII show that f o r each RNase the sum of the separate a c t i v i t i e s of each subcellular f r a c t i o n i s s i g n i f i c a n t l y greater than the t o t a l a c t i v i t y expressed i n the i n i t i a l i s o t o n i c sucrose homogenate. T o t a l recovery of each RNase a c t i v i t y i n a l l the subc e l l u l a r f r a c t i o n s assayed at pH 6 . 7 , pH 9 . 5 and pH 7 . 8 was 1 9 2 $ , 1 6 7 $ and 2 0 5 $ respectively of the a c t i v i t y expressed i n the isoto 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 to the rehomogenisiation of the separated subcellular 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 pf&considerable RNase a c t i v i t y which was i n i t i a l l y present i n the is o t o n i c sucrose whole homogenate i n a latent, non-functional form either bound to or compartmentalized within organelles. The t o t a l recovery of each RNase a c t i v i t y i n a l l the subcellular f r a c t i o n s assayed at pH 6 . 7 , pH 9 . 5 , and pH 7 . 8 was, however, only 5 6 $ , - 1 1 7 -6 3 % , and 7 6 % respectively of the a c t i v i t y expressed i n 0.1% T r i t o n X100 homogenates. This indicates that homogeniza-t i o n of the separated subcellular p a r t i c u l a t e f r a c t i o n s i n isot o n i c sucrose does not l i b e r a t e a l l of the t o t a l detergent-extractable RNase a c t i v i t y . Table X shows that the pH 6 . 7 RNase a c t i v i t y which i s expressed under these conditions i s predominantly l o c a l i z e d i n the crude mitochondrial and cytosol f r a c t i o n s . Of the t o t a l recovered pH 6 . 7 RNase a c t i v i t y , 41% was present i n the crude mitochondrial f r a c t i o n and 3 1 % i n the cytosol f r a c t i o n . a c t i v i t y The s p e c i f i c ~ o f t h i s enzyme i n the cytosol f r a c t i o n was three-f o l d greater than that of any of the other subcellular f r a c -t i o n s . S t u d i e s 2 9 5 , 3 0 5 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 of acid RNase i n rat l i v e r by d i f f e r e n t i a l and sucrose density gradient centrifugation have concluded that the acid RNase a c t i v i t y associated with the crude mitochondrial f r a c t i o n i s l o c a l i z e d i n lysosomes. In view of the s i m i l a r i t i e s of the pH 6 . 7 RNase a c t i v i t y reported i n the present study to that of Acid RNase of rat l i v e r , i t seems l i k e l y that the large amount of pH 6 . 7 Rttase a c t i v i t y of rat brain which i s recovered i n the crude mitochondrial f r a c t i o n may also be l o c a l i z e d within the lysosomes of t h i s f r a c t i o n . TABLE X. INTRACELLULAR DISTRIBUTION OF pH 6 .7 RNase ACTIVITY pH 6 .7 RNase A c t i v i t y Enzyme Preparation S p e c i f i o T o t a l a o t i v i t y A c t i v i t y (units/mg (units.) . protein) $> of sucrose homogenate a c t i -v i t y recovered % of the sum o f T o t a l a c t i v i t y a c t i v i t i e s r e - (units) recovered covered i n a l l the per gram wet weight s u b c e l l u l a r f r a c - of whole b r a i n t i o n s • 0,1# T r i t o n X100 homogenate 420.0 Isotonic sucrose homogenate 120.9 800 x g p e l l e t (nuclear f r a c t i o n ) 40.0 8,000 x g p e l l e t (crude mitochondrial f r a c t i o n ) 94.8 105,000 x g p e l l e t (microsomal f r a c t i o n ) 23 .8 105,000 x g supernatant ( c y t o s o l f r a c t i o n ) 73.2 0.55 0.16 >0.23 0.29 0.21 0.62 '347 100 33 78 20 61 181 52 17 41 10 32 70.0 20.2 6 .6 15.8 4 . 0 12.2 In the experiments recorded i n the above and following Tables, 60 mis of a 10% (wet wt. b r a i n / f i n a l volume) i s o t o n i c sucrose homogenate of adult (4-month-old) r a t whole brains wa3 f r a c -tionated according to the procedure described i n s e c t i o n 2.12 of Methods. P e l l e t e d sub-c e l l u l a r f r a c t i o n s were resuspended by homogenization i n O.32 M sucrose, brought to f i n a l volume with 0*32 M sucrose, and a l i q u o t s of each f r a c t i o n were assayed i n 50 mM Tr i s - H C l b u f f e r at the appropriate pH. c - 119 -Although the studies on r a t l i v e r found that l i t t l e a cid RNase was present i n the cytosol f r a c t i o n , De Lamirande and A l l a r d found that acid RNase of i n t e s t i n a l mucosa and kidney, i n contrast with 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 to what extent such apparent t i s s u e - s p e c i f i c differences 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 of t h i s RNase a c t i v i t y may represent differences i n the f r a g i l i t y of lysosomes or other subcellular p a r t i c l e s and, hence, i n the release of enzyme into the so l u b l e , f r a c t i o n during the i s o l a t i o n of subcellular p a r t i c l e s . I t i s also possible that a large portion of t h i s enzyme, at le a s t i n brain, may be a normal constituent of 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 t h e i r i s o l a t i o n . Such adsorption phenomena are a well known 2 9 6 source of a r t i f a c t s i n subcellular d i s t r i b u t i o n studies. However, i n rat l i v e r , Rahman * found no evidence of the adsorption of soluble RNases to subcellular p a r t i c l e s . Table XI shows that pH 9»5 RNase a c t i v i t y i s also found predominantly i n the crude mitochondrial and cytosol f r a c t i o n s . However, proportionally more of t h i s enzyme a c t i v i t y i s recovered i n the crude mitochondrial f r a c t i o n (57$) than i n the cytosol (20$) . 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 that of the nuclear 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 acti-vity recovered io of the sum?of activities re-covered in 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 Isotonic sucrose homogenate 93*0 0.12 800 x g pellet (nuclear fraction) 21.8 0.13 8,000 x g pellet 93.6 0.29 (crude mito-chondrial fraction) 105,000 x g pellet (microsomal 10 .1 0.09 fraction) 105,000 x g super-natant (cytosol fraction) 29.4 0.28 263 100 23 101 11 32 158 60 14 60 19 40.7 15.5 3.6 15.6 1.7 4.9 to O - 1 2 1 -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 the recovered pH 9 « 5 RNase a c t i v i t y thus resembles that of pH 6 . 5 RNase except that a larger portion of t h i s a c t i v i t y i s obtained i n the crude mitochondrial f r a c t i o n and les s i n the microsomal and cytosol f r a c t i o n s . This d i s t r i b u t i o n d i f f e r s from that reported by Rahman f o r pH 9*5 RNase of, rat l i v e r . In l i v e r , r e l a t i v e l y more of the t o t a l c e l l u l a r content of pH 9 . 5 RNase a c t i v i t y was recovered 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 cytosol f r a c t i o n . Table XII shows that the largest amount 6 3 % of re- •. covered free pH 7 . 8 RNase a c t i v i t y and the highest s p e c i f i c a c t i v i t y of t h i s enzyme was found i n the crude mitochondrial f r a c t i o n . However, assuming that 5 6 $ of the optimal a c t i v i t y of. pH 9 » 5 RNase i s retained i n assays at pH 7 . 8 , 8 . 7 units (per gram wet weight) of the t o t a l RNase a c t i v i t y expressed i n the crude mitochondrial f r a c t i o n assayed at pH 7 . 8 ( 1 3 . 2 units/g wet weight) can be accounted f o r i n terms of pH 9 * 5 RNase a c t i v i t y expressed at pH 7 * 8 . This leaves a remainder of 4 . 5 units((per gram wet weight) which must represent the actual a c t i v i t y contributed by free pH 7 « 8 RNase. It i s u n l i k e l y that i n the in t a c t c e l l the soluble c e l l sap contains any free pH 7 * 8 RNase a c t i v i t y i n view of the large excess of free RNase i n h i b i t o r found i n the cytosol. TABLE XII. INTRACELLULAR DISTRIBUTION OF FREE pH 7.8 RNase ACTIVITY Free pH 7.8 RNase Activity Enzyme Preparation Total activity [units, Specific activity (units/mg jrotein f<> of sucrose homogenate acti-vity recovered # of the sum of activities re-covered in a l l the subcellular fractions Total activity (units) recovered per gram wet weight of whole brain 0.1# Triton X100 homogenate 163.2 0.21 Isotonic sucrose homogenate 60.9 0.08 800 x g pellet (nuclear fraction) 24.4 0.15 8,000 x g pellet 78.9 0.24 (crude mito-chondrial fraction) 105,000 x g pellet 10.6 0.10 (microsomal fraction) 105,000 x g supernatant (cytosol fraction) 11.0 0.09 268 100 40 130 17 18 131 49 20 63 27.2 10.2 4.0 13.2 1.8 1.8 ro ro - 1 2 3 -The 9 $ of free RNase a c t i v i t y assayed at 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 of pH 9 » 5 RNase a c t i v i t y expressed at pH 7 . 8 . Table XIII shows that 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 was mostly recovered i n the cytosol f r a c t i o n , and the s p e c i f i c a c t i v i t y of t h i s f r a c t i o n was 5 - to 1 0 - f o l d greater than that of the other subcellular f r a c t i o n s . From the difference between free and t o t a l pH 7 * 8 RNase a c t i v i t y i t can be calculated that an apparent 8 8 $ of the latent, inhibitor-bound pH 7 » 8 RNase i s present i n the cytosol (Table XIV). The pH 7 * 8 RNase a c t i v i t y i n the crude mitochondrial f r a c t i o n assayed i n the presence of 0 . 2 mM pCMB was 3 8 $ lower than that assayed without pCMB, in d i c a t i n g that pCMB has a net i n h i b i t o r y 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 f r a c t i o n (see F i g . 1 2 a ) . From the f a i l u r e of pCNB to stimu-l a t e RNase a c t i v i t y assayed at pH 7 * 8 i n the crude mito-chondrial f r a c t i o n , i t would appear that t h i s f r a c t i o n contains no inhibitor-bound pH 7 * 8 RNase despite the fact that t h i s f r a c t i o n contains 6 3 $ of the t o t a l recovered free pH 7 « 8 RNase a c t i v i t y and 2 7 $ of the t o t a l recovered free RNase i n h i b i t o r a c t i v i t y (Table XV). - 124 -TABLE XIII. INTRACELLULAR DISTRIBUTION OF TOTAL pH 7.8 RNase ACTIVITY Total pH 7.8 RNase Activity Enzyme Preparation Total activity (units) Specific activity (units/mg protein) % of sucrose homogenate acti-vity recovered fo of the sum of Total activity activities recovered (units) recovered in a l l the sub- per gram wet weight cellular fractions of whole brain O.ljfr..Triton X100 homogenate 244.2 Isotonic sucrose homogenate 192.6 800 x g pellet 38*0 (nuclear fraction) 8,000 x g pellet 49.0 (crude mito-chondrial fraction) 105,000 x g pellet « i , 5 (microsomal fraction) 105,000 x g super- 128.2 natant (cytosol fraction) 0.32 0.25 0.22 0.15 0.11 1.08 127 100 20 25 67 107 S85 17 22 56 40.7 32.1 6.3 8.2 2.1 21.4 - 125 -TABLE XIV. INTRACELLULAR DISTRIBUTION OF LATENT pH 7.8 RNase ACTIVITY Latent ,.P,H ,7.8 RNase Activity* m i$> of'the sum of Enzyme P f o n a r a t 1 on Total a c t i v i t y (units) Specific a c t i v i t y (units/mg protein) 0.1J6 Triton X100 homogenate 81.0 0.11 Isotonic sucrose homogenate 131.7 0.17 800 x g pellet 13*6 (nuclear fraction) 0.08 8,000 x g pellet -29.9a i> of sucrose homogenate activity recovered ac t i v i t i e s recovered in a l l the sub-cellular fractions Total a c t i v i t y (units) recovered per gram wet weight of whole brain 62 100 10 61 99 10 0 chondrial fraction) 105,000 x g pellet (microsomal fraction) 1.9 105,000 x g super- 117.2 natant (cytosol fraction) 0.02 0.98 89 88 13.5 21.9 2.3 0 0.3 19.5 •Latent or inhibitor-bound pH 7.8 RNase ac t i v i t y i s defined as the acti v i t y released upon treatment with pCMB, and was calculated by subtracting the free pH 7.8 RNase act i v i t y (Table XII) from the to t a l pH 7.8 RNase activity assayed i n the presence of 0.2 mM pCMB (Table XIII). aThis negative value r e f l e c t s the net inhibitory effect of pCMB on RNase activity assayed at pH 7*8 i n the crude mitochondrial fraction. This value was taken as zero i n calculating t o t a l recovery. ro - 126 -TABLE XV. INTRACELLULAR DISTRIBUTION OF FREE RNase INHIBITOR ACTIVITY Free RNase Inhibitor Activity* % of sura of Total activity % of total % of sucrose activities (units recovered Specific protein homogenate recovered in per gram wet Total Activity recovered in- activity a l l sub- weight of whole Enzyme Activity (units/mg fraction recovered cellular brain Preparation (units) protein) fractions Isotonic sucrose 6,660 8.54 100 100 100 1,110.0 homogenate 800 x g pellet 217.8 1.26 3 22 3 36.3 (nuclear fraction) 1 8,000 x g pellet 1,800.0 5.56 27 42 27 300.0 (crude mitochondrial 0 fraction) 1 105,000 x g pellet 315.0 2.84 5 14 5 52.5 (microsomal fraction) 105,000 x g supernatant4,317.5 36.34 65 X-5 65 719.6 (cytosol fraction) •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 Is u n l i k e l y that these r e s u l t s can be explained i n terms of d i f f e r e n t i a l compartmentalization of the free pH 7»8 RNase a c t i v i t y and the free RNase i n h i b i t o r a c t i v i t y since the rehoraogenization to which t h i s f r a c t i o n was subjected and the hypotonic condition of assay would be expected to l a r g e l y remove the constraints to i n t e r a c t i o n between segregated molecular species. The data suggests that the crude mitochondrial f r a c t i o n contains an a l k a l i n e RNase d i f f e r i n g from that §f the cytosol 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 to i n h i -b i t i o n fey the free RNase i n h i b i t o r endogenous to the crude mitochondrial f r a c t i o n . In r a t l i v e r , both R o t h 1 9 5 and Shortraan 2 1 3 found the mitochondrial pH 7.8 RNase a c t i v i t y to be sensitive to i n h i b i -t i o n by the free RNase i n h i b i t o r present i n the c y t o s o l . S@tfe R o t h 1 8 7 i n f e r r e d that the predominant f r a c t i o n of mitochondrial pH 7,8 RNase a c t i v i t y must hence be present i n the free form. He also r e p o r t e d 1 9 0 evidence f o r the presence of some i n h i b i t o r -bound pH 7.8 RNase i n the crude mitochondrial f r a c t i o n of r a t l i v e r . The simplest explanation f o r the apparent absence of detectable inhibitor-bound pH 7«8 RNase i n the crude mito-chondrial f r a c t i o n of brain l i e s i n the fac t that i n h i b i t o r -bound pH 7.8 RNase i s normally i n d i r e c t l y i n f e r r e d from the - 128 -activating effect of pCMBf but due to the high pH 9.5 RNase content of the crude mitochondrial fraction, pCMB activation of inhibitor-bound pH 7*8 RNase i s more than counterbalanced by the inhibitory effect of pCMB on the residual pH 9.5 RNase activity expressed in assays at pH 7 .8 . The results thus represent a composite effect reflec-ting the i n a b i l i t y of the assay conditions to clearly dis-criminate between the RNase act i v i t i e s present in the crude mitochondrial fraction. The RNase activity measured at a given pH i s not due solely to a single enzyme but i s the result of the combined actions of the multiple enzyme species present in this subcellular fraction. Table XV shows that free RNase inhibitor activity assayed as described in section 2.144 of Methods was found predominantly i n the cytosol (66% of total recovered activity) and crude mitochondrial fraction (27% of total recovered a c t i v i t y ) . The small amount of inhibitor activity recovered i n the nuclear fraction probably represents the degree to which this fraction i s contaminated with adhering cytoplasmic material. The intracellular distribution of free RNase inhibitor activity i n brain closely resembles that reported for rat l i v e r . Roth 1® 7 found RNase inhibitor activity to be high i n the cytosol and low i n the microsomal and nuclear fractions 129 -of r a t l i v e r . Roth £" L 5' has more recently reported the absence of any detectable free RNase i n h i b i t o r i n p u r i f i e d nuclei from r at l i v e r . I t i s of special i n t e r e s t that the sum of the free RNase i n h i b i t o r a c t i v i t i e s of the subcellular f r a c t i o n s was founfl to be equal to the t o t a l free RNase a c t i v i t y i n the isot o n i c sucrose whole homogenate. This component of the RNase enzyme system thus exhibits no subcellular structure-linked latency and t h i s may be c r i t i c a l to the functioning of t h i s protein i n t i t r a t i n g the l e v e l of a c t i v i t y of pH 7.8 RNase i n the i n t a c t c e l l . 3.5 Characterization of RNase a c t i v i t i e s i n separated subcellular f r a c t i o n s In view of the i n a b i l i t y of the assay conditions 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 contribution of each of the multiple enzyme species to the RNase a c t i v i t y measured at a given pH, a more r e l i a b l e determination of the a c t i v i t y con-tributed by each component RNase seemed to require t h e i r more thorough characterization and separation s t a r t i n g from a p a r t i c u l a r subcellular f r a c t i o n . Also, i n view of the rather unremarkable s i m i l a r i t i e s of the three RNases detected i n brain to those previously reported i n rat l i v e r , a more detailed study of the RNase a c t i v i t i e s of 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 to detect novel 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 detectable - 1 3 0 -i n previous studies of whole homogenates and extracts. The RNase a c t i v i t y of each subcellular f r a c t i o n , i s o -homogenates , , lated from i s o t o n i c sucrose^of adult rat brain, was assayed over a range of hydrogen ion concentrations with and without 1 mM EDTA, 0 . 2 mM pCMB or 2 . 0 M urea. The control pH curves of a l l four subcellular f r a c t i o n s i n the absence of any added reagents exhibited higher a c t i v i t y i n the acid pH region than i n the al k a l i n e region. The control pH optimum f o r acid RNase a c t i v i t y was pH 6 . 7 i n Tris-HCl buffer and pH 6 . 3 i n NH14, acetate buffer as for the whole c e l l homogenate. However, each subcellular f r a c t i o n d i f f e r e d from the whole homogenate i n the alk a l i n e region of the control a c t i v i t y versus pH p r o f i l e . The control pH curve of the nuclear f r a c t i o n has a bimodal appearance suggestive of a c t i v i t y maximae at pH 8 and 8 . 6 . The crude mitochondrial f r a c t i o n exhibits a broad plateau of 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 at pH 8 „ 9 . The pH curve of fr e s h l y prepared cytosol f r a c t i o n exhibits a deep trough between pH 7 and 8 . 5 with a c t i v i t y minima occurring at pH 7 « 5 » Above pH 8 . 5 the cytosol f r a c t i o n exhibits a broad a c t i v i t y plateau with no d i s t i n c t maxima (see F i g . 14 on page /iff )• - 131 The e f f e c t of 0.2 mM pCMB on the RNase a c t i v i t i e s of the nuclear and mitochondrial f r a c t i o n s i s shown i n Figures 11a and 12a respectively. This sulfhydryl blocking agent stimulated a c t i v i t y i n the nuclear f r a c t i o n between pH 7 and 8.5 with a maximal stimulation of 53$ occurring at the pH minima of control a c t i v i t y . A s l i g h t i n h i b i t i o n of a c t i v i t y was observed above pH 8.6 and below pH 6.7* The mitochondrial f r a c t i o n RNase a c t i v i t y , however, showed a consistent i n h i b i t i o n of about kQ% by 0.2 mM pCMB through-out the entire pH range tested. The magnitude .of the measured a c t i v i t y i s the net ef 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 of the various enzyme species present i n t h i s f r a c t i o n , and i s interpreted to indicate that 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 considerably higher than that f o r nuclear and cytosol 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 of t h i s putative explanation w i l l require the separation of these enzyme components from each other s t a r t i n g from the crude mitochondrial f r a c t i o n ^ . The e f f e c t of 1 mM EDTA on RNase a c t i v i t y i n nuclear and mitochondrial f r a c t i o n s i s shown i n Figures llb'o and 12b r e s p e c t i v e l y . This concentration of EDTA completely abolishes mitochondrial a l k a l i n e RNase a c t i v i t y whereas about 50$ of the control a l k a l i n e RNase a c t i v i t y i s retained i n the nuclear f r a c t i o n . - 1 3 2 -.5 ^ 4 E o .3 Fiqure 11 a / 7 \ FIGURE 11. The RNase a c t i v i t y i n the nuclear f r a c t i o n i s o l a t e d from adult rat whole brain (a) i n the presence of 2 . 0 M urea and 0.2 mM pCMB, and (b) i n the presence of 1.0 mM EDTA. Aliquots of 0.2 ml of the nuclear f r a c t i o n were assayed i n 50 mM buffer i n the absence of any added reagents ( N H4-acetate—*—; Tris-HCl — • — ), and i n the presence of either 2 . 0 M urea (NH4-acetate - v - - ; T r i s - H C l " - o - - ) , or 0.2 mM pCMB (Tris-HCl ), or 1.0 mM EDTA (NH4-acetate --v-- i Tris-HCl - - o - - ) . Incubation was for 60 minutes. - 133 -FIGURE 12. The RNase a c t i v i t y i n the crude mitochondrial f r a c t i o n i s o l a t e d from adult rat whole braini (a) ef f e c t of 0.2 mM pCMB, and (b) e f f e c t of 1.0 mM EDTA and 0.5 mM MgC12. Aliquots of 0.1 ml of the crude mitochondrial f r a c t i o n were assayed i n 50 mM buffer i n the absence of any added reagents (NH4-acetate—*— j Tris-HCl — • — ) and i n the presence of eit h e r 0.2 mM pCMB (Tris-HCl ••+••), or 1.0 mM EDTA (NH4-acetate — v — ; Tris-HCl - - 0 - - ) f or 0.5 mM MgC12 ( T r i s - H C l — • ). Incubation was for 60 minutes. - 13 k -acetate In ammoniumAbuffer, both mitochondrial and nuclear acid RNase activity i s slightly stimulated by 1 mM EDTA, with maximal stimulation occurring above pH 6.0. In Tris -HCl buffer, however, the acid RNase activity assayed below pH 6.7 i s slightly inhibited by 1 mM EDTA in both mitochon-d r i a l and nuclear fractions. In Tris-HCl buffer above pH 6.7, acid RNase activity i s stimulated in the nuclear fraction and unaffected in the mitochondrial fraction. The effect of 2.0 M urea on RNase activity i n the nuclear and microsomal fractions i s shown i n Figures 11a and 13 respectively. Acid RNase activity i s markedly stimulated 140$ and 100$ in nuclear and microsomal fractions respectively and the pH optimum of this enzyme activity i s shifted to pH 5.8. In the alkaline region of the pH curve, 2.0 M urea stimulates RNase activity maximally (193$) at pH 8 in the nuclear fraction. Urea-activation of RNase activity assayed at this pH cannot be completely accounted for i n terms of the release of inhibitor-bound pH 7.8 RNase activity since the level of activity evoked in the presence of urea i s 2-fold greater than that obtained in the presence of an optimal con-centration (0.2 mM) of pCMB. Hence, the activation by urea and pCMB of RNase activity assayed at pH 7.8 must be effected at least i n part by different mechanisms. - 135 -.5 c >> CD -•—' O .4 > i_ ao CO .3 CO £ CD \ .2 CO C/J co ••—< .1 z E CC 3 Figure 1 3 j I i L 7 p H 10 FIGURE 1 3 . E f f e c t of 2 . 0 M urea on the RNase a c t i v i t y of the microsomal f r a c t i o n . Aliquots of 0 . 2 ml of the microsomal f r a c t i o n were assayed i n the absence of any added reagents (NH4-acetate — • — ; Tris-HCl — • — ) and i n the presence of 2 . 0 M urea (NH4-acetate - - v - - j Tris-HCl — o — ) . Incubation was f o r 60 minutes. - 136 -Urea stimulates a l k a l i n e RNase a c t i v i t y i n the micro-somal f r a c t i o n to a l e s s e r extent (100%) than i n the nuclear f r a c t i o n . In addition to the a c t i v i t y maxima at pH 8, a second a c t i v i t y maxima at pH 8.6 i s observed i n t h i s f r a c -t i o n . This a c t i v i t y may correspond to the microsome-bound ooo oo'c o ft A RNase reported by other workers J " * t D* and known to be activated by urea. Thus, both acid and a l k a l i n e RNase a c t i v i t e s are markedly stimulated i n the presence of 2.0 M urea. The stimulating e f f e c t of urea on pH 9»5 RNase i s lea s t marked. The optimal concentration of urea was not determined. The increased y i e l d of acid-soluble oligoribfonuc-leotides i n the presence of ureail&spprb&ably nut dueato a d i r e c t stimulation of the RNases since the d i r e c t a c t i o n of urea on these enzymes would be expected to unfold t h e i r structure and i n h i b i t t h e i r a c t i v i t y . Rather, the stimu-l a t i n g e f f e c t of urea may be due to ( l ) ; d i s s o c i a t i o n of non-specific protein-RNase and protein-RNA complexes and aggregates thereby f a c i l i t a t i n g enzyme-substrate i n t e r a c t i o n ! (2) disruption of the H-bonded secondary structure of the RNA thereby making the substrate more vulnerable to enzyme action, and/or (3) since RNases may produce single-stranded endonueteolytic breaks i n RNA, leaving oligoribonucleotide sequences H-bonded to the i n t a c t strand, urea may f a c i l i t a t e - 137 -the release of those products of the enzyme action which remain H-bonded. Eluc i d a t i o n of the precise contribution of each of these mechanisms to theobserved composite e f f e c t of urea requires further experiments. A si m i l a r enhancement of pancreatic RNase A a c t i v i t y by urea has been reported by 2 9 7 Kalnitsky et a l . ' These workers found that one-third of the increase could be accounted f o r by the s o l u b i l i z i n g e f f e c t of urea on the products of the reaction; i . e . , f a c i -l i t a t i o n of the release of H-bonded base pairs into a c i d -soluble form. This 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 subcellular f r a c t i o n s suggests the presence of an additional 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 previously de-tected i n the whole c e l l homogenates and extracts. This micro-somal al 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 was presumably masked by the pre-dominance of pH 7.8 and pH 9«5 RNase a c t i v i t i e s i n assays of whole c e l l homogenates and extracts. I t may be possible to take advantage of 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 various RNases and reduce the complexity of the system under observation by attempting to separate and p u r i f y the various enzyme a c t i v i t i e s present within a p a r t i c u l a r subcellular f r a c t i o n , rather than s t a r t i n g from the whole c e l l homogenate. - 1 3 8 -D) E J 1 1 i I i ! i I I 1_ 5 6 7 8 9 10 P H FIGURE 14. RNase a c t i v i t y as a function of pH i n f r e s h l y prepared and aged cytosol f r a c t i o n . Aliquots of 0.2 ml of cytosol were assayed immediately upon preparation (NH4-acetate —f—~ j Tris-HCl — • — ) and a f t e r two weeks storage at 0° ( T r i s - H C l - - * - — ). Incubation was for 60 minutes. - 1 3 9 -3 . & Developmental Changes i n RNase A c t i v i t i e s and i n RNase Inh i b i t o r A c t i v i t y i n Rat Whole Brain Figures 1 5 , 1 6 and 1 7 show the s p e c i f i c a c t i v i t y l e v e l s of RNase assayed at pH 6 . 7 . 9 . 5 and 7 . 8 i n 0 . 1 $ T r i t o n X100 whole brain homogenates of rats at various stages of postnatal maturation. The s p e c i f i c a c t i v i t y of RNase assayed at pH 6 . 7 i s highest at b i r t h , begins to decline gradually at about day 7 , then more ra 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. The s p e c i f i c a c t i v i t y at day 3 2 i s 6 0 $ of that at day 1 . However, on a per unit wet weight of brain basis the a c t i v i t y progressively i n -creases from b i r t h , peaks at day 2 2 ( 1 2 0 $ of day 1 level) and f a l l s r a p i d l y thereafter ( 1 . 5 - f o l d decrease) to reach adult l e v e l s by day 32 ( 8 7 $ of day 1 l e v e l ) . Both the s p e c i f i c a c t i v i t y and the a c t i v i t y per gram wet weight brain of RNase assayed at pH 9 . 5 are minimal at b i r t h , r i s e r a p i d l y from day 7 to peak at day 2 2 , and subsequently exhibit a gradual but d i s t i n c t decline throughout adulthood. S p e c i f i c a c t i v i t i e s at day 2 2 and 8 months are 224$ and 1 5 2 $ respectively of that at day 1 . The developmental p r o f i l e of free pH 7 . 8 RNase a c t i v i t y i s s i m i l a r to that of RNase a c t i v i t y assayed at pH 9 * 5 except that the lag i n the increase up to age 7 days i s not observed - 1 3 9 a --I 1 1 1 1 1 1 ft I I L 5 10 15 20 25 30 days 3 months 4 5 P o s t - n a t a l Age FIGURE 15, Changes i n acid RNase a c t i v i t y during postnatal development, RNase a c t i v i t y i n 0.1% T r i t o n X100 homo-genates of adult rat whole brain was assayed at pH 6 , 7 i n 50 mM Tris-HCl buffer. Aliquots of 0,05 ml of homogenate were used for assay. Incubation was f o r 30 minutes. -139b -i i ? i \ CO -*—• "c 3 • 4 - » o CO CD CO CO z rr Figure 16 60 H 50Y 40 30 2o[*- ' o-10h i I1 i c o o a cf) E / A "0-- A . .5 c> c 3 .3 .t: > -*—• .2 ° - N 5 10 15 20 25 30 days Post natal (0 CU CO CO Z DC 3 months 4 age FIGURE 1 6 . Postnatal developmental changes i n pH 9 * 5 RNase a c t i v i t y . Incubation was f o r 6 0 minutes. A l l other conditions were as i n legend to Figure 15» except the assay was conducted at pH 9 . 5 . - T39c-z5 I I I I I 1 I 1 ff. I L 0 5 10 15 20 25 30 days 3 months 4 Post-natal Age FIGURE 17. Postnatal 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 the absence of pCMB expressed i n units/mg p r o t e i n - - ^ — a n d i n units/gram wet weight brain — o — , Total pH 7«8 RNase a c t i v i t y assayed i n the presence of 0.2 mM pCMB expressed i n units/mg protein — ¥ — and i n units/grate wet weight brain • — » — . Incubation was f o r oO minutes. A l l other conditions were as i n legend to Figure 15* - 1 4 0 -and the rate of increase i s more gradual. The magnitude of the net increase, however, i s nearly the same. The specific activity observed at age 22 days and 5 months is 221% and 150% respectively of that at day 1. Free pH 7 . 8 RNase specific activity also undergoes a similar gradual decline throughout adulthood with the specific activity at 5 months being 68% of the peak specific activity at age 22 days. Total pH 7 * 8 RNase activity (assayed in the presence of 0.2 mM pCMB) exhibits a rapid 2 . 3-fold increase in specific activity between day 10 and day 22 after which i t remains relatively constant throughout adulthood. Parachloro-mercuribenzoate i s inhibitory at a l l ages prior to day 20. 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 to.talAis interpreted as an index of the inhibitor-bound pH 7 . 8 RNase/pH 9.5 RNase activity ratio. Thus, this ratio i s relatively low at birth and decreases to a minimum at 10 days due to a proportionally greater increase in pH 9.5 RNase specific activity during this period. It then increases and attains day 1 values by age 1 8 days due to a propor-tionally greater increase during this time in free RNase inhibitor and hence i n inhibitor-bound pH 7 . 8 RNase. The fact that after age 20 days the net effect of pCMB is a stimu-lation of RNase activity assayed at pH 7 . 8 indicates that this ratio has become sufficiently large so that the inhibitory - 141 -eff e c t of pCMB on pH 9 * 5 RNase a c t i v i t y expressed i n assays at pH 7 . 8 i s masked by the overriding stimulatory e f f e c t of pCMB due to i t s i n a c t i v a t i o n of i n h i b i t o r and release of inhibitor-bound pH 7 . 8 RNase a c t i v i t y . The difference between the s p e c i f i c a c t i v i t y curves of RNase assayed at pH 7 . 8 i n the absence or presence of pCMB does not represent the absolute amount of inhibitor-bound, latent pH 7 . 8 RNase a c t i v i t y . Whereas from day 2 2 onward, the t o t a l pH 7 . 8 RNase s p e c i f i c a c t i v i t y remains constant, the amount of i n h i b i t o r -bound pH 7 . 8 RNase a c t i v i t y per mg protein apparently i n -creases progressively from 0% at day 2 0 to 6 $ at day 2 2 , to 3 0 $ at 3 months, of the t o t a l pH 7 . 8 RNase a c t i v i t y . This progressive increase i n pCMB 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 would appear to indicate an increase i n the concentration of RNase inhibitor-pH 7 . 8 RNase complexes and such an int e r p r e t a t i o n i s consistent with the observed con-current decline i n free pH 7 . 8 RNase s p e c i f i c a c t i v i t y . However, no sim i l a r concurrent decline i n s p e c i f i c a c t i v i t y of free RNase inh i b i t o r i s observed. Hence, the amount of free RNase i n h i b i t o r must increase at such a rate as to replenish that u t i l i z e d i n the formation of complexes with free pH 7 * 8 RNase i n order to maintain a constant amount of free RNase i n h i b i t o r throughout adulthood. Howeyer, the favored i n t e r p r e t a t i o n of the progressive increase i n pGMB a ac t i v a t i o n of RNase a c t i v i t y assayed at pH 7 . 8 i s that i t i s 142 -due to a further increase in the inhibitor-bound pH 7.8 RNase/pH 9 » 5 RNase ratio which i s due not to an increase in the amount of inhibitor-bound pH 7.8 RNase but, rather, an age-dependent decline in pH 9 « 5 RNase activity and hence a concurrent decline in the contribution of the inhibitory effect of pCMB to the total RNase activity assayed at pH 7.8. It follows from this interpretation that the amount of inhi-bitor-bound pH 7«8 RNase remains constant throughout adult-hood, whereas the amount of free pH 7«8 RNase activity and pH 9 . 5 RNase activity concurrently decline after 2 2 days. In summary, on a per gram wet weight basis a l l three RNase act i v i t i e s attain their maximal values at age 2 2 days and subsequently decline. This decline i s gradual and con-tinuous throughout adulthood in the case of pH 7.8 and pH 9 . 5 RNase a c t i v i t i e s . Acid RNase activity, however, exhibits a rapid decline in activity between age 2 2 and 32 days and, subsequently, remains comparatively constant throughout adulthood. The specific activity of free pH 7«8 RNase inhibitor i n the whole brain of newborn rats i s about 5 0 $ that of adults. There i s a rapid increase in free RNase inhibitor activity between age 10 and 18 days and the level of activity attained by 18 days remains constant throughout adulthood. Comparison of the developmental profiles of free RNase - 143 -~ 100 h o 90 h CO < 80 h CD CO * 70 CC o 60 h co ^ 50 o 2 40 Q. ° 30 c o '£ 20 JO £ 10 Figure is JL 10 15 20 25 30 days 3 months 4 P o s t - n a t a l Age FIGURE 18. Postnatal developmental changes i n free RNase i n h i b i t o r a c t i v i t y . Homogenates of 0 . 1 % T r i t o n X 1 0 0 were di l u t e d with i c e - c o l d . 0 2 M Tri s - H C l , pH 7«8 buffer to a protein concentra-t i o n of 2 . 0 mg/mlt and 0 . 2 ml aliquots containing 0 r 2 mg protein were assayed for i n h i b i t i o n of the a c t i v i t y of 0 . 5 ng. pancreatic RNase A i n the presence of 1 . 0 mM EDTA. Incubation was f o r 60 minutes. - 144 -i n h i b i t o r a c t i v i t y and free pH 7»8 RNase a c t i v i t y indicates a rapid increase i n the free RNase i n h i b i t o r a c t i v i t y / f r e e 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 since free RNase i n h i b i t o r a c t i v i t y has attained i t s adult l e v e l by 18 days whereas free pH 7»8 RNase a c t i v i t y continues to increase up to 22 days. A f t e r age 22 days t h i s r a t i o again increases and continues to increase throughout adulthood due to the gradual decline i n free pH 7.8 RNase a c t i v i t y . 285 Suzuki and Takahashi have studied the developmental change i n free RNase i n h i b i t o r a c t i v i t y i n the cytosol f r a c -t i o n of rat cerebral cortex. These investigators found that t h i s component of the RNase enzyme system exhibits a sharp peak i n s p e c i f i c a c t i v i t y between the 5th and 10th day a f t e r b i r t h , f a l l s to near neonatal l e v e l s by day 13 and remains r e l a t i v e l y constant thereafter. However, i n the present study using whole c e l l homogenates of whole brain,free 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 exhibited low l e v e l s up to age 10 days a f t e r which i t increased r a p i d l y and plateaued at adult l e v e l s by 18 days. Two of the most obvious explanations f o r t h i s apparent discrepancy are that (1) there occurs an age-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 of free RNase i n h i b i t o r such that the developmental p r o f i l e of t h i s a c t i v i t y i n the - 145 cytosol i s not c h a r a c t e r i s t i c of the developmental p r o f i l e of free 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) there are re g i o n - s p e c i f i c differences i n the developmental pattern of free 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 using whole rat brain may not be representative of the developmental changes i n free 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. I f t h i s second explanation i s the correct one, then there 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 difference between the cerebral cortex and the res t of the brain i n the developmental p r o f i l e of free 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 of developmental changes i n t h i s component of the RNase enzyme system i n d i f f e r e n t regions of the brain such as the cerebral cortex, cerebellum and brainstem may y i e l d further i n s i g h t into the ro l e of RNase i n h i b i t o r i n RNA metabolism and into the functional importance of i t s presence i n such high concen-t r a t i o n i n brain. Takabashi and Suzuki^-* have reported s i g n i f i c a n t differences i n the s p e c i f i c a c t i v i t y of free RNase i n h i b i t o r i n d i f f e r e n t regions of the adult rabbit brain. I t must be emphasized that a developmental increase i n enzymatic a c t i v i t y measured i n v i t r o does not prove increased enzyme function i n vivo where enzyme a c t i v i t y may be regulated by substrate a c c e s s i b i l i t y and other control f a c t o r s . Develop-mental increases i n enzyme a c t i v i t y do . not necessarily 146 -indicate a s p e c i f i c increase i n enzyme amount since increased a c t i v i t y could also r e s u l t from a non-specific increase i n the rate of protein synthesis, a c t i v a t i o n of precursors ( a c t i v a t i o n of bound or latent formsoof the enzyme) or to a decrease i n the rate of degradation of the enzyme. Whether the observed changes i n enzyme a c t i v i t y during development s i g n i f y an actual increase i n enzyme protein due to an enhanced rate of de novo enzyme synthesis, or to developmental changes i n factors regulating the a c t i v i t y of a constant amount of enzyme, remains to be shown. Also, i t must be stressed that l e v e l s of 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 of the t o t a l RNase capacity of the t i s s u e . Functional l e v e l s of 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 of a c t i v i t y ex-pressed i n isotonic sucrose homogenates, «*»*»** IV. DISCUSSION The presence of multiple enzymes responsible f o r the degradation of RNA i n brain i s consistent with the hetero-geneity of t h i s enzyme system i n other mammalian tissues and i n procaryotes where i n the case of wild-type E. c o l i at least seven d i s t i n c t RNases have been reported. The true complexity of t h i s enzyme system i n mammalian tissues has yet to be elucidated. I t i s l i k e l y that the RNases characterized i n the present study, as well as those characterized by other investigators i n other mammalian tissues, are the most r e a d i l y detectable components of an enzyme system consisting of many other additional enzymes of various s p e c i f i c i t i e s capable of cleaning phosphodiester bonds i n RNA. A l l of the enzymes i d e n t i f i e d i n the present invest i g a t i o n probably function i n the exo- and/or endonu-c l e o l y t i c cleavage of RNA. Highly s p e c i f i c endoribonucleases which are thought to p a r t i c i p a t e i n the maturational proces-sing 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 RNA molecules would not contribute to the findings reported here since such a c t i v i t i e s would not be expected to y i e l d s u f f i c i e n t acid-soluble products to be detectable by the assay employed i n the present study. 147 - 148 -4.0 Regulation of the I M J Vivo Function of RNases i n brain The f a c t that a large f r a c t i o n of the detergent-extractable RNase a c t i v i t y i s not expressed i n assays of iso t o n i c sucrose homogenates indicates that considerable amounts of the maximal l e v e l s of a l l three RNases are present i n the c e l l s of the brain i n a laten t , non-functional state. In s i t u , latent RNase may be present bound to and/or compartmentalized within subcellular organelles thus rendering the enzymes inaccessible to and inactive against substrate* This 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 studies which have shown that while the crude mitochondrial f r a c t i o n contains only 1 6 $ of the t o t a l c e l l u l a r RNA,-^11 5 2 $ of 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 f r a c t i o n . Moreover, the microsomal f r a c t i o n which contains 3 7 $ of the t o t a l c e l l u l a r RNA^11 contains comparatively l i t t l e of the t o t a l expressed c e l l u l a r RNase a c t i v i t y . These observations imply that the functional l e v e l s of a c t i v i t y of large amounts of the c e l l u l a r RNA-depolymerizing enzymes i s controlled 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 to the segregation of enzyme and substrate molecules into d i f f e r e n t i n t r a c e l l u l a r membrane compartments. I t also follows that modulations of the steady state rate of RNA catabolism i n the i n t a c t c e l l may be affected by factors a l t e r i n g organelle membrane permeability or s h i f t i n g the r a t i o of bound to free enzyme. 149 -Several workers y - ? t J -> have reported that acid RNase i n r a t l i v e r i s associated with lysosomes and exhibits the c h a r a c t e r i s t i c structure-linked latency of other lysosomal acid hydrolases. A s i g n i f i c a n t f r a c t i o n of the t o t a l c e l l u l a r pH 7.8 RNase and pH 9«5 RNase a c t i v i t i e s have also been found i n lysosomes i n l i v e r . In view of the s i m i l a r i t i e s of the three RNase a c t i v i t i e s found i n the present study to those of rat l i v e r , i t seems l i k e l y that a large portion of the t o t a l a c t i v i t y which i s recovered i n the crude mitochondrial f r a c -t i o n of rat brain may also be associated with the lysosomal components of t h i s f r a c t i o n . The compartmentalization of acid hydrolases within lysosomes may serve to regulate t h e i r a c t i v i t y and prevent the indiscriminate action of these p o t e n t i a l l y destructive enzymes. In view of the protective function of the lysosomal membrane any in j u r y to the membrane r e s u l t i n g i n increased permeability would be expected to enhance the rate of cata-bolism of cytoplasmic constituents. That segregation of acid RNase within lysosomes i s an important mode of regu-l a t i n g the a c t i v i t y of 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 of acid RNase i n the c e l l , the absence of any known s p e c i f i c i n h i b i t o r s of acid RNase, and the i n a c t i v i t y toward t h i s enzyme of pH 7*8 RNase i n h i b i t o r . Hence, a discussion of the regulation of the i n vivo a c t i v i t y of acid RNase must take into consideration the mode of action - 15© -of lysosomal enzymes and the regulation of lysosomal function. Although the presence of acid hydrolases within lysosomes i s well established, t h e i r s i t e of function i s s t i l l i n dispute. Two hypotheses have been formulated as to how lysosomes perform t h e i r digestive functions i n the intac t c e l l . According to one, material i s encapsulated by membrane to form endocytotic vacuoles which then fuse with lysosomal membranes and empty t h e i r contents into the lysosome where they are degraded and the degradation products recycled to thescytosol. According to t h i s model lysosomal contents normally remain continuously delimited by an uninterrupted membrane which shields the res t of the cytoplasm against attack by lysosomal enzymes. This hypo-thesis i s supported by observations that ( l ) phagocytized and pinocytized material i s found i n lysosomes{ (2) primary lysosomes fuse r e a d i l y with endocytotic v a c u o l e s - ^ 2 ' a s well as with other lysosomes (but not with mitochondria or nuclei)f314,315 a n d ^ a n intralysosomal s i t e of action f o r lysosomal enzymes, most of which have acid pH optima, i s compatible with normal intralysosomal pH which has been reported to be 6.34 f o r r a t l i v e r lysosomes. According to the second hypothesis, the enzymes com-partmentalized within lysosomes are normally inactive and pa r t i c i p a t e i n the catabolism of c e l l constituents only upon - 1 5 1 -317 release into the soluble c e l l sap.-' f Thus, only lysosomal enzymes which occur free i n the cytosol represent metabo-l i c a l l y available enzyme. Support f o r t h i s hypothesis comes from several l i n e s of i n v e s t i g a t i o n . I t has been observed 3"*" 8"" 3 2 1 that increased f r a g i l i t y of lysosomes i s correlated with enhanced l e v e l s of various acid hydrolases i n the cytosol. This implies release or leakage of lysosomal acid hydrolases into the cytosol and t h i s process appears to be more pronounced under conditions of c e l l stress than during normal c e l l functioning. Some forms of stress that have resulted i n an increase i n acid RNase a c t i v i t y of the 322 cytosol i n l i v e r c e l l s include hypophysectomy,-^ adrena-lectomy, 3 2 2 and c a r c i n o g e n e s i s . 1 9 ^ Furthermore, the release and a c t i v a t i o n of acid hydrolases into the cytosol has been reported to precede and contribute to c e l l degeneration during v i r a l i n f e c t i o n . 3 2 9 An increase i n the r a t i o of free to bound lysosomal enzyme a c t i v i t y during tissue degeneration has also been r e p o r t e d . 2 5 6 - 2 5 8 , 3 2 3 - 3 2 5 F i n a l l y , several r e p o r t s 3 2 ^ " " 3 2 8 indicate that the a c t i v i t y of lysosomal enzymes increase with c e l l age. Lysosomal membrane l y s i s and/or acid hydrolase induction under conditions of extreme c e l l u l a r (metabolic and func-t i o n a l ) stress may thus be important mechanisms i n the process of c e l l aging and a u t o l y s i s . The r o l e of lysosomal - 1 5 2 -RNases and other acid hydrolases i n tumor regression and c e l l degeneration may, however, be secondary to a general enhancement i n the rate of c e l l or tissue catabolism. Although acid hydrolases would be expected to have much reduced a c t i v i t y at the normal physiological pH of the cytosol, some conditions of c e l l u l a r stress under which fragmentation of lysosomes may occur are accompanied by a concomitant acid s h i f t i n the normal pH of the cytosol (e.g., hypoxia, eschemia,-^ 0 acidosis, neoplasm-^**) thus r e s u l t i n g i n more favorable conditions for the degradation of cytoplasmic constituents by lysosomal enzymes. A method that has been used to gain information r e -garding the function and s i t e of^ action of lysosomal enzymes has been the physiological perturbation of i n t a c t animals. Thus, Pontremoli et a l . - ^ 1 have reported the occurrence of increased numbers of lysosomes, changes i n t h e i r morphology including membrane breaks, as well as release into the cytosol of several lysosomal enzymes i n the hepatocytes of starved or cold-stressed r a b b i t s . I f such a phenomenon represented a general mechanism of metabolic response to c e l l u l a r stress, i t would be expected that cytosol l e v e l s of acid RNase would also be increased i n starvation or cold-stressed c e l l s . However, such r e s u l t s have not been reported. De Lamirande 2 0 2 and A l l a r d have, i n f a c t , reported that f a s t i n g s l i g h t l y - 1 5 3 -lowers the l e v e l s of both acid 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 d i s t r i b u t i o n 332 of these enzymes i n rat l i v e r . Deckers-Passau et a l . ^  have reported d i f f e r e n t i a l reactions of several lysosomal enzymes to changes i n phys i o l o g i c a l state of the animal. Dynamic changes i n membrane permeability may be of c r i t i c a l importance i n the regulation of i n vivo RNase a c t i v i t y since such changes w i l l determine the a c c e s s i b i l i t y of these enzymes to t h e i r substrates. The key to under-standing the ro l e of lysosomal enzyme function 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 factors res-ponsible f o r maintaining the s t r u c t u r a l and functional i n t e g r i t y of lysosomal membranes or a f f e c t i n g lysosomal membrane permeability thereby f a c i l i t a t i n g or i n h i b i t i n g the release of lysosomal enzymes into the cytosol. In t h i s regard, Demin and Nechaeva 3 3 3 have shown that adrenalin at 3 . 3 x 1 0" 3M causes a 27% a c t i v a t i o n of acid RNase a c t i v i t y i n cerebral cortex crude mitochondrial f r a c t i o n incubated i n isoton i c sucrose, whereas acetylcholine at 6 x 10""^ M i n h i b i t s the normal leakage of latent acid RNase from t h i s subcellular f r a c t i o n . In leucocyte lysosomes, however, catecholamines have been shown-^ to i n h i b i t lysosomal enzyme release 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 of i n t r a c e l l u l a r cAMP. C y c l i c AMP has also been reported to increase the permeability of lysosomal membranes - 15 k -with respect to acid phosphatase and 3-glucuronidase i n rat se d\ 256 l i v e r ^ 0 and to acid RNas during degeneration of hormone-dependent mammary tumors. Some of the discrepancies between these findings may be explained i n terms of t i s s u e - s p e c i f i c differences i n the response of lysosomes to these reagents. Also, since lyso-201 341 342 somes have been shown • % J to be heterogeneous i n t h e i r size and enzyme content, i t i s possible that variable a c t i v a -t i o n or release of lysosomal enzymes may be due to selec t i v e membrane permeability changes i n s p e c i f i c classes of lysosomes or to differences i n permeability of the lysosomal membrane to s p e c i f i c acid hydrolases. The implication of cAMP i n the regulation of lysosomal membrane permeability combined with reports of neurotrans-mitter-stimulated adenyl cyclase i n brain suggests a pla u s i b l e mechanism by which the a c t i v a t i o n of post synaptic c e l l sur-face neurotransmitter receptors may modulate neuronal RNA metabolism. O s c i l l a t o r y changes i n neuronal RNA content accompanying electrophysiological a c t i v i t y 1 - ^ may thus be attrib u t a b l e to the i n t r a c e l l u l a r release of a receptor-transduced metabolic demand signal which modulates the rate of RNA degradation by regulating RNase a c c e s s i b i l i t y to substrate. 1 5 5 -There have been consistent findings of a p o s i t i v e c o r r e l a t i o n between elevated RNase a c t i v i t y or decreased RNase i n h i b i t o r to pH 7.8 RNase a c t i v i t y r a t i o and either peduced rates of protein synthesis or a general increase i n catabolic a c t i v i t y . This i s i n agreement with the view that functional l e v e l s of these enzymes may l i m i t the a v a i l a b i l i t y and frequency of t r a n s l a t i o n of mRNA molecules. Furthermore, the f i n d i n g that reduced l e v e l s of free RNase i n h i b i t o r or enhanced l e v e l s of free pH 7*8 RNase a c t i v i t y i s correlated with enhanced polysome breakdown, decreased capacity of ribosomes f o r accepting or tr a n s l a t i n g mRNA, decrease i n functional mRNA, and general impairment i n the functioning of the t r a n s l a t i o n apparatus suggests that RNase i n h i b i t o r may play a s i g n i f i c a n t r o l e i n regulating protein synthesis by preserving the s t r u c t u r a l and functional i n t e g r i t y of polysomes. Several workers have investigated the disaggregation of is o l a t e d polysomes. Eker and P i h l 3 ^ 3 have reported that the disaggregation of polysomes i n v i t r o and the time-dependent a c t i v a t i o n of a latent RNase a c t i v i t y associated with polysome preparations i s prevented by 1 . 0 mM d i t h i o -t h r e i t o l or 1 . 0 mM glutathione. The ef f e c t of these compounds was not due to 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 since 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 of - 156 -p a r t i a l l y p u r i f i e d RNase preparations which exhibit neither stimulation by pCMB nor i n h i b i t i o n of bovine pancreatic RNase A. I t has also been reported that rat l i v e r cytosol s t a b i l i z e s polysomes,344-346 m RNAf345,347 a n d HnRNA3^8  i n v i t r o . RNase i n h i b i t o r has been demonstrated by several workers222»345*357 ^ 0 s t a b i l i z e polysomes and prevent t h e i r disaggregation. The presence of excess free RNase i n h i b i t o r i n the cytosol has thus been assigned the physiological 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 preventing the degradation of polysomal RNA. The release and a c t i v a t i o n of inhibitor-bound pH 7.8 RNase could be effected by any endogenous factor capable of d e s t a b i l i z i n g the inhibitor-RNase i n t e r a c t i o n and disso-c i a t i n g the inhibitor-RNase complex. Factors such as reduced sulf h y d r y l compounds capable of modulating the i n t e r a c t i o n between free RNase i n h i b i t o r and free pH 7«8 RNase could s h i f t the dynamic equilibrium between RNase-inhibitor complexes, free pH 7*8 RNase, and free RNase i n h i b i t o r , thereby regulating the rate of RNA degradation. The e q u i l i -brium between these molecular species could also be con-t r o l l e d by constraints to 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 indicate that the compartmentalization of free RNase i n h i -b i t o r may be of importance i n determining the d i f f e r e n t i a l - 157 -rates of RNA degradation i n the c e l l nucleus and cytoplasm. The absence of free RNase i n h i b i t o r a c t i v i t y i n the nucleus indicates that the nucleo-cytoplasmic envelope functions as a permeability b a r r i e r which prevents the large excess of free RNase i n h i b i t o r i n the cytosol from e q u i l i b r a t i n g with the nucleoplasm. I t must be emphasized that the l e v e l s of RNase a c t i v i t y measured under optimized conditions i n v i t r o cannot be taken as a r e l i a b l e index of enzyme function i n the i n t a c t c e l l . The a c t i v i t y of these enzymes under normal physiological The c o n t r i b u t i o n s of each of the conditions i s not known.A Shree RNases i d e n t i f i e d i n t h i s study to c e l l u l a r RNA degradation under normal p h y s i o l o g i c a l conditions i s also not apparent. Information as to the pre-f e r e n t i a l substrate s p e c i f i c i t i e s of each of the three enzymes with respect to d i f f e r e n t classes (e.g., rRNA, tRNA, HnRNA) of brain RNA may provide a better understanding of t h e i r function i n vivo. In view of the facts that ( l ) there i s no detectable free pH 7.8 RNase a c t i v i t y i n the cytosol; (2) pH 6.7 RNase exhibits n e g l i g i b l e a c t i v i t y at pH 7A and i s markedly i n h i -bited by physiological t o n i c i t y above pH 6, and (3) pH 9*5 RNase exhibits only 68% of i t s optimal a c t i v i t y when assayed at pH 7«8 and i s also markedly i n h i b i t e d by ph y s i o l o g i c a l i o n i c strength, i t would seem that i n t r a c e l l u l a r RNA would - 1 5 8 -be protected from the action of these enzymes under normal physiological conditions. The measured l e v e l s of RNase a c t i v i t i e s hence probably represent t o t a l c e l l u l a r capacity f o r RNA degradation and do not r e f l e c t functional l e v e l s of 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 the breakdown of RNA molecules under normal ph y s i o l o g i c a l conditions i n vivo. I t i s , nevertheless, c l e a r that c e r t a i n perturbations i n the steady-state p h y s i o l o g i c a l conditions could r e s u l t i n an ac t i v a t i o n of the latent RNA-degrading capacity of brain c e l l s and hence i n an enhanced rate of RNA degradation. 4 . 1 Correlation of Developmental Changes i n the Content, Synthesis and Degradation of RNA i n brain Despite previously mentioned reservations, i t seems worthwhile to make some tentative inferrences as to the possible functional significance of the developmental changes i n RNase a c t i v i t i e s observed under the conditions of the present study. How are these changes correlated with develop-mental changes i n RNA synthesis and c e l l u l a r RNA content? Figure 1 9 i s a composite developmental p r o f i l e of the t o t a l RNA degradative capacity of whole brain constructed by summing the experimentally measured a c t i v i t i e s of each i n d i v i d u a l RNase (pH 6 . 7 RNase, pH 9 . 5 RNase, and free p i 7 . 8 RNase) at a given age. The increase i n t o t a l c e l l u l a r RNA hydrolytic capacity per gram wet weight of whole brain between 1 0 and 2 0 days - 1 5 9 -5 10 15 20 25 30 35 days Pos t -na ta l A g e 5 months FIGURE 19. Total RNA degradative capacity of whole brain as a function of the postnatal age of r a t . This i s a t h e o r e t i c a l curve constructed from data shown i n Figures 1 5 • 16 and 17 by summing the a c t i v i t y of each i n d i v i d u a l RNase assayed at pH 6.7. 9.5 and 7.8 (free pH 7.8 RNase a c t i v i t y ) at each age. - 1$9 -would be expected to correspond to an increase i n the i n vivo rate of RNA degradation, unless i t i s accompanied by a pro-portional increase i n regulatory mechanisms r e s t r i c t i n g the a c t i v i t y of RNases. This increase i n brain RNA degradative capacity d i r e c t l y p a r a l l e l s the concurrent increase i n organ and c e l l u l a r RNA content f o r i t has been shown that the t o t a l SNA content of both the cerebral cortex and whole brain continues to increase up to age 20 days at which time t h i s accumulation ceases. Hence, the rate of t o t a l RNA synthesis exceeds the rate of t o t a l RNA degradation by a decreasing increment which approaches zero by age 20 days when a steady state balance between the rates of RNA synthesis and degrada-t i o n i s attained and a constant organ content of RNA i s maintained. Possibly the most r e l i a b l e data on the rate of t o t a l RNA synthesis i s that obtained with tissue s l i c e s . Unfor-tunately, developmental data for the rate of t o t a l RNA synthesis i n whole brain s l i c e s i s not a v a i l a b l e . However, developmental studies of the rate of RNA synthesis i n rat 46 349 cerebral cortex s l i c e s * J 7 indicate that the rate of synthesis declines (undergoing a 3 to 4 - f o l d decrease) a f t e r age 10 days and attains adult steady state l e v e l s by about 20 days. - 161 -Since the rate of t o t a l RNA synthesis declines during the time when organ RNA content i s accumulating and the t o t a l RNA degradative capacity (per gram wet weight whole brain) i s increasing, i t follows that any concomitant increase i n the rate of RNA degradation i s l i m i t e d by the f a c t that the t o t a l rate of RNA degradation does not exceed the d e c l i n i n g rate of RNA synthesis. Since the steady state adult rate of RNA synthesis and the f i n a l organ and c e l l u l a r RNA content i s attained by age 20 days and subsequently sustained, i t also follows that the rate of t o t a l RNA degradation and hence the functional l e v e l of RNase a c t i v i t y must also reach steady state conditions by t h i s age. 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 capacity of whole brain subsequently declines up to age 30 days. Since between 20 and 3° days there i s no net accumulation i n organ RNA, and since the rate of RNA synthesis remains constant, i t must be t e n t a t i v e l y concluded that the decline i n maximal RNA-degradative capacity on maximal RNase a c t i v i t y i s not accompanied by a p a r a l l e l decline i n the functional contribution of these enzymes to the rate of t o t a l RNA degradation i n vivo. Thus, although the t o t a l RNA-degradative capacity declines between age 20 and 30 days, compatibility with the other events observed during t h i s time requires that the amount of t h i s capacity which i s functional i n vivo must remain constant a f t e r 20 - 162 -days. Control factors must regulate and maintain a constant level of RNase activity after age 20 days despite the decline in total RNase capacity. Hence, an increasingly greater fraction of the total RNA-degradative capacity or maximal RNase activity i s functional in vivo. For the functional or expressed level of RNase activity to remain constant while the maximal RNase activity measured in vitro decreases, there must occur either a preferential dropout i n the amount of non-functional, latent, RNase activity and/or a proportional decline of both functional and non-functional RNase (activity) concomitant with a progressive relaxation of control factors normally restricting the expression of RNase activity. This might be thought of as increasing the in vivo specific a c t i v i t y of the enzymes. Elucidation of the precise relationship of the develop-mental changes in the measured levels of RNase activity to the changes in the physiologically functional levels of activity of these enzymes w i l l require a developmental study of the rate of turnover of total RNA i n whole brain. Such data would provide a more reliable index of the in vivo rate of total RNA degradation. The difference curve between the develop-mental profiles for total RNA turnover and for total RNA-degradative capacity would then represent developmental changes in control factors regulating the expression of maximal RNase activity. § 163 -4.2 Regional Differences in the Metabolism of RNA and i n the Functional Roles of RNases in brain The developmental pattern of RNA depolymerase act i v i t i e s can be correlated with the occurrence of both quantitative and qualitative 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 activity than adult rat brain. Older rats have higher levels 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 proliferation stage of brain development suggests a possibly specific role for this enzyme in the catabolism of RNA during c e l l division. Presumably, RNA metabolism during c e l l differentiation entails a rapid degra-dation of RNA and the synthesis of new species of RNA mole-cules. The rise in alkaline RNase activ i t i e s between day 7 and day 22 may be associated with the replacement of the mRNA molecules coding for mitosis and c e l l differentiation by RNA molecules required for the maintenance functions of non-dividing brain c e l l s . Thus, these enzymes may participate in the turnover of the RNA content of fully-differentiated c e l l s . This line of reasoning gives rise to the expectation that significant differences in the developmental profiles - 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 brain regions. Unlike the cerebral cortex i n which c e l l d i v i s i o n i s almost complete at b i r t h , the cerebellum i n the rat exhibits rapid c e l l p r o l i f e r a t i o n up to 1 6 to 2 0 days a f t e r b i r t h . k ? » 3 0 8 , 3 0 9 Hence, since acid RNase a c t i v i t y i s highest during the c e l l p r o l i f e r a t i o n stage of brain development, the decline i n s p e c i f i c a c t i v i t y of acid RNase may occur at a l a t e r postnatal age i n the cerebellum than i n the cerebral cortex. Also, 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 to begin at a l a t e r age i n the cerebellum and/or to be more prolonged than i n the cerebral cortex. Such regional differences i n the le v e l s of the various components of the RNase enzyme system during maturation may account f o r the fi n d i n g that developmental changes i n RNA metabolism i n the cerebral cortex are not representative of whole brain. The available information indicates that the t o t a l RNA content of the cerebral cortex, unlike the cerebellum and whole brain, a c t u a l l y declines a f t e r a t t a i n i n g peak values at 18 to 2 0 days of age, and t h i s decline appears 24 to be due to a p r e f e r e n t i a l depletion of rRNA. Since i t 46 349 has been shown 7 that the rate of RNA synthesis i n r a t cerebral cortex s l i c e s declines up to 2 0 days and subsequently remains constant, the decline i n RNA content of th i s brain region a f t e r age 2 0 days must be due to an increase i n the rate of RNA degradation. - 165 -This conclusion i s i n agreement with the difference i n the developmental p r o f i l e s of free 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 for whole brain and that reported by Takahashi and Suzuki D f o r cerebral cortex. The lower free 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 cerebral cortex of adult r a t s as compared to 10 day old animals i s consistent with an enhancement i n the rate of RNA degradation i n t h i s brain region. In the adult r at the cerebral cortex contains more RNA per average c e l l J and has a 2-6 f o l d greater rate of if6 RNA synthesis than the cerebellum. I t follows that at t h i s age RNA turnover and the t o t a l RNA degradative a c t i v i t y w i l l be proportionally greater i n the cerebral cortex as compared to the cerebellum. The difference i n the rate of RNA degra-dation between these two areas w i l l be greater than the difference i n the rate of RNA synthesis such as to allow a net depletion of RNA content i n the cerebral cortex a f t e r age 20 days. Direct t e s t i n g of these conclusions w i l l require a comparative study of the developmental changes i n RNA turn-71 over i n these brain regions. Dawson' studied the rate of rRNA turnover i n 35 day old rat brain a f t e r an intracerebral i n j e c t i o n of l a b e l l e d uridine and found that the l a b e l incor-porated into rRNA decayed with a h a l f - l i f e of 6 days. Studies - 166 -using other routes of precursor administration have shown that i n adult r a t , whole brain rRNA turns over with a h a l f l i f e of 12 d a y s . ^ ' - ^ 1 I t seems l i k e l y that i n t r a c e r e b r a l i n j e c t i o n of precursor i n Dawson's study may have resulted i n the p r e f e r e n t i a l l a b e l l i n g of rRNA i n the cerebral cortex, and hence, the higher turnover rate of rRNA reported by Dawson may r e f l e c t a higher rate of rRNA synthesis and degradation i n the cerebral cortex as compared to other regions of the adult r a t brain. The f a c t that for whole brain the rate of rRNA turnover i n the newborn rat i s twice that of the a d u l t ^ 0 i s consistent with the developmental pattern of free RNase i n h i b i t o r a c t i v i t y observed f o r whole brain i n the present study. I f free pH 7,8 RNase p a r t i c i -pates i n the degradation of rRNA, the higher l e v e l of free RNase i n h i b i t o r i n the brain of the adult would l i m i t the a c t i v i t y of t h i s enzyme thus affording a longer h a l f - l i f e of RNA. Although i t was desirable to obtain an o v e r a l l picture of the RNase a c t i v i t i e s of the whole brain, i t must be stressed that the data thus obtained may mask s i g n i f i c a n t regio n - s p e c i f i c differences. In view of the organizational complexity of the brain and the d i f f e r e n t i a l rates of develop-ment of d i f f e r e n t brain regions, a regional d i s t r i b u t i o n study w i l l be required to test some of the speculations which have emerged i n the foregoing discussion. Moreover, the data - 167 -obtained i n the present study of r a t whole brain represents composite r e s u l t s f o r a complex mixture of c e l l s (primarily neurons and g l i a ) each of which may have quite d i f f e r e n t <co degradation capacities with respect to RNA. ? For example, 90 Watson has reported that RNA turnover proceeds more ra 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 depending on the environ-mental status of the animal. I t i s hence desirable that measurements of the functional l e v e l s of the various components of the RNase system be extended to l o c a l i z e d brain regions and homogeneous c e l l populations of discrete c e l l types. 4,3 T i s s u e - s p e c i f i c Differences i n RNA Turnover and RNase A c t i v i t i e s , i n the adult animal In the adult r a t rRNA of whole brain turns over at h a l f the rate of l i v e r rRNA, This i s consistent with the observation that when expressed on a per gram wet weight basis the l i v e r content of RNase a c t i v i t y i s 10-fold higher than brain and i t s inhibitor-bound pH 7,8 RNase i s 2-fold lower than brain. Although the l e v e l of free RNase i n h i b i t o r a c t i v i t y i n r a t l i v e r was not measured i n the present study, R o t h 1 8 7 has reported that i n adult rats the s p e c i f i c a c t i v i t y of free - 168 -RNase i n h i b i t o r of the cytosol f r a c t i o n i s 15$ higher i n whole brain than i n l i v e r s Roth also found that the s p e c i f i c a c t i v i t y of free RNase i n h i b i t o r i n the cytosol of l i v e r was 2-fold greater than that 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 free pH 7.8 RNase i n the cytosol of kidney to l i v e r was k0»l. 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 of t o t a l pH 7*8 RNase (assayed i n the presence of pCMB) was con-2 02 siderably lower ( k i l ) than t h i s . De Lamirande and A l l a r d i n a comparative study of free pH 7«8 RNase and acid RNase a c t i v i t i e s i n d i f f e r e n t tissues of the adult r a t found lowest l e v e l s of both a c t i v i t i e s i n the cerebral cortex (3-f o l d lower than i n l i v e r ) . Greenstein et al.^52 a n d R 0-t n and M i l s t e i n ^ 3 have also reported that the acid RNase a c t i v i t y of whole brain i s amongst the lowest of a l l r at 215 tissues studied. Ellem and Colter J studied the l e v e l s of acid RNase and free pH 7«8 RNase a c t i v i t i e s i n various mouse ti s s u e s . The a c t i v i t y of acid RNase per gram wet weight tissue was 3-^old lower f o r brain than f o r l i v e r and the a c t i v i t y of free pH 7«8 RNase per gram wet weight tissue was 4-fold lower f o r brain as compared to l i v e r . The l e v e l of both enzyme a c t i v i t i e s on a per gram wet weight tissue basis was higher i n kidney than l i v e r and lower i n muscle than brain. - 169 -The significance of these t i s s u e - s p e c i f i c differences i n RNase a c t i v i t i e s i s not 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 brain, as compared to l i v e r of adult r a t s , may be relevant to the f a c t that i n the adult rat rRNA of whole brain turns over at h a l f the rate of l i v e r rRNA. A better understanding of the functional significance of the higher l e v e l of inhibitor-bound pH 7»8 RNase and possibly also of free RNase i n h i b i t o r a c t i v i t y i n brain, as compared to l i v e r , must await further information. 4.4 Concluding Remarks The present study has elucidated some of the gross features of the enzyme system which p a r t i c i p a t e s i n the de-gradation of RNA i n brain. The RNase enzyme system i n brain i s s i m i l a r i n i t s broad and general outline to that studied i n other mammalian tissues and may d i f f e r from other organs only i n the so p h i s t i c a t i o n and v e r s a t i l i t y of the regulatory mechanisms by which i t s a c t i v i t y i s controlled and co-ordinated with other enzyme systems. The i n i t i a l intent of t h i s study was to explore the RNase system i n brain f o r any disti n g u i s h i n g features which might be s p e c i f i c a l l y r e l a t e d to the specialized functions of t h i s t i s s u e . However, a preliminary characterization of the properties of t h i s enzyme system i n whole brain was required due to the lack of information as to i t s RNase content and composition. A - 170 -deeper l e v e l of analysis w i l l be required to evaluate the physiological function of these enzymes and t h e i r respective r o l e s i n the integrated metabolism of RNA i n brain. The e f f e c t of a number of variables influencing the determination of the component 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 information thus provides the necessary groundwork f o r the in v e s t i g a t i o n of variations i n the functional l e v e l s of the various components of t h i s enzyme system following a l t e r a t i o n s i n the milieu of the animal or a l t e r a t i o n s i n s p e c i f i c brain functions. 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 physiological stresses such as learning experience, sensory deprivation, electroschock, malnutrition, as well as during c e l l degeneration following axonal section or e l e c t r o l y t i c l e s i o n s . Such measurements of the functional l e v e l s and subcellular d i s t r i b u t i o n of the various components of the RNase system under conditions known to be accompanied by changes i n c e l l u l a r RNA content may provide a clearer understanding of the contribution of each of the component 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 content which have been reported to occur i n brain. This would r e s u l t i n a clear d e f i n i t i o n of the complex rela t i o n s h i p between the a c t i v i t y of each of - 171 -the i d e n t i f i e d components of t h i s enzyme system to neuronal functional states. Further information i s required to determine (1) the significance of the high l e v e l of RNase i n h i b i t o r i n brain and how i t i s geared to s p e c i f i c tissue function; (2) how regulatory mechanisms c o n t r o l l i n g RNA catabolism are coupled to the electrophysiological a c t i v i t y of neurons, and (3) how these mechanisms respond to changes i n the neuron's functional demand for RNA. To achieve a clear picture of integrated RNA function and metabolism i n the i n t a c t c e l l , changes i n the rate of RNA degradation must also be correlated with changes i n the rate of de novo RNA synthesis, conversion 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 RNA, and nucleocytoplasmic transport. The detection of changes i n these parameters i n response to enhanced electrophysiological a c t i v i t y may be c r i t i c a l to the i d e n t i f i c a t i o n of those molecular events which underlie the modification of a neuron's functional r e l a t i o n s h i p with other neurons. The present inves t i g a t i o n provides a descriptive picture of the baseline RNase composition of r a t whole brain under states of normal brain function. I t i s hoped that the information reported herein w i l l provide guidelines - 172 -f o r prospective investigations into the more basic questions of the nature of the molecular mechanisms and control points through which RNA metabolism i s regulated and the underlying molecular processes by which changes i n c e l l u l a r RNA metabolism r e s u l t i n a l t e r a t i o n s i n integrated brain c e l l functions. - 173 -V- BIBLIOGRAPHY 1. Gross, P.R. Ann. Rev. Biochem. .22, 6 3 1 , ( 1 9 6 8 ) 2. Scott, R.B. and Bell, E. 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