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Studies on heart muscle lipases and studies on 3', 5'-cyclic nucleotide phosphodies-terase Yamamoto, Masanobu 1966

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-  X -  STUDIES ON HEART MUSCLE LIPASES AND STUDIES ON 3«,5t-CYCLIC NUCLEOTIDE PHOSPHODIESTERASE by MASANOBU YAMAMOTO  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  In the Department of Pharmacology  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1966  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements  for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study,  I f u r t h e r agree that permission-for  extensive  copying of t h i s  t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives.  I t i s understood that copying  or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada  Supervisor:  G. I . Drummond.  MASANOBU YAMAMOTO. STUDIES ON HEART MUSCLE LIPASES AND STUDIES ON y,5'-CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.  ABSTRACT PART I  STUDIES ON HEART MUSCLE LIPASES The study of the r o l e of l i p i d s i n supplying the energy  requirements of the heart has attracted widespread p a r t i c u l a r l y within the past decade.  attention,  I t i s now known that  the heart, under normal conditions, oxidizes l i p i d s as i t s main source of energy. Numerous investigators have studied the Jjn vivo and i n v i t r o uptake and u t i l i z a t i o n of exogenously supplied l i p i d s i n the form of t r i g l y c e r i d e s , free f a t t y acids and ketone bodies.  However, very few have studied the u t i l i z a t i o n of  endogenous l i p i d s by the working heart. We have examined the r e l a t i v e importance of both endogenous glycogen and t r i g l y c e r i d e s f o r supplying the c a l o r i c needs of the isolated beating r a t heart, and found that under the perfusion conditions used, endogenous glycogen appears to supply the i n i t i a l source of energy. A lipase i n r a t cardiac tissue was also examined.  The  enzyme had a pH optimum near 6.8, and was strongly inhibited by 0.2 M NaF and by 2 x 10~^M  diisopropylfluorophosphate.  Most  of the a c t i v i t y was found i n the nuclear f r a c t i o n of tissue homogenates.  The enzyme hydrolyzed both monoolein and mono-  s t e a r i n , and possessed much less a c t i v i t y against t r i p a l m i t i n . The enzyme also r a p i d l y hydrolyzed the monostearin component of E d i o l  (a commercial coconut o i l emulsion widely used i n  l i p a s e studies), and the implications of these findings are discussed.  I t was concluded from these studies that a lipase  other than l i p o p r o t e i n l i p a s e e x i s t s i n r a t myocardium.  - iii PART I I  STUDIES ON CYCLIC 3», 5 -NUCLEOTIDE PHOSPHODIESTERASE 1  In recent years, the study of the role of c y c l i c 3»,5»adenosine monophosphate ( c y c l i c 3*,5»-AMP) i n the regulation of several b i o l o g i c a l  reactions and processes has received  widespread attention. The presence of a p h y s i o l o g i c a l mechanism for terminating the action of c y c l i c 3,5.*-AMP i n t  biological  systems would therefore be expected.  enzyme, c y c l i c 3*,5"-nucleotide phosphodiesterase shown to exist  has been  i n most mammalian tissues which have been  studied for i t s a c t i v i t y . cularly  Indeed, an  The c e n t r a l nervous system, p a r t i -  the cerebral cortex, possesses a very high a c t i v i t y  of this enzyme. In this study, c y c l i c 3 ,5'-nucleotide 1  was p a r t i a l l y p u r i f i e d were studied. was  inhibited  phosphodiesterase  from rabbit brain and i t s properties  The enzyme required Mg+^ions for a c t i v i t y and by 2 x lO'^M  theophylline. C y c l i c  3*,5*-dAMP,  c y c l i c 3*,5»-GMP and c y c l i c 3*,5«-dGMP were hydrolyzed by the brain diesterase at approximately one-half the rate at which c y c l i c 3*,5»-AMP was hydrolyzed.  L i t t l e a c t i v i t y against  c y c l i c 3>,5t-CMP, c y c l i c 3»,5«-dCMP and c y c l i c 3«,5«-TMP was detected, although c y c l i c 3 ,5«-UMP was hydrolyzed at ,  approximately 13% of the rate at which c y c l i c 3*,5*-AMP was hydrolyzed.  The brain diesterase therefore possessed a high  s p e c i f i c i t y for c y c l i c 3 , 5'-nucleotides with purine bases. f  Optimum enzyme a c t i v i t y was observed near pH 7.0, a c t i v i t y was The K  and the  stimulated about 1.5-fold by 0.06 M imidazole.  value of the enzyme with c y c l i c  3*,5}-AMP  as substrate  - iv was  approximately 0.8  x 10"^M.  The properties of the part-  i a l l y p u r i f i e d phosphodiesterase from brain were thus very similar to the diesterases which have been p u r i f i e d from beef and dog  hearts.  A study of the 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 brain diesterase indicated that about 50% of the a c t i v i t y was ted i n the 105,000 x g supernate.  loca-  The microsomal and mito-  chondrial f r a c t i o n s also contained considerable amounts of diesterase a c t i v i t y , but l i t t l e a c t i v i t y was nuclear  located i n the  fraction.  A survey of c y c l i c 3*,5*-nucleotide phosphodiesterase a c t i v i t y i n several available specimens of the plant kingdom indicated the absence of this enzyme a c t i v i t y i n these organisms.  However, appreciable levels of diesterase a c t i v i t y  were detected  i n E. c o l i .  - V -  TABLE OF CONTENTS Page PART I  STUDIES ON HEART MUSCLE LIPASES  1  INTRODUCTION  2  EXPERIMENTAL PROCEDURE  16  Materials Methods  16 I II  Perfusion Studies  18  Cardiac Lipase Studies  23  Lipase Assay Preparation of Substrates Measurement of T r i p a l m i t i n - l - C ^ Hydrolysis Preparation of Enzyme Extract  23 27  1  27 28  RESULTS  30  I  Perfusion Studies  30  Cardiac Lipases  36  II  A.  Existence of NaF-inhibited Lipase  38  B.  Preparation of P a r t i a l l y P u r i f i e d Extract from Heart Tissue  49  C.  Properties of Monoglyceride-hydrolyzing Enzyme  DISCUSSION  51  1.  Albumin Requirement  54  2.  pH Optimum  54  3. 4.  Temperature Optimum E f f e c t of Physical State of Substrate  5.  Inhibitor Studies  59  6.  Intracellular Localization  63  57 ;  57  65  - v iTABLE OF CONTENTS (cont'd.)  Page PART I I STUDIES ON 3',5'-CYCLIC NUCLEOTIDE PHC^PWD lE^TEiRASE  1  "  :  :  79  INTRODUCTION  80  EXPERIMENTAL PROCEDURE  94  Materials Methods - Standard Diesterase Assay - P a r t i a l P u r i f i c a t i o n of Brain Diesterase RESULTS  94 94 99 103  1.  Preliminary  103  2.  P a r t i a l P u r i f i c a t i o n of Brain Diesterase  106  3.  Properties of Brain Diesterase (a) Metal Requirement (b) E f f e c t of Imidazole, pH Curve (c) E f f e c t of Theophylline (d) C y c l i c 3«,5'-dAMP/Cyclic 3«,5'-AMP A c t i v i t y Ratios (e) Hydrolysis Rates of Purine and Pyrimidine C y c l i c 3»^'-Nucleotides (f) Further Studies on S p e c i f i c i t y of Brain Diesterase C e l l u l a r D i s t r i b u t i o n of Brain Diesterase  108 108 108 111  4. 5.  Survey of Diesterase i n Human Brain, Dog Nervous System, Marine Organisms, Plants and Microorganisms  111 113 116 121  125  DISCUSSION  132  BIBLIOGRAPHY  140  -' V l l -  LIST OF TABLES  No.  Title  Page  PART I STUDIES ON HEART MUSCLE LIPASES I II III IV V  E f f e c t of Rat Serum on T r i p a l m i t i n Hydrolysis  44  P a r t i a l P u r i f i c a t i o n of Cardiac Monoglyceride-hydrolyzing Enzyme  52  I n h i b i t i o n of Monoglyceride-hydrolyzing Enzyme by Various Compounds  61  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 Monoglyc e r i d e - s p l i t t i n g Enzyme  64  Relative Rates of Hydrolysis of Monoglycerides and Triglycerides  72  PART II STUDIES ON 3'5'-CYCLIC NUCLEOTIDE MQSPtilOblES^ERA^r — : — VI VII VIII IX X XI XII XIII XIV  Relative A c t i v i t i e s of 5'-Nucleotidase Diesterase i n Snake Venom  and 105  P a r t i a l P u r i f i c a t i o n and Y i e l d of Diesterase  109  C y c l i c 3',5'-dAMP/Cyclic 3'i5'-AMP A c t i v i t y Ratios  114  Relative Hydrolysis Rates of Purine and Pyrimidine C y c l i c 3',5'-Nucleotides  115  C e l l u l a r D i s t r i b u t i o n of Diesterase i n Rabbit Brain  122  D i s t r i b u t i o n of Diesterase A c t i v i t y i n Human Brain  127  Diesterase A c t i v i t y i n Various Areas of Dog Nervous System  128  Diesterase A c t i v i t y i n Various Marine Organisms  129  Diesterase A c t i v i t y i n Plants and Microorganisms  130  - viii -  LIST OF FIGURES  No.  Title PART I  Page  STUDIES ON HEART MUSCLE LIPASES  1  Glycogen Standard Curve  21  2  T r i p a l m i t i n Standard Curve  24  3  Palmitic Acid Standard Curve  26  4  Tissue Glycogen Content - Substrate Free Perfusion  32  Tissue T r i g l y c e r i d e Content - Substrate Free Perfusion  35  6  Time Course of L i p o l y t i c A c t i v i t y  40  7  I n h i b i t i o n of L i p o l y t i c A c t i v i t y i n Crude Homogenates by NaF  41  8  E f f e c t of NaF on Hydrolysis of Monostearin  5  and T r i p a l m i t i n 9 10  Hydrolysis of E d i o l  43 R  Components  46  Enzyme Concentration Curve - Monostearin as Substrate  53  11  E f f e c t of Albumin on L i p o l y t i c A c t i v i t y  55  12  pH Curve of Monoglycerlde-hydrolyzing Enzyme E f f e c t of Temperature on Cardiac Monoglyceride-hydrolyzing L i p o l y t i c Activity E f f e c t of Monoolein Concentration on Enzyme Activity  13 14 15  E f f e c t of Fasting on Myocardial Glycogen Level  56 58 60 67  PART I I STUDIES ON -3' ,5'-CYCLIC NUCLEOTWE PHOSFHQD IESTERASE ~ "— 1  16  Structural Formula of C y c l i c 3' ,5'-AMP  82  -  LIST OF FIGURES  XX  -  (cont'd.)  No.  Title  17  Mediation of C y c l i c 3«,5'-AMP i n the Glycogenolytic Response of Liver to Epinephrine  85  18  Inorganic Phosphate Concentration Curve  96  19  Spectrophotometrie terase  98  20  Hydrolysis of 5*-AMP by Snake Venom (Crotalus adamanteus)  104  Phosphodiesterase A c t i v i t y as a Linear Function of Protein Concentration  107  pH Curve of Brain Diesterase and of Imidazole  110  21 22 23  Page  Assay of Phosphodies-  Effect  I n h i b i t i o n of Diesterase by Theophylline in vitro  112  Hydrolysis of C y c l i c 3«,5»-AMP by Brain Diesterase  117  25  Hydrolysis of C y c l i c 3«,5»-dAMP by Brain Diesterase  118  26  Hydrolysis of C y c l i c 3«,5'-GMP by Brain Diesterase  119  27  Hydrolysis of C y c l i c 3«,5«-dGMP by Brain Diesterase  120  28  Absence of 5'-Nucleotidase A c t i v i t y i n P a r t i a l l y P u r i f i e d Preparation  123  Absence of C y c l i c 2«,3«-AMP Hydrolytic A c t i v i t y i n P a r t i a l l y P u r i f i e d Preparation  124  The Two-Messenger Concept for the Expression of Hormonal Control  133  Structural Formulae of Methyl Xanthines  137  24  29 30 31  -  X -  LIST OF ABBREVIATIONS FFA  Free f a t t y acid  DFP  D iisopropylfluorophosphate  EDTA 5«-AMP ATP UDPG  Ethylenediaminetetraacetic  acid  Adenosine 5»-phosphate Adeno s ine  5«-triphosphate  Uridine diphosphate glucose  C y c l i c 3« ,5t -AMP  Adenosine 3*,5*-phosphate  C y c l i c 3» ,5« -UMP  Urid ine 3 *,5 -phosphate 1  C y c l i c 3« ,5« -dAMP  Deoxyadenosine 3 ,5 *-phosphate  C y c l i c 3« ,5« -dGMP  Deoxyguanosine 3 ,5 *-phosphate  C y c l i c 3« ,5« -GMP C y c l i c 3« ,5t -dCMP  1  1  Guanosine 3*,5»-phosphate Deoxycytidine  3*,5*-phosphate  C y c l i c 3« ,5t -TMP  Thymidine 3»,5*-phosphate  C y c l i c 2« ,3« -AMP  Adenosine 2«,3*-phosphate  Pi  Inorganic phosphate  - xi -  ACKNOWLEDGEMENT I am deeply grateful to Dr. George I . Drummond, Department of Pharmacology, U. B. C. f o r frequently taking time o f f h i s busy schedule to give me much valuable advice and h e l p f u l c r i t i c i s m throughout the course of t h i s work. I also wish to thank him f o r making available Medical Research Council grants which made my graduate work possible. I thank Dr. James G. Foulks, Head of the Department of Pharmacology, U. B. C. f o r the frequent use of h i s laboratory facilities.  My thanks also to Dr. Hans-Peter Baer,  Mrs. Loverne Duncan, and Mrs. E. Hertzman f o r the occasional technical assistance, many i n t e r e s t i n g discussions, and personal encouragement. In addition, I am grateful to Dr. L. Druehl and Mr. M. McLaren of the Department of Botany, U. B. C., f o r making available several specimens of plants, and to Dr. D. Duncan (B. C. Research Council) and Dr. W. J . Polglase (Department of Biochemistry, U. B. C.) f o r the generous r  supply of microorganisms.  I am p a r t i c u l a r l y indebted to  Mr. G. Kent and Mr. R. Smith of t h i s department f o r frequently giving me a helping hand when needed throughout these past few years.  PART I  STUDIES ON HEART MUSCLE LIPASES  2. INTRODUCTION I t i s widely known that during,starvation, the mammalian organism derives v i r t u a l l y a l l of i t s energy requirements from f a t catabolism.  Less widely r e a l i z e d , perhaps, i s the  f a c t that even under normal p h y s i o l o g i c a l conditions, mammals depend to a large extent on the oxidation of f a t s for energy. For example, Fredrickson and Gordon (20) measured the expired C^02  a f t e r i n j e c t i o n of C ^ - l a b e l l e d albumin-bound long-chain  f a t t y acids into man  and indicated that up to 50% of the  energy could have been derived from f a t t y acids during the post-absorptive  state.  Various tissues and organs have been  examined f o r their a b i l i t y to oxidize l i p i d s jLn vivo and i n vitro.  In 1930,  Richardson et al.(65) found that the res-  p i r a t o r y quotients of incubated, excised renal and muscular tissues were c o n s i s t e n t l y intermediate between 0.7 and suggested that f a t s may tive process.  and  1.0,  have participated i n the oxida-  Artom (66), Volk et al.(68) and Geyer and  associates (69) demonstrated that when carboxyl-labelled C ^ - f a t t y acids were incubated with kidney, l i v e r ,  spleen,  heart, lung, brain, s k e l e t a l muscle and t e s t i s s l i c e s , a l l of these tissues were capable of o x i d i z i n g the f a t t y acids to C^o^.  The i n v i t r o uptake of f a t t y acids by the isolated  diaphragm was measured by Wertheimer and Ben-Tor (70), and the oxidation of octanoic acid by t h i s tissue was by Hansen (71).  According  demonstrated  to Neptune et al.(90,91),  isolated  diaphragms possess the a b i l i t y to u t i l i z e endogenous l i p i d s . When r a t diaphragms were incubated  f o r 4 hours i n a substrate-  3.  free medium, there was  a s l i g h t net decrease i n tissue free  f a t t y acids, t r i g l y c e r i d e s and e s p e c i a l l y i n t o t a l phospholipids,  F r i t z and co-workers (72) measured the uptake and  oxidation of a c e t a t e - l - C ^ , octanoate-l-C ^ 14 1  1-C  1  and  palmitate-  by the isolated diaphragm and s k e l e t a l muscle at r e s t  and at work.  Their data indicated that e l e c t r i c a l l y stimu-  lated s k e l e t a l muscles oxidized twice as much palmitate r e s t i n g muscles i n the presence or absence of added  than  glucose.  Freidberg et ai.(73) showed that a f t e r 5 minutes of moderate exercise, the plasma FFA concentration i n man approximately 0.82 about 1.2 mM  to 0.61  mM,  then increased sharply to  a f t e r cessation of a c t i v i t y .  tions were recorded  dropped from  Similar observa-  by Issekutz and M i l l e r (74) i n dogs, who  noted that the decrease i n plasma FFA by a 5-fold increase i n oxygen uptake.  l e v e l s was  accompanied  F r i t z (63)  has  suggested, therefore, that during moderate, sustained work, i f the oxygen supply i s adequate, s k e l e t a l muscles can o x i dize a considerable amount of l i p i d s f o r energy,  Indeed, 14  when Havel and h i s associates (21) infused palmitate-l-C intravenously into human subjects walking on a treadmill at 3-4 miles per hour, they found a rapid mobilization of f a t t y acids (presumably from adipose t i s s u e ) , and an increased rate of oxidation of p a l m i t a t e - l - C ^ . 1  The uptake and  oxidation  of l a b e l l e d long-chain f a t t y acids by s k e l e t a l muscle i n vivo was measured at r e s t and during e l e c t r i c a l stimulation by Spitzer and Gold (40).  Their data agreed with the concept  that FFA are oxidized by s k e l e t a l muscle at r e s t , and  that  4.  FFA oxidation increases during muscular a c t i v i t y .  Andres  et a l o ( 7 5 ) measured the differences i n arteriovenous concentrations of 0^, CO^,  glucose and l a c t a t e i n forearms of human  subjects at r e s t , and concluded that under basal conditions the oxidation of glucose could account f o r only 7% of the oxygen uptake, arm muscle was  Since the mean r e s p i r a t o r y quotient of fore0;80,  these investigators suggested that the  major non-carbohydrate material which served as f u e l under the conditions of t h e i r experiment was  lipid.  From the fore-  going, i t i s c l e a r that both diaphragm and s k e l e t a l muscles are capable of o x i d i z i n g f a t t y acids d i r e c t l y for energy. Recently, Masoro et ;al»(92) found that no decrease i n endogenous t r i g l y c e r i d e s or i n any of the muscle phospholipids had occurred i n monkey s k e l e t a l muscles which had been stimulated f o r 5 hours i n s i t u .  They concluded, therefore,  that s k e l e t a l muscles are capable of o x i d i z i n g only  exo-  genously supplied l i p i d s . The u t i l i z a t i o n of f a t t y acids for energy by the brain has been studied both _in vivo and jLn v i t r o .  According  to  Gordon and Cherkes (77) and Quastel and Wheatley (78), the brain does not u t i l i z e l i p i d s for energy.  On the other hand,  a number of investigators have indicated that the brain i s capable of o x i d i z i n g l i p i d s to a limited extent (68, 69, 80).  However, i n view of a number of e a r l i e r  on the R.Q.  79,  observations  of the brain (81), i t would appear that although  the brain possesses the enzymes f o r o x i d i z i n g l i p i d s ,  the  amount of energy derived from t h i s source i s i n s i g n i f i c a n t  5.  compared to that contributed by the oxidation of glucose. Other tissues which have been examined f o r t h e i r a b i l i t y to oxidize l i p i d s are adipose tissue (82) and l i v e r (66, 68, 69).  Isolated mitochondria from the l a t t e r tissue a c t i v e l y  oxidize l i p i d s , e s p e c i a l l y i n the presence of c a r n i t i n e (83, 84, 85). I t i s w e l l known that the heart functions l a r g e l y a e r o b i c a l l y and one might expect therefore that t h i s organ would be p a r t i c u l a r l y adapted f o r the oxidation of l i p i d s . Perhaps $wo  of the e a r l i e s t investigators to suggest that the  heart must u t i l i z e some substrate other than carbohydrates were Visscher and Mulder  (18) i n 1930.  Using the isolated  heart-lung preparation, these investigators discovered that even a f t e r 6 hours of work, the same amount of glycogen was found as i n the normal, unworked hearts.  Furthermore, they  suggested that since a l l the carbohydrates i n the heart-lung system could not account f o r the t o t a l energy requirements of the heart, some other non-carbohydrate  substrate must have  been u t i l i z e d during the 6 hours of work.  Eight years l a t e r ,  Visscher (19) presented quantitative evidence that the noncarbohydrate source may be f a t . Again using the heart-lung preparation, he showed that during 3 hours of cardiac work, the t o t a l f a t content of v e n t r i c u l a r muscle decreased from 3.71 i 0.76  to 3.18 "± 0.68  g per 100 wet weight of cardiac  tissue. In spite of Visscher*s investigations (18, 19) and Cruikshank«s review i n the 1930«s (94) on myocardial  6. metabolism, i n t e r e s t i n cardiac l i p i d metabolism appears to have subsided  f o r almost two decades, u n t i l Bing and h i s  associates (22, 86, 87) i n 1953 and 1954 reported on the i n vivo uptake of f a t t y acids by the myocardium i n human subjects.  They found that at blood f a t t y acid l e v e l s of 1.105-  0.286 mEq/100 ml, the extraction of f a t t y acids was 0.016 ±. 0.013 mEq/100 ml. Their 4aturn i s not highly impressive, but their reports on the i n vivo uptake of f a t t y acids by the human heart appear.: to have attracted the i n t e r e s t of numerous investigators to the area of cardiac l i p i d metabolism. measuring the transport  :  While  ; of plasma FFA, Gordon (88) also  noted the myocardial extraction of FFA jLn v i v o . dance with e a r l i e r observations,  In accor-  the uptake of FFA by the  myocardium i n vivo was also demonstrated by Ballard e t a l . (89).  Their data indicated that i n f a s t i n g dogs, free f a t t y  acids accounted for only 23% of the t o t a l f a t t y acids extracted by the heart, while the e s t e r i f i e d f a t t y acids made up the other 777..  Similar conclusions were reached by Scott and co-  workers (24) who measured the myocardial removal of FFA under normal and pathological conditions i n dogs.  I t i s perhaps  pertinent to mention here that when Bragdon and Gordon (93) injected C ^ - l a b e l l e d chylomicrons into fasted r a t s and 1  analyzed  the various tissues f o r r a d i o a c t i v i t y , the tissues  with the highest s p e c i f i c a c t i v i t y were the l i v e r and heart, which accounted f o r about 50% and 25% respectively of the t o t a l a c t i v i t i e s found.  More recently, Rothlin and Bing (23)  showed that o l e i c acid was extracted by the heart to a greater  7.  extent than any other long-chain f a t t y acids i n a r t e r i a l blood.  Similar r e s u l t s have been obtained with the isolated  perfused heart (98). I t must be emphasized that these i n vivo studies, though important, nevertheless gave only an i n d i c a t i o n that l i p i d s may have been u t i l i z e d by the heart, since only their uptake by the myocardium was measured.  The strongest evidence that  l i p i d s i n the form of FFA and t r i g l y c e r i d e s are not only taken up but also oxidized by the heart f o r energy has come from more recent studies u t i l i z i n g the isolated perfused heart.  Evans and h i s associates (27) used a closed perfusion  system to demonstrate that isolated r a t hearts converted about 90% of the p a l m i t a t e - l - C ^ i n the perfusion medium to C 0 l£i  i n  6 0  2  minutes.  That short-chain f a t t y acids (C ^1  labelled acetate, propionate, n-butyrate, n-octanoate) were also r e a d i l y oxidized d i r e c t l y by isolated dog hearts to 14 C >  O2 was  shown e a r i l i e r by Cavert and Johnson (95).  Opie  et'al»(26), Shipp (97) and Shipp and h i s associates (96) found that whereas palmitate-l-C"^ was  taken up and oxidized  by hearts obtained from both fed and fasted r a t s , glucose14 U-C  was oxidized only by hearts obtained from fed r a t s .  Furthermore, when both substrates were made a v a i l a b l e to the isolated hearts from either fed or fasted r a t s , palmitate-1was - p r e f e r e n t i a l l y taken up and oxidized over glucose-U-C^. Studies with free f a t t y acids as substrates f o r the myocardium demonstrated unequivocally their importance as an energy source.  However, the question as to whether c i r c u -  8.  l a t i n g t r i g l y c e r i d e s (which a c t u a l l y represent the majority of the t o t a l c i r c u l a t i n g f a t t y acids i n vivo) are taken up and oxidized by the myocardium remained unanswered u n t i l recent years.  The r e l a t i v e importance of e s t e r i f i e d f a t t y  acids i n supplying the energy demands of the heart suggested e a r l i e r by Ballard et al»(89), who  was  found that 777.  of the t o t a l f a t t y acids extracted by the heart i n vivo in the e s t e r i f i e d form.  was  The uptake and oxidation of t r i -  glycerides by the isolated perfused r a t heart were studied by Gousios and co-workers (33), 307. of the t o t a l d < 1.006  These investigators found that  lipoproteins (labelled with  C ^1  t r i p a l m i t i n ) i n the perfusion medium was extracted by hearts from starved rabbits and that 107. of the extracted cerides was  oxidized to C^02.  Hearts obtained  rabbits extracted 157. of the available C  trigly-  from fed  - t r i p a l m i t i n and 14 oxidized 87. of the extracted t r i g l y c e r i d e to C Olivecrona (31) and Olivecrona and Belfrage (32) injected C ^1 Z h  1  3  -  glycerol-H -palmitate-labelled chylomicrons intravenously into r a t s and examined the d i s t r i b u t i o n of the labels i n a number of tissues, including l i v e r , heart and adipose t i s s u e s . They concluded from t h e i r studies that the heart (and adipose tissue) took up chylomicron  t r i g l y c e r i d e s i n t a c t , and  that  extensive and rapid hydrolysis of t h i s glyceride occurred probably near the plasma membrane. of d < 1.006 heart was who  The uptake and oxidation  l i p o p r o t e i n t r i g l y c e r i d e s by the isolated r a t  also demonstrated by Delcher and associates  found that about 407.-907. of the t o t a l C0  2  was  (34)  derived  9 .  from the exogenously  supplied t r i g l y c e r i d e s , and thus con-  cluded that l i p o p r o t e i n - t r i g l y c e r i d e f a t t y acids were the primary"source of f a t t y acids f o r the heart. I t should be mentioned b r i e f l y that other substrates have also been shown to be r a p i d l y metabolized by the heart. Williamson and Krebs (29), Williamson (30) and H a l l  (99)  demonstrated that acetoacetate, acetate and ^-hydroxybutyrate were, as might be expected, oxidized r a p i d l y by the perfused r a t heart i n preference to glucose. In summary, the evidence presented by numerous i n v e s t i gators demonstrates c l e a r l y that a v a r i e t y of mammalian tissues are capable of o x i d i z i n g l i p i d s f o r energy.  The heart, i n  p a r t i c u l a r , appears to depend p r i m a r i l y upon l i p i d s as i t s source of energy.  The importance  of FFA i n myocardial  metabolism has been reviewed recently by Evans (28) and by Bing (100).  The comparative  aspect  of muscle metabolism,  with s p e c i a l emphasis on the importance of l i p i d metabolism i n insects, birds and f i s h e s has been reviewed by Drummond and Black (76).  ,  The i n t r a c e l l u l a r fate of FFA»s which are transported to the myocardium (either complexed with serum albumin or as chylomicron t r i g l y c e r i d e s ) has been examined, and the e v i dence indicates that not a l l of the FFA s are d i r e c t l y f  oxidized by the myocardium;  a portion  to t r i g l y c e r i d e s and phospholipids.  i s re-esterified  For example, Shipp  found that when r a t hearts were perfused with 0.5 mM tate-1-C  14  f  o  r  (97)  palrai-  30 minutes, 4.69 ± 0.16 umoles of palmitate  were taken up per gram weight of tissue, 2.34 - 0.24 recovered as C tissue l i p i d s .  14  umoles  . 0^, and 1.68 - 0.06 umoles recovered as  In hearts perfused for 1 hour with 0.5  p a l m i t a t e - l - C , 65.4 ± 2.3% of the C 14  1 4  mM  i n tissue l i p i d s  was  recovered as t r i g l y c e r i d e , 14.1 ± 1.1% as phospholipids, 6,6 i 0.5% as FFA, and 13.9-  1.7% as c h o l e s t e r o l .  The  : -  synthesis of t r i g l y c e r i d e s i n r a t hearts 'jn vivo from i n j e c ted palmitate-l-C''" was 4  Olivecrona (101).  shown e a r l i e r by Borgstrom and  According to Stein and Stein (103), the  isolated perfused r a t heart incorporated  \ palmitate-l-C" " 1  into tissue l i p i d s , the t r i g l y c e r i d e s accounting f o r 70-75%, and the phospholipids, 25-30%.,of the l a b e l incorporated. Q u a l i t a t i v e l y , similar observations have been recorded by Shipp et al*(35) and by Olson (102).  Hence i t i s reasonable  to assume that FFA*s which are taken up i n excess of the immediate energy requirements are r e - e s t e r i f i e d and stored in the form of phospholipids or t r i g l y c e r i d e s .  Indeed, elec-  tron micrographs of cardiac muscle c e l l s often show abundant amounts of l i p i d droplets often adhering to the mitochondria. The u t i l i z a t i o n of these endogenous l i p i d s f o r energy by the myocardium was  investigated by Shipp and h i s asso-  c i a t e s (35, 36, 104) and also by Denton and Randle (37). E s s e n t i a l l y , the observations made by Shipp et al»were that when glycogen-depleted r a t hearts (whose i n t r a c e l l u l a r l i p i d s had been pre-labelled with C^" _in vivo) were perfused i n a closed system with substrate-free buffer, the production of C 0 o gave d i r e c t evidence that endogenous l i p i d s were l4  4  11.  oxidized.  Furthermore, they stated that the net decrease i n  endogenous C ^ - l a b e l l e d phospholipid content alone could account f o r oyer 757. of the t o t a l metabolic C0 these conditions.  2  formed under  However, i n d i r e c t contrast to these  observations, Denton and Randle (37) showed that a f t e r 60 minutes of substrate-free perfusion, the t r i g l y c e r i d e content f e l l from 18.7 - 0.8  to 8.7 ± 0.7  umoles per gram dry weight  of tissue, and there was no change i n endogenous phosphol i p i d levels.  These contradictory observations  present much  d i f f i c u l t y i n assessing the exact r o l e of endogenous l i p i d s as p o t e n t i a l f u e l f o r the working heart muscle, and  therefore  this p a r t i c u l a r aspect of cardiac metabolism must s t i l l considered  be  open for further i n v e s t i g a t i o n .  The r o l e of endogenous l i p i d s i n supplying the energy requirements of the heart may  be uncertain, but as mentioned  e a r l i e r , the importance of exogenously supplied chylomicron t r i g l y c e r i d e s i n t h i s respect cannot be over-emphasized. Complete agreement e x i s t s among researchers  (31, 32, 33,  34)  that chylomicron t r i g l y c e r i d e s are taken up i n t a c t by the myocardium and subsequently hydrolyzed These observations  immediately suggest that l i p o l y t i c enzymes  must e x i s t i n heart c e l l s to hydrolyze and g l y c e r o l .  r a p i d l y to FFA.*s.  t r i g l y c e r i d e s to FFA  Indeed, an active enzyme, l i p o p r o t e i n l i p a s e ,  was characterized i n cardiac tissue i n 1955 This enzyme was  also detected  by Korn (38,  i n l i v e r , kidney, spleen, aorta,  lung, s k e l e t a l muscle and adipose tissue, by Korn (38, as w e l l as i n post-heparin  39).  47),  plasma (42) and diaphragm (50).  12.  Lipoprotein lipase has been extensively studied by numerous investigators, and since i t has been the subject of a f a i r l y recent review (41), only the s a l i e n t features of t h i s enzyme w i l l be presented.  Lipoprotein lipase has been reported p u r i -  f i e d from post-heparin plasma 1480-fold by H o l l e t t and Meng (42) using i s o e l e c t r i c p r e c i p i t a t i o n and ammonium sulfate f r a c t i o n a t i o n procedures.  The p u r i f i e d preparation was  optimally active at pH 8.5 and i t s a c t i v i t y destroyed heating f o r 5 minutes at 50°.  by  The natural substrate f o r l i p o -  protein l i p a s e appears to be chylomicrons, which are composed of about 90% t r i g l y c e r i d e s , some phospholipids, c h o l e s t e r o l esters and about 2% protein.  Coconut o i l emulsions and other  a r t i f i c i a l t r i g l y c e r i d e preparations are attacked at only a slow rate by the enzyme, unless small quantities of serum are present (39).  A unique property of l i p o p r o t e i n lipase  i s i t s sudden appearance i n the c i r c u l a t i o n after heparin administration, as f i r s t observed  by Hahn (105).  Indeed, the  enzyme i s eluted within minutes from adipose tissue and heart when these organs are perfused with buffer containing heparin and serum (43, 44, 45, 46).  The pH optimum for t h i s enzyme  i s near 8.5 and i t s a c t i v i t y i s inhibited 100% by 0.2  to 1.0 M  NaCl (38, 43, 45, 48) and 30-60% by protamine sulfate, 20 mg/ml (43, 48). 0.2 M NaF  The enzyme i s very s l i g h t l y (0-7%) inhibited by (43, 48, 49).  Hollenberg (50) and A l o u s i and Mallov  (17) have noted a 2-3 f o l d increase i n lipoprotein l i p a s e a c t i v i t y i n hearts obtained from 3-4 day fasted r a t s .  A simi-  l a r increase i n enzyme a c t i v i t y was observed by N i k k i l a est a l .  13.  (51, 52) i n the myocardium of rats which had been to moderate exercise f o r 90 minutes.  subjected  The mode of action of  lipoprotein lipase has been investigated by Borgstrom and Carlson  (53), Carlson and Wadstrom (54) and most recently,  Payza et al.(106).  Carlson and Wadstrom»s data (54)  by  indicates  c l e a r l y that chylomicron t r i g l y c e r i d e s are hydrolyzed r a p i d l y to monoglycerides, but the hydrolysis of the l a t t e r glyceride occurred very slowly.  This was  i l l u s t r a t e d by the rapid  (150-fold) increase i n monoglyceride content within the  first  5 minutes of incubation, accompanied by decreases i n t r i glyceride and diglyceride l e v e l s .  Payza and co-workers  (106)  have s i m i l a r l y shown that monoglycerides accumulated when an a r t i f i c i a l coconut emulsion (Ediol^) was  used as substratei  I t i s reasonable to conclude, therefore, that monoglycerides are not hydrolyzed to any extent by l i p o p r o t e i n l i p a s e , and that i t s action i s s p e c i f i c f o r t r i g l y c e r i d e s , and may extend to d i g l y c e r i d e s .  even  The physiological importance of  l i p o p r o t e i n lipase i n cardiac energy metabolism cannot be underestimated. In adipose tissue, a lipase possessing properties considerably d i f f e r e n t from that of l i p o p r o t e i n lipase shown to: e x i s t by Rizack (107). he showed that the enzyme was 7% by 8 x 1 0 " %  observed near pH 6.5  R  as  substrate,  inhibited 16% by 0.6 M NaCl,  EDTA, 66% by 0.2 M NaF,  protamine sulfate, 300 jug/ml.  lipase.  Using E d i o l  was  but not inhibited by  The optimum a c t i v i t y  as compared with pH 8.5  was  for l i p o p r o t e i n  The most i n t e r e s t i n g feature of t h i s lipase  was  14.  that i t could be re-activated when incubated with and  epinephrine  tissue sediment, suggesting an important means o f c o n t r o l -  l i n g free f a t t y acid release from adipose t i s s u e .  I t was  l a t e r reported by Rizack (108) that the enzyme could be a c t i vated  i n v i t r o by 2 x 1 0 " % c y c l i c 3* ,5*-PiSGP.  Much i n t e r e s t  i s c u r r e n t l y being directed toward the p o s s i b i l i t y that t h i s enzyme i s under hormonal c o n t r o l and therefore regulates the output of free f a t t y acids from adipose t i s s u e . Recently,  Bjorntorp and Furman (13) reported that a  l i p o l y t i c a c t i v i t y , s i m i l a r to that observed by Rizack (107) i n adipose tissue, existed i n r a t hearts.  Using E d i o l  R  as  substrate and crude extracts as the enzyme source, these investigators indicated that the l i p o l y t i c a c t i v i t y was o p t i mal near pH 6.8, s l i g h t l y i n h i b i t e d (8%) by 0.5 M NaCl but strongly inhibited (68-100%) by 0.2 M NaF. bited by protamine s u l f a t e , 400 ug/ral.  I t was not i n h i -  Furthermore, they  reported that when heart tissue from fasted r a t s bated i n the presence of epinephrine,  was  incu-  1 ug/ml, the a c t i v i t y  increased from 12.70 ± 1.30 to 13.60 i 1.60 (uraoles FFA released/g tissue/hour).  They concluded that i n addition to  l i p o p r o t e i n l i p a s e , another l i p o l y t i c component existed i n r a t cardiac tissue, whose function was perhaps analogous to that of the lipase found i n r a t adipose tissue by Rizack (107). Evidence supporting  the concept that l i p i d s play a major  r o l e i n supplying the energy demands of the mammalian heart has been presented.  I t may be concluded that the heart i n  vivo derives part of i t s energy from the d i r e c t oxidation of  15.  albumin-bound free f a t t y acids which are taken up from the arterial circulation.  However, the f u e l for muscle metabolism  i s to a much greater extent derived from exogenously supplied chylomicron t r i g l y c e r i d e s and very low density l i p o p r o t e i n s . I t follows, therefore, that cardiac lipases must play a v i t a l r o l e i n providing a source of oxidizable f a t t y acids for the myocardium.  The question as to whether endogenously stored  cardiac l i p i d s are r e a d i l y mobilized and u t i l i z e d f o r energy has not been unequivocally answered. cribed i n t h i s thesis was  The work to be des-  undertaken to further study the  nature of l i p o l y t i c a c t i v i t i e s i n heart muscle.  Special  attention has been directed toward l i p o l y t i c a c t i v i t y other than l i p o p r o t e i n l i p a s e a c t i v i t y . interested by the suggestion  We have been p a r t i c u l a r l y  that a cardiac l i p a s e may e x i s t  which i s activated by epinephrine.  An investigation to pro-  vide additional i n s i g h t into the possible u t i l i z a t i o n of endogenous t r i g l y c e r i d e s by isolated perfused also reported.  r a t hearts i s  16.  EXPERIMENTAL PROCEDURE Materials Glycogen was obtained from N u t r i t i o n a l Biochemical Company.  Diazyme , an amyloglucosidase preparation, was •a  purchased from Miles Chemical Company.  Glucostat^ reagent,  which contains glucose oxidase and horseradish peroxidase, was purchased  from Worthington  Biochemical Corporation.  R  E d i o l , which was  generously provided by Dr. Martin Rizack  of the Rockefeller I n s t i t u t e , New York, contains coconut o i l 507., sucrose 12.5%, g l y c e r y l monostearate 1.5%, oxyethylene sorbitan monostearate Bovine serum albumin  and poly-  2.0%.  (Fraction V) was purchased  from  Sigma Chemical Company and p u r i f i e d before use by the method of Goodman (1) as follows:  50 g of the crude albumin  was  dissolved i n 200 ml of g l a s s - d i s t i l l e d water by simply placing the albumin powder over the water and allowing i t to dissolve overnight. philized;  The resultant dark amber solution was  then l y o -  the residue was powdered with mortar and pestle,  covered with anhydrous 2,2,4-trimethylpentane  containing 5%  acetic acid, and f i n a l l y placed i n the cold room overnight. As much as possible of the acetic acid-trimethylpentane extraction solvent was  then aspirated, and the albumin washed  twice with anhydrous trimethylpentane.  A g i t a t i o n of the  albumin suspension during the extraction process with organic solvents was kept to a minimum to reduce the extent of protein denaturation.  A f t e r aspiration of the trimethylpentane, the  albumin was again covered with the anhydrous 57. acetic a c i d trimethylpentane mixture, and stored i n the cold room overnight.  The removal of the acetic acid-trimethylpentane  mixture and washing with anhydrous trimethylpentane was repeated. the  The organic solvent was removed under vacuum, and  powder obtained was taken up i n a suitable volume of  g l a s s - d i s t i l l e d water.  To remove the l a s t trace of acetic  acid, the albumin solution was dialyzed by continuous flow for  3 days against a t o t a l volume of 60 l i t e r s of demineral-  ized water, followed by 20 l i t e r s of g l a s s - d i s t i l l e d water. The solution was then l y o p h i l i z e d and the extracted albumin stored i n the deepfreeze u n t i l required.  Commercial albumin  (Fraction V) contains about 0.60 eq FFA/mole.  A f t e r extrac-  t i o n by the method of Goodman (1) j u s t described, the content of FFA i s reduced to about 0.14-0.18 eq/mole. Monoolein (Calbiochem, "907. pure") was made free of trace t r i g l y c e r i d e contaminant by adsorption on 80-200 mesh s i l i c i c acid, followed by e l u t i o n with chloroform:methanol, 2:1.  T r i p a l m i t i n was obtained from Eastman Organic chemicals  and p u r i f i e d ( > 997.) by s i l i c i c acid chromatography.  Tri-  palmitin-1-G ^ (967. pure) was purchased from Nuclear-Chicago 1  Corporation.  Commercial monostearin was obtained from the  Faculty of Pharmacy, U. B. C , from hot ethanol before use.  and r e - c r y s t a l l i z e d twice S i l i c a gel GF (Merck) was  secured from Canadian Laboratories.  18.  Methods I.  Perfusion Studies Normal fed and 3-day fasted female Wistar rats weighing  between 275 and 325 grams were used.  The animals were stunned  by a blow on the head, their hearts removed immediately and attached to a cannula of a Langendorf perfusion apparatus. The apex of the hearts was secured  to a Stratham Force D i s -  placement Transducer, a 5-gram tension applied, and the rate and strength of contractions recorded on a Grass Model 5D polygraph..  The flow rate was adjusted as required for maxi-  mal e f f i c i e n c y of the heart, usually between 5 and 8 mis per minute.  The perfusion medium was carbogenated Krebs-Ringer  bicarbonate  solution a t pH 7.4, 37°.  When epinephrine was  added to the perfusion f l u i d , i t was injected with a Lambda Pump Driver at the rate of 0.2 to 1.0 jug per minute.  When  heparin was used, i t was injected at the rate of 60 ug per minute. At the end of the perfusion period, the hearts were removed from the apparatus and the a u r i c l e s cut away and d i s carded.  The v e n t r i c l e s were c a r e f u l l y blotted to remove  excess water and divided i n two i n such a way as to provide approximately equal parts of the l e f t and r i g h t v e n t r i c u l a r tissues for subsequent glycogen and t r i g l y c e r i d e  analyses.  Samples thus obtained were immersed i n l i q u i d nitrogen within one minute following termination of perfusion, then assayed on the following day. Tissue Glycogen —  Tissue glycogen was assayed enzymatically  19.  according to Johnson et jal (2). A sample, of v e n t r i c u l a r tissue; weighing between 150-250 mgwas placed i n a graduated 12-ml centrifuge tube: containing 1.0 ml of 30% KOH.  The  tuber was t placed i n b o i l i n g water f o r 20 minutes, the contents cooled, and 1.25 ml 95% ethanol was added to p r e c i p i tate the glycogen.  The contents of the tube were mixed  thoroughly with a glass rod.  The tube was  chilled i n ice  for 15 minutes, then the contents heated to a b o i l i n a water bath.  The p r e c i p i t a t e was collected by centrifugation  for 15 minutes, using a bench top Model H International centrifuge.  The supernatant f l u i d was decanted and the pre-  c i p i t a t e dissolved i n 1.0 ml g l a s s - d i s t i l l e d water. 1.25 ml  Then  957. ethanol was added to r e - p r e c i p i t a t e the glycogen,  and the tube  c h i l l e d and centrifuged as before.  was taken up i n 2.0 ml  The  sediment  of g l a s s - d i s t i l l e d water, and usually  an 0.2 ml aliquot was taken f o r glycogen determination. The amyloglucosidase solution used f o r the glycogen assay was prepared by mixing 200 mg Diazyme  with 100 ml  0.1 M potassium phosphate buffer, pH 6, and f i l t e r i n g .  The  enzyme solution was stored at 4°, and discarded a f t e r one week.  The glucose oxidase-horseradish peroxidase reaction  mixture (Glucostat^ x 4) was prepared by f i r s t d i s s o l v i n g the contents of the smaller (chromagen) v i a l i n 4.0 ml methanol.  The contents of the larger (enzyme) v i a l ware then  dissolved i n about 380 ml  of buffered g l y c e r o l (4 volumes  g l y c e r o l plus 6 volumes 0.04 M potassium phosphate  buffer,  pH 7 ) , the chromagen solution added, and made up to 400 ml  with buffered g l y c e r o l .  This preparation was stored i n the  deepfreeze i n small i n d i v i d u a l quantities, and was stable to repeated freezing and  thawing.  The incubation mixture for glycogen determination consisted of the following:  0.2 ml aliquot of the glycogen  solution, 1.0 ml of G l u c o s t a t reagent, 1.0 ml of Diazyme R  solution, and 0.8 ml g l a s s - d i s t i l l e d water.  R  The mixture was  incubated f o r 1 hour at 37° and the reaction stopped by the addition of 0.5 ml 2 N HC1.  The o p t i c a l density was read at  400 mu i n a Beckman DU spectrophotometer, using a l i g h t path of 1.0 cm.  The standard curve for glycogen i n the range,  5-100 ug i s i l l u s t r a t e d i n F i g . 1.  Recovery  experiments  indicated 90-95% recovery of added glycogen. Tissue T r i g l y c e r i d e s  Tissue t r i g l y c e r i d e s were extracted  by the method of Folch e_t a l (7). Ventricular tissues weighing between 150 and 250 mg were minced and homogenized f o r at least 7 minutes i n a glass mortar (with a loose f i t t i n g Teflon motor-driven pestle) with 18 volumes of chloroformmethanol mixture (2:1).  The f l a k y suspension was  filtered  through paper into a 12-ml centrifuge tube, using 2 volumes of the chloroform-methanol mixture as a f i n a l r i n s e .  Four  volumes of g l a s s - d i s t i l l e d water was then added, the tube shaken by hand, and centrifuged.  The upper phase was care-  f u l l y removed with a pipette, and a 2.0 ml aliquot of the lower phase transferred to a screw-capped  tube and evaporated  to dryness under a gentle stream of nitrogen. The r e s i d u a l l i p i d was assayed f o r t r i g l y c e r i d e s by the o r i g i n a l  21.  FIG. 1  Glycogen concentration versus o p t i c a l density*  The assay of glycogen was performed with the coupled amyloglucosidase-glucose oxidase technique of Johnson e_t a l . (2) as described i n the text, except that the quantities of glycogen were v a r i e d .  22.  method of Van Handel and Zilversmit (3) as modified by Jagannathan (4), except that s i l i c i c acid and diisopropyl ether were substituted f o r z e o l i t e and chloroform, r e s p e c t i v e l y . To the l i p i d residue was added 1.2 g activated s i l i c i c acid followed  by  :  7.5 ml diisopropyl ether, and the contents  shaken on a mechanical shaker f o r 30 minutes.  After centri-  fugation at low speed, a 4.0 ml aliquot was transferred to another screw-capped  tube and the organic solvent evaporated  to dryness under a gentle stream of nitrogen.  The rate of  evaporation was iiincjiieasel by immersing the tube i n a water bath at 60°.  To t h i s residue was added 1 drop of 2.5%  KOH  and 1.0 ml of aldehyde-free ethanol, and the t r i g l y c e r i d e s hydrolyzed to g l y c e r o l and f a t t y acid s a l t s by heating f o r 30 minutes at 60°.  Two drops of 6% acetic acid were then  added, and the contents evaporated to dryness i n an oven at 55° using a gentle stream of a i r to hasten the process. the  To  dry contents were added 10.0 ml petroleum ether (b.p, 35-  60°) and 2.0 ml 0.7 N H S0^. 2  and inverted 25 times.  The tube was then capped  tightly  The petroleum ether layer was removed  by aspiration and discarded. To remove the l a s t traces of the  organic solvent, the tube was heated i n an oven f o r 15  minutes at 60° under a gentle stream of a i r .  Three drops of  25 mM sodium metaperiodate solution were then added, and the contents of the tube mixed thoroughly. A f t e r 10 minutes, 0.2 ml of f r e s h l y prepared sodium b i s u l f i t e (10% w/v) t i o n was added and the contents mixed thoroughly.  solu-  Ten ml of  chromotropic acid solution were f i n a l l y added, the tubes  shaken and heated i n a b o i l i n g water bath f o r 30 minutes. A f t e r cooling to room temperature, 1.0 ml of 5% thiourea solution was added, the contents mixed and the i n t e n s i t y of the  colour was read at 570 raji i n Beckman DU spectrophoto-  meter using a l i g h t path of 1.0 cm. Aldehyde-free ethanol was prepared by heating 1000 mis ethanol under r e f l u x f o r 60 minutes with 20 g zinc dust and 20 g KOH.  The ethanol was then d i s t i l l e d , discarding the  head and t a i l f r a c t i o n s .  Chromotropic acid reagent was  prepared i n subdued l i g h t by f i r s t d i s s o l v i n g 2.24 g chromotropic acid i n 200 mis H^O, then adding t h i s solution to 900 mis of s u l f u r i c acid solution (300 mis H^O plus 600 mis concentrated H^SO^).  The reagent was stored i n the dark and  prepared fresh every two weeks. The standard curve f o r t r i p a l m i t i n i s shown i n F i g .  2.  Recovery experiments c a r r i e d through from the s i l i c i c acid extraction step indicated recoveries i n the range 95-98%. Both glycogen and t r i g l y c e r i d e tissue l e v e l s are expressed as mg/g dry weight of v e n t r i c u l a r tissue. the  This i s based on  observation that the dry/wet weight r a t i o s were 25.4%  and 20.5% f o r non-perfused hearts, and hearts perfused over 5 minutes, respectively. II.  Cardiac Lipase Studies Lipase Assay -- Standard l i p a s e assays were performed i n  screw-capped  tubes at 37° on a Dubnoff metabolic shaker.  Incubation time was 30 minutes.  The incubation mixture con-  tained 60 mM potassium phosphate buffer, pH 6.8, 20 mg  24.  E  0  100  200  300  400  500  600  TRIPALMITIN Ipv)  FIG.  2  T r i p a l m i t i n concentration  versus o p t i c a l density.  The assay was performed as described i n the text except that the i n i t i a l s i l i c i c acid extraction step was omitted. Recovery experiments indicated 95-98% recovery of added t r i palmitin . when c a r r i e d through the s i l i c i c acid extraction step.  25.  p u r i f i e d bovine serum albumin, enzyme preparation, the approp r i a t e substrate, and g l a s s - d i s t i l l e d water to make a f i n a l volume of 1.0 ml.  When E d i o l  served as substrate i n the  standard assay, 0.1 ml of a 1:19 d i l u t i o n was used. The amount of monoolein as substrate was either 5 or 15 ueqs per reaction mixture.  T r i p a l m i t i n was used at a concentration  of 2.5 mg per ml of reaction mixture.  L i p o l y t i c a c t i v i t y was  measured by the method of Duncombe (5), as modified by Vaughan et al<>(6) as follows:  The reaction was stopped by the addi-  t i o n of 1.0 ml of a mixture containing 0.9 M triethanolamine, 0.1 N acetic acid, and 5% cupric nitratetSH^O.  The purpose  of this treatment i s to convert the FFA formed to the chloroform-soluble copper soaps.  Chloroform,  6.0 ml, was added  and the tubes shaken on a mechanical shaker f o r 15 minutes. A f t e r b r i e f centrifugation, the aqueous copper solution and the denatured protein were removed by suction.  An aliquot  (0.2-2.0 ml) of the chloroform layer was removed, made up to a f i n a l volume of 2.0 ml with chloroform, and 0.25 ml of f r e s h l y prepared  0.1% diethyldithiocarbamate (prepared i n n-  butanol) was added.  The i n t e n s i t y of the colour was read  i n a Beckman DU spectrophotometer a t 440 mu using a l i g h t path of 1.0 cm. shown i n F i g . 3.  The standard curve f o r palmitic acid i s In the author*s opinion, t h i s method i s  very much superior for long chain f a t t y acid determination Lto. : the m i c r o t i t r i m e t r i c method of Dole (117) which has been used for many years. The conditions f o r the assay u t i l i z i n g t r i p a l m i t i n - 1 - C ^  4  26.  e  u O*  •40  a.  E  O  o  •30  >-  CO Z •20 Ul o -J  <  o  •10  o. o  0  100 PALMITIC  FIG. 3 density.  200  300  ACID  (mjie'q)  Palmitic acid concentration  versus o p t i c a l  To 1.0 ml of mixture containing 20 mg albumin and 60 mM phosphate buffer, pH 6.8, was added 6.0 ml chloroform cont a i n i n g the indicated amounts o f palmitic a c i d . Extraction and subsequent assay were performed as described i n the text.  \  \  27.  were e s s e n t i a l l y i d e n t i c a l to the standard assay which contained E d i o l  R  as substrate, except that the volume of E d i o l  R  (1:19) used was 0.05 ml instead of 0.1 ml. One mp u n i t of enzyme a c t i v i t y i s defined as that amount which produced 1.0 mjj equivalent  FFA/60 min at 37°.  Specific  a c t i v i t y i s defined as the number of mueqs FFA produced/mg protein i n 60 minutes at 37°. Protein was determined by the biuret method (14) and o p t i c a l l y by the method of Warburg and C h r i s t i a n (61). Preparation of Substrates —  Monoolein suspension was prepared  by heating 570 mg monoolein i n 32 ml 0.25 M sucrose containing 5% acacia (pH 7) at 70°. The mixture was homogenized i n a S e r v a l l Omnimixer at maximum v e l o c i t y f o r 30 seconds at 70°, then slowly cooled with the omnimixer operating at a lower velocity.  This procedure gave a s a t i s f a c t o r y  suspension  which remained stable f o r a considerable length of time. S i m i l a r l y , very stable suspensions of t r i p a l m i t i n (25 mg/ml)... and monostearin (50 Lieq/ml) were prepared at pH 7.0. The substrate preparations were stored at room temperature. Ediol  R  l a b e l l e d with t r i p a l m i t i n - l - C ^ " was prepared by 4  c a r e f u l l y evaporating a suitable a l i q u o t of toluene containing about 5 u c u r i e s a c t i v i t y , and mixing the residue with 2.5 ml of a 1:19 d i l u t i o n of E d i o l minutes on a mechanical shaker.  R  at 60° f o r at least 60  Shaking was continued  while  the mixture was cooled to room temperature. Measurement of Hydrolysis of T r i p a l m i t i n - l - C ^ — Hydrolysis 1  of t r i p a l m i t i n - l - C  1 4  was followed by removing an a l i q u o t of  28.  the chloroform layer obtained during the standard FFA assay, and evaporating i t to dryness i n a centrifuge tube.  The  residue was quantitatively taken up i n small volumes of chloroform and spotted on a thin layer chromatograph plate, using s i l i c a g e l GF (Merck) as the adsorbent.  The plates  were developed with f r e s h l y d i s t i l l e d chloroform, then exposed to iodine vapour to allow detection of the FFA and glycerides. The t r i g l y c e r i d e spots were consistently and c l e a r l y defined, moving j u s t behind the solvent f r o n t .  The lower glycerides  and the f a t t y acids did not separate consistently nor comp l e t e l y to allow an i n d i v i d u a l quantitative analysis of these components.  Therefore these products of t r i g l y c e r i d e hydro-  l y s i s were scraped o f f together and counted as one component. F i f t e e n mis o f L i q u i f l u o r (Nuclear-Chicago) containing 0.4% PPO  (2,5-diphenyloxazole) and 0.05% POPOP (p-bis [2-(5-  phenyloxazolyl)J -benzene) i n toluene was added to the counting v i a l s , the contents thoroughly swirled and the r a d i o a c t i v i t y counted counter.  i n a Nuclear-Chicago  scintillation  Correction for quenching was made for each v i a l by  the channels r a t i o method. Preparation of Enzyme Extract —  A l l v e n t r i c u l a r tissues used  for the preparation of extracts were obtained from fed, female Wistar rats weighing between 200 and 300 grams.  Unless other-  wise indicated, a l l hearts were perfused for 5 minutes with Ringer-Tyrode  solution, pH 7.4, containing heparin, 20 ug per  ml, before homogenization.  This procedure e f f e c t i v e l y removes  blood from the tissues and also elutes  : a considerable  amount of l i p o p r o t e i n lipase from the heart. Robinson and Jennings  According to  (46), about 50% of the l i p o p r o t e i n  l i p a s e a c t i v i t y of the heart i s eluted i n 60 minutes, and the rate of enzyme release i s highest during the f i r s t  few  minutes. Ventricular tissues pooled from 10 to 15 r a t hearts were homogenized for three 1-minute periods at 0° i n 5 v o l umes of 0.25 M sucrose containing 0.05 M T r i s , pH 7, using a S e r v a l l Omnimixer.  The homogenate was centrifuged at  105,000 x g for 60 minutes, and the sediment thus obtained was re-homogenized i n 507. of the o r i g i n a l volume of 0.25 sucrose solution, pH 7, containing 0.17. Triton X-100. homogenate was  M The  again centrifuged at 105,000 x g for 60  minutes at 0° and the supernatant f l u i d s combined. sodium acetate per ml were added to the combined  Four mg  supernatant  f r a c t i o n s and the pH was adjusted to 5.9 with the dropwise addition of 1.0 N acetic a c i d .  Following e q u i l i b r a t i o n i n  an ice bath f o r 20 minutes, the p r e c i p i t a t e was c o l l e c t e d by centrifuging f o r 60 minutes at 37,000 x g at 0° and discarded. The supernatant f l u i d was taken to  pH  5.2 with 1.0 N acetic  acid and equilibrated i n an i c e bath f o r 30 minutes before centrifuging at 37,000 x g f o r 60 minutes at 0°. p i t a t e was  The p r e c i -  taken up i n 0.25 M sucrose, pH 7.  The a c t i v i t y of the pH 5.2-5.9 f r a c t i o n was quite unstable to freezing and thawing, about 50% of the a c t i v i t y being l o s t a f t e r overnight storage at -20°.  Therefore much  of the work described l a t e r i n the text was performed with f r e s h l y prepared pH 5.2-5.9 enzyme extracts.  30.  RESULTS I  Perfusion Studies I t i s common knowledge that the isolated mammalian heart  continues to function for hours when perfused with "physiol o g i c a l " solutions (e.g. Tyrode's) which contain glucose as energy source.  This extremely useful technique has been  employed by numerous investigators to study the various biochemical aspects of cardiac metabolism, and has recently been used widely i n studies on l i p i d metabolism by the heart. f i r s t studies were designed fused without  Our  to determine whether hearts per-  substrate were capable of u t i l i z i n g endogenous  t r i g l y c e r i d e s as energy source. During the course of t h i s work, Shipp et al»(35) reported that phospholipids were u t i l i z e d to a much greater extent than were t r i g l y c e r i d e s by the isolated perfused r a t heart perfused without  substrate.  This seemed rather u n l i k e l y , considering  the generally accepted view that phospholipids play p r i m a r i l y a s t r u c t u r a l r o l e i n mammalian tissues.  Furthermore, Denton  and Randle (37) have reported recently that no decrease i n phospholipid levels occurred during substrate-free perfusions, whereas tissue t r i g l y c e r i d e contents decreased.  In the  present studies, phospholipid levels were not measured. Instead an attempt was made to compare the r e l a t i v e rates of u t i l i z a t i o n of endogenous t r i g l y c e r i d e s with that of glycogen under varying experimental conditions. Fasting has been shown to increase the glycogen  content  31.  of r a t hearts (8) and the t r i g l y c e r i d e content of guinea p i g hearts (118)•  Fasting also increases l i p o p r o t e i n l i p a s e  a c t i v i t y i n r a t hearts (50).  We have included studies on  hearts from animals fasted f o r 3 days before s a c r i f i c e . purpose of f a s t i n g the animals was  two-fold:  The  F i r s t , to pro-  vide a larger store of endogenous substrates so that the hearts could be perfused f o r longer periods of time, arid second, to examine the p o s s i b i l i t y that increased cardiac lipase a c t i v i t y due to f a s t i n g might increase the rate of tissue t r i g l y c e r i d e breakdown and u t i l i z a t i o n during perfusion. The e f f e c t s of epinephrine and heparin upon the u t i l i zation of endogenous substrates were also investigated. The glycogenolytic action of epinephrine i s well known. However, the p o s s i b i l i t y that epinephrine might also cause an increase i n l i p o l y t i c a c t i v i t y i n cardiac tissue, as i t does i n adipose tissue seemed most a t t r a c t i v e , and was used i n the perfusion studies to test t h i s p o s s i b i l i t y .  The release of  l i p o p r o t e i n lipase a c t i v i t y from cardiac tissue s l i c e s and from isolated heart by heparin has been demonstrated (46). This suggested,  then, that the perfusion of r a t hearts with  heparin might cause a reduction i n tissue lipase content, r e s u l t i n g i n a decreased rate of disappearance  of endogenous  triglycerides. I n i t i a l v e n t r i c u l a r l e v e l s of glycogen i n the hearts of fed r a t s were 10.1 - 1.3 mg/g  dry weight, and 20.9 - 0.8  mg/g  dry weight i n hearts from 3-day fasted r a t s ("CONTROL" panel, F i g . 4).  The glycogen l e v e l s i n hearts from fed r a t s decreased  0 5 10 15 PERFUSION  20 25 3 0 TIME (min.)  FIG. 4 Tissue glycogen contents i n hearts-from fed (——) and fasted (——) r a t s during sub strate-free perfusion. Control hearts were perfused f o r the indicated period of time with substrate-free KrebsRinger bicarbonate media, pH 7.4 at 37°. Epinephrine, 0.2-1.0 ug/min, and heparin, 60 ug/min, were injected into the perfusion system j u s t above the cannula. Tissue glycogen contents are shown as the means ± standard e r r o r of. the mean of the number of observations i n parenthesis ( v e r t i c a l b a r s ) . The h o r i z o n t a l bars represent the mean time and range over which hearts were c o l l e c t e d .  33.  at a faster rate than those from fasted r a t s when perfused with substrate-free medium.  In contrast to hearts from  fasted r a t s , hearts from fed animals generally developed arrhythmia and decrease i n c o n t r a c t i l e force within 10 minutes of perfusion. maintained  On the other hand, hearts from fasted rats  good rhythm and contractions f o r much longer periods  of time, often longer than 25 minutes. As might be expected, the addition of epinephrine to the perfusion medium caused a rapid depletion of tissue glycogen, which was e s p e c i a l l y noticeable i n the hearts from fasted animals ( see "EPINEPHRINE" panel, F i g . 4).  Furthermore,  epinephrine appeared to have caused a more complete depletion of tissue glycogen, as compared with the control s e r i e s .  In  f a c t , the glycogen content a f t e r 10 minutes of perfusion i n some of the epinephrine treated (fed) r a t hearts was barely detectable. was  The e f f e c t of epinephrine on cardiac function  to increase both rate and force for the f i r s t 3-5 minutes,  followed by a rapid decline i n cardiac function. Within  8-12  minutes, most of the epinephrine treated hearts were v i r t u a l l y non-functional.  Arrhythmias were very frequently encountered  in the presence of epinephrine.  I t should perhaps be noted  that when the perfusate from the epinephrine-treated fasted series was assayed f o r glucose, none could be detected. Addition of heparin to the perfusion medium appeared to have l i t t l e e f f e c t on the rates of glycogen depletion i n both fed and fasted r a t hearts ("HEPARIN" panel, F i g . 4). Whereas f a s t i n g caused a s i g n i f i c a n t increase i n cardiac  34.  glycogen content, there was no s i g n i f i c a n t increase i n tissue t r i g l y c e r i d e l e v e l i n hearts obtained from fasted r a t s .  This  observation i s i n contrast to the observations of Wittels (118) who found increased t r i g l y c e r i d e levels i n hearts of fasted guinea pigs.  Perfusion of about 30 minutes duration ( F i g . 5)  appeared to cause some decrease i n t r i g l y c e r i d e l e v e l s i n both fed and fasted r a t s .  However, considering the r e l a t i v e l y  large v a r i a t i o n i n the tissue t r i g l y c e r i d e values, and the small population of rats used, i t i s suggested  that i f any  changes i n t r i g l y c e r i d e l e v els did occur, these changes were probably not very s i g n i f i c a n t during the 27-30 minutes of perfusion.  No decrease i n t r i g l y c e r i d e levels were noted i n  hearts perfused with epinephrine or with heparin.  In f a c t ,  an apparent increase i n t r i g l y c e r i d e levels appeared.  This  apparent increase i n tissue t r i g l y c e r i d e l e v e l s i n the epinephrine and heparin perfused series ("EPINEPHRINE" and "HEPARIN" panels, F i g . 5) i s most d i f f i c u l t to interpret.  I t i s incon-  ceivable that t r i g l y c e r i d e s were synthesized under these perfusion conditions.  I t seems more l i k e l y that some factor  may have been produced as a d i r e c t r e s u l t of perfusion with epinephrine and heparin, giving r i s e to anomalously high values f o r t r i g l y c e r i d e s . The apparent decrease i n tissue t r i g l y c e r i d e l e v e l s observed during perfusion of the control series must be substantiated with more data.  Since more p o t e n t i a l energy i s  contained per weight of t r i g l y c e r i d e s than i s contained i n glycogen, the decrease i n t r i g l y c e r i d e s w i l l probably not be  CONTROL  22  20  V- 20  *• r  HEPARIN  22  18  ^ 16 E . 14 UJ  (4) (8)  o  £ |(i2) T u .-> 10 -I ,2  T  o  DC  8.11®'  6  UJ  3  (6)  (8)  (6)  CO  <2 l-  4 .  2 0  10  15  20  25  30  0 5 10 PERFUSION  FIG. 5 Tissue t r i g l y c e r i d e contents during substrate-free perfusion.  15 20 TIME  25 30 (min.)  i n hearts from fed (  Perfusion conditions as described under Fig* 4.  10  15  20  25  30  ) and fasted (- — -) r a t s  36.  evident immediately conditions.  under these r e l a t i v e l y short perfusion  Hence, i n order to demonstrate conclusively that  tissue t r i g l y c e r i d e s do decrease during substrate-free perfusions, i t would l i k e l y be necessary to perfuse hearts f o r longer periods, perhaps up to 60 minutes.  However, i t was  found extremely d i f f i c u l t to maintain hearts i n good working order for periods much longer than 30 minutes under the perfusion conditions used i n these experiments.  These r e s u l t s  unfortunately shed l i t t l e l i g h t upon the r e l a t i v e importance of endogenous t r i g l y c e r i d e s to the isolated perfused heart. I t i s tentatively concluded  that the immediate endogenous  source of myocardial energy i s derived from the breakdown of tissue glycogen when hearts are perfused with substrate-free media, at least during the i n i t i a l phases of perfusion. haps decreased  Per-  levels of t r i g l y c e r i d e s become apparent a f t e r  longer periods of time. II  Cardiac Lipases Triglycerides  i n blood ( i n the form of chylomicrons  and  very low density lipoproteins) are taken up i n t a c t , i . e . without p r i o r hydrolysis, by cardiac c e l l s .  When one considers  that perhaps 90% of the c i r c u l a t i n g l i p i d s which are extracted by the heart i s i n the form of t r i g l y c e r i d e s , i t i s obvious that the r o l e of cardiac lipases must be extremely  important  for providing a constant source of f r e e - f a t t y acids f o r o x i dation by the myocardium. In many tissues, the sequence of t r i g l y c e r i d e hydrolysis to f a t t y acids proceeds enzymatically i n the following manner:  37. triglycerides  »-diglycerides  monoglycerides  where the i n i t i a l step i s considered r a t e - l i m i t i n g .  »- FFA, In cardiac  tissues, an enzyme system e x i s t s which hydrolyzes t r i g l y c e r i d e s to diglycerides.  This triglyceride-hydrolyzing enzyme,  l i p o p r o t e i n lipase, probably hydrolyzes diglycerides to monoglycerides as w e l l .  Another lipase, similar to the epinephrine-  sensitive lipase of adipose tissue (107), has been reported to e x i s t i n r a t hearts (13).  The existence of a lipase which  i s s p e c i f i c for diglycerides has not yet been shown i n heart or i n any other tissue, although there i s some evidence to strongly indicate that l i p o p r o t e i n lipase also attacks d i g l y cerides.  On the other hand, the presence of monoglyceride-  s p l i t t i n g lipases has been demonstrated i n a number of tissues, including adipose tissue (6, 16), l i v e r (55), and  the  intes-  t i n a l mucosa (56-59), but i t s presence has not been shown i n cardiac The  tissue. general procedure for studying any enzyme i s to  determine f i r s t whether i t s a c t i v i t y e x i s t s i n whole homogenates or i n i n t a c t systems.  The next step i s to p u r i f y i t  as a discrete e n t i t y so that i t s properties may  be  better  studied without the problem of contamination by other enzymes possessing similar properties.  This general approach  followed i n the present work for investigating the  was  lipolytic  a c t i v i t i e s i n r a t hearts. Although numerous enzymes have been extensively  puri-  f i e d , and many even c r y s t a l l i z e d , most lipases have r e s i s t e d purification.  In f a c t , no lipase has yet been c r y s t a l l i z e d .  38.  Therefore, studies on most lipases up to the present time have usually been done on crude extracts of tissues, leading to much confusion i n the l i t e r a t u r e with respect to some of the properties of l i p a s e s .  Another major obstacle i n the  study of lipases i s the technical d i f f i c u l t y of preparing suitable substrates for the enzymes.  For example, there are  no standard t r i g l y c e r i d e substrates for triglyceride-hydrolysing lipases. substrate may  The f a t t y acid moiety of a t r i g l y c e r i d e  be saturated, unsaturated, long, medium or  short chain, and the p h y s i c a l nature of the substrate may  be  an oil-in-water emulsion or a crude glyceride suspension i n a suitable aqueous buffer.  F i n a l l y , a l l known methods of  assaying l i p o l y t i c a c t i v i t y are considerably more laborious and t e c h n i c a l l y cumbersome than most commonly used enzyme assays.  A l l these reasons contribute to the fact that the  study of lipases has lagged f a r behind the study of enzymes i n other areas of the b i o l o g i c a l system. In spite of the d i f f i c u l t i e s anticipated, an attempt was nevertheless made to study the lipases of cardiac tissue, with p a r t i c u l a r emphasis on lipases other than lipoprotein lipase. A.  EXISTENCE OF NaF-INHIBITED LIPASE Rat hearts were perfused for 5 minutes with  Krebs-Ringer  bicarbonate solutions containing 20 ug/ml heparin.  The ven-  t r i c u l a r tissues were pooled and homogenized i n 10 volumes of 0.25 M sucrose, pH 7.0 at 0-4° with the aid of a PotterElvehjem Homogenizer.  The crude homogenate was  filtered  39. through cheesecloth and used d i r e c t l y f o r assay of l i p o l y t i c a c t i v i t y at pH 6.8, using E d i o l  R  (1:19) as substrate as  described i n the Experimental section.  A rapid l i b e r a t i o n  of free f a t t y acids occurred, as may be seen i n F i g . 6, and the enzyme a c t i v i t y was proportional to incubation time up to 30 minutes. When similar experiments were performed i n the presence and absence of 0.2 M NaF, again using E d i o l  R  as substrate,  i t was noted that a constant i n h i b i t i o n of about 40% occurred throughout the course of the reaction ( F i g . 7).  This experi-  ment was performed at a pH suboptimal f o r l i p o p r o t e i n lipase and without pre-activating the E d i o l  with serum.  Under  these conditions, the a c t i v i t y of l i p o p r o t e i n lipase would be minimized.  Since NaF i s known not to be an i n h i b i t o r of  l i p o p r o t e i n lipase, the observed 407. i n h i b i t i o n i n the crude system suggested that there indeed was an active lipase or lipases present i n cardiac tissue i n addition to l i p o p r o t e i n lipase.  I n h i b i t i o n by NaF would indicate that the enzyme  might be similar to the non-lipoprotein lipase of adipose tissue described by Rizack (107). At t h i s point, i t must be emphasized  that Ediol - was 8  used as substrate f o r measuring l i p o l y t i c a c t i v i t y i n these experiments.  The major advantage of t h i s emulsion i s that  i t i s an extremely smooth and stable oil-in-water t r i g l y ceride preparation. Ediol  R  However, a serious disadvantage of  as substrate i s that i t contains monostearin as a  s t a b i l i z i n g agent, and this monostearin could well serve as  40.  INCUBATION  FIG. 6  TIME  (mln.)  Time course of l i p o l y t i c a c t i v i t y .  The reaction mixture contained 60 mM phosphate buffer, pH 6.8, 20 mg p u r i f i e d bovine serum albumin, 0.10 ml of 1:19 d i l u t i o n of E d i o l , 0.04 ml whole homogenate i n a t o t a l volume of 1.0 ml. Incubation was at 37° i n a Dubnoff Metabolic Shaker. Each reaction mixture contained 0.42 mg protein.  41.  FIG. 7 I n h i b i t i o n of l i p o l y t i c a c t i v i t y i n crude heart homogenates by NaF. The incubation mixture contained a l l components.' of the standard assay as described i n the text, except that 0.15 ml of 1:19 d i l u t i o n of E d i o l was used as substrate. Curve "A" - c o n t r o l . Curve "B" - NaF, 0.2 M included in reaction mixture. Each reaction mixture contained 0.21 mg protein. The homogenate was stored overnight at -20° and thawed before use.  42.  substrate f o r a l i p o l y t i c enzyme.  Experiments were therefore  performed i n order to explore further the nature of the NaFinhibited l i p o l y t i c a c t i v i t y i n cardiac tissue by using substrates prepared from monoglycerides or t r i g l y c e r i d e s only, When monostearin (suspended i n 5% acacia solution) was used as substrate, i t was r a p i d l y hydrolyzed by the heart homogenate ( F i g . 8, Curve A ) . t i o n a l to time.  A c t i v i t y again was propor-  Of p a r t i c u l a r interest was the observation  that i n the presence of 0.2 M NaF, a constant 40% i n h i b i t i o n of l i p o l y t i c a c t i v i t y was obtained (Curve B).  This degree  of i n h i b i t i o n was almost i d e n t i c a l to that observed when s i m i l a r experiments using E d i o l  R  was performed ( F i g . 7), thus  strongly suggesting that the NaF-inhibited l i p o l y t i c a c t i v i t y measured with E d i o l ^ as substrate may have been due to the hydrolysis of i t s monostearin component.  Furthermore, when  t r i p a l m i t i n alone was used as substrate, the l i p o l y t i c  acti-  v i t y was so small as to make accurate and r e l i a b l e l i p o l y t i c measurements exceedingly d i f f i c u l t (Curve C).  NaF (0.2 M) did  not appear to i n h i b i t the hydrolysis of t r i p a l m i t i n , although the low a c t i v i t i e s observed made the interpretation of the data d i f f i c u l t (Curve D).  When, however, the incubation  mixture was supplemented with r a t serum, a rapid hydrolysis of t r i p a l m i t i n occurred. The data i n Table I shows a greater than 12-fold increase i n l i p o l y t i c a c t i v i t y when the t r i p a l m i t i n substrate was presented to the enzymes i n the form of a l i p o p r o t e i n complex.  This experiment c l e a r l y indicated  not only the presence of lipoprotein lipase a c t i v i t y i n  43.  INCUBATION  TIME  (mln.)  FIG. 8 Hydrolysis of monostearin and t r i p a l m i t i n , and e f f e c t of NaF. Line "A" represents the time course of l i p o l y t i c a c t i v i t y of the whole homogenate when the substrate was monostearin (5 ueq/ml) i n the standard assay. Line "B" represents i n h i b i t i o n of monostearin hydrolysis by 0.2 M NaF. Line "C" represents the l i p o l y t i c a c t i v i t y of the homogenate (same volume used as i n "A") when the standard incubation mixture contained 2.5 mg t r i p a l m i t i n as subs t r a t e . Line "D" represents the hydrolysis of t r i p a l m i t i n i n the presence of 0.2 M NaF. The hydrolysis of t r i p a l m i t i n was consistently so small that accurate l i p o l y t i c measurements were extremely d i f f i c u l t . Each reaction mixture contained approximately 0.42 mg protein.  TABLE I  E f f e c t of r a t serum on the hydrolysis of t r i p a l m i t i n by p a r t i a l l y p u r i f i e d extract of cardiac t i s s u e . The complete system contained 20 mg p u r i f i e d bovine serum albumin, 50 mM T r i s buffer, pH 8.5, 2.5 mg t r i palmitin which had been pre-incubated f o r 30 minutes at 37° with 0.1 ml r a t serum, 0.1 ml of enzyme extract, and s u f f i c i e n t water to make a f i n a l volume of 1.0 ml.  Reaction mixture  Lipolytic Activity mueq FFA/60 min at 37°  Complete system  99  Complete system without serum  8  45.  cardiac extracts, but also demonstrated the marked dependence of t h i s enzyme on serum-activated t r i g l y c e r i d e s . tant, the data provided  More impor-  additional evidence that an enzyme  other than l i p o p r o t e i n l i p a s e e x i s t s i n r a t hearts.  The  enzyme appeared to hydrolyze monoglycerides with greater f a c i l i t y than t r i g l y c e r i d e s .  The a b i l i t y of the l i p a s e to  l i b e r a t e free f a t t y acids from E d i o l  R  i s l i k e l y due to i t s  action on the monostearin component of t h i s preparation.  The  enzyme already seemed s i m i l a r to that reported by Bjorntorp and Furman (13) who used E d i o l  R  as substrate.  The p o s s i b i l i t y  existed that the a c t i v i t y these authors had measured was a c t u a l l y the hydrolysis of monoglyceride.  Further  studies  were therefore necessary to c l a r i f y the source of f a t t y acids a r i s i n g from E d i o l . One could accomplish t h i s by following the hydrolysis of either C - l a b e l l e d monoglycerides or C ^ l 4  t r i g l y c e r i d e s when these glycerides were added to E d i o l subjected  to lipase a c t i v i t y i n heart extracts.  R  and  Since labelled  long-chain monoglycerides were not r e a d i l y a v a i l a b l e , t r i p a l mitin- 1-C* was used. 4  The rate of hydrolysis of t r i p a l m i t i n -  1-C"*" and the rate of t o t a l f a t t y acids released were measured 4  simultaneously.  I t was assumed that f a t t y acids produced i n  the absence of t r i p a l m i t i n - 1 - C ^ hydrolysis must have been 4  derived from the monoglyceride component of E d i o l .  Further-  more, i t was thought that the e f f e c t s of known l i p a s e i n h i b i t o r s could be more c l e a r l y observed i n such a system.  As  may be seen i n F i g . 9, the hydrolysis of t r l p a l m i t i n - l - C ^ in E d i o l  R  occurred  4  at an appreciable rate i n the presence of  46.  FIG. 9 Time c o u r s e o f E d i o l h y d r o l y s i s i n the presence of lipase i n h i b i t o r s . L i n e s "A", "B", and "C" r e p r e s e n t the t i m e c o u r s e o f t o t a l f a t t y a c i d s produced d u r i n g the e x p e r i m e n t a l c o n d i t i o n s as o u t l i n e d below. L i n e s "a""and "b" r e p r e s e n t t h e r a t e o f t r i p a l m i t i n - 1 - C l 4 h y d r o l y s i s d u r i n g t h e same i n t e r v a l . L i n e s "A" and " a " - The r e a c t i o n m i x t u r e c o n t a i n e d 120 mg b o v i n e serum albumin, 50 mM T r i s b u f f e r , pH 8.5, 0.3 ml r a t serum, 0.3 m l o f 1:1.9 d i l u t i o n E d i o l c o n t a i n i n g t r i p a l m i t i n l - C ^ (0.5 p c u r i e / n i l ) , 0.6 ml 6000 x g r a t h e a r t s u p e r n a t e , and s u f f i c i e n t water t o make 6.0 m l . I n c u b a t i o n was performed a t 37° i n a Dubnoff M e t a b o l i c Shaker and 1.0 m l a l i q u o t s removed a t t h e i n d i c a t e d time i n t e r v a l s f o r t h e a s s a y o f t o t a l f r e e f a t t y a c i d s produced ( l i n e "A") and a l s o t r i p a l m i tin-l-C hydrolyzed ( l i n e " a " ) . L i n e s "B'Vand "b" - R e a c t i o n c o n d i t i o n s were e s s e n t i a l l y 1  1 4  47.  the same as indicated above f o r "A" and a " , except that 50 mM T r i s buffer pH 7.0 was used and the system also contained NaCl, 0.5 M. Line "C" - Reaction conditions were as f o r "B" and "b", except that the system also contained"NaF, 0.2 M. - . The rate of t r i p a l m i t i n - l - C l 4 hydrolysis ( l i n e "a") was proportional to incubation time f o r 90 minutes. w  48.  rat  serum ( l i n e "a").  The t o t a l f a t t y acids produced during  t h i s period was also appreciable ( l i n e "A").  In the presence  of 0.5 M NaCl (and absence of serum), the hydrolysis of t r i palmitin- 1-C  14  was completely  abolished ( l i n e "b").  This  indicated that under these conditions ( i . e . , 0.5 M NaCl, no serum), l i p o p r o t e i n lipase a c t i v i t y against t r i g l y c e r i d e s was completely  inhibited.  However, even under conditions where  the hydrolysis of t r i p a l m i t i n - 1 - C  14  was completely  inhibited,  FFA release was s t i l l evident i n the system ( l i n e "B").  Hence,  t h i s ' l i p o l y t i c a c t i v i t y was attributed to the hydrolysis of monostearin i n E d i o l . R  I f t h i s were the case, the addition  of 0.2 M NaF to the reaction mixture should r e s u l t i n further i n h i b i t i o n of l i p o l y t i c a c t i v i t y , and as may be seen, t h i s was indeed observed ( l i n e "C").  This experiment therefore gave  additional support to the o r i g i n a l observation that when Ediol  R  was used as substrate i n the "non-activated"  state, a  large proportion of free f a t t y acids liberated was derived from the hydrolysis of i t s monoglyceride component.  I t also  supported the view that i t i s the hydrolysis of the monoglyceride component of E d i o l  R  which i s inhibited by NaF.  I t was noted that the rate of monoglyceride hydrolysis ( l i n e "B") did not change a f t e r about 25 minutes of incubation. This consistent observation i n these experiments was due to the fact that under the conditions of the assay, the amount of monoglyceride was l i m i t i n g the r e a c t i o n . approximately 1.5% monostearin. stearin i s equal to 40 peq/ml.  Ediol  R  contains  This concentration of monoIn those experiments u t i l i z i n g  49.  t r i p a l m i t i n - l - C ^ , each 1.0 ml of reaction mixture contained 1  0.05 ml of a 1:19 d i l u t i o n of E d i o l . R  Hence the t o t a l amount  of monostearin available as substrate was about 100 mueqs. Considering the noticeable and inevitable shrinkage i n volume of E d i o l  R  during storage over a two year period, i t i s not  unreasonable to assume that about 150 mpeqs of monostearin were perhaps available as substrate per reaction tube.  This  would explain the consistent plateauing of reaction rate observed at around 25-30 minutes incubation i n those experiments which were designed s p e c i f i c a l l y to show only monoglycerides being hydrolyzed. The r e s u l t s of these experiments c l e a r l y demonstrated that the l i p o l y t i c a c t i v i t y i n r a t hearts possessing propert i e s d i f f e r e n t from lipoprotein lipase was an active NaFinhibited enzyme which r a p i d l y hydrolyzed the monostearin component of E d i o l . R  the  Since E d i o l  R  has been so widely used i n  study of l i p a s e , e s p e c i a l l y i n adipose tissue, one wonders  i f r e s u l t s have not been occasionally misinterpreted. B.  PREPARATION OF PARTIALLY PURIFIED -EXTRACT FROM HEART TISSUE The presence of a NaF-inhibited monoglyceride-hydrolyzing  l i p o l y t i c system i n r a t hearts having been established, i t was considered e s s e n t i a l that the enzyme be isolated so that i t s properties could be better understood.  As anticipated, the  s a t i s f a c t o r y p u r i f i c a t i o n of t h i s enzyme proved extremely difficult. Some of the problems encountered during the attempt to  50.  p u r i f y the enzyme w i l l be noted here, but a detailed discussion w i l l be reserved f o r the f i n a l Discussion section.  F i r s t , about  75-85% of the enzyme a c t i v i t y was bound to the p a r t i c u l a t e f r a c tions of the c e l l s .  Attempts to s o l u b i l i z e the enzyme using  a number of standard techniques were i n most instances unsuccessful.  For example, preparations of acetone powders,  repeated freezing and thawing, deoxycholate treatment, sonication at 9 and 20 k i l o c y c l e s f o r various periods of time, sonication combined with deoxycholate treatment were t r i e d i n vain.  The technique which gave reasonably s a t i s f y i n g r e s u l t s  was the use of 0.1% Triton X-100, a non-ionic synthetic detergent.  When a 105,000 x g p e l l e t was re-homogenized with  T r i t o n X-100, a 2-to 3-fold increase i n the amount of enzyme a c t i v i t y was noted i n the subsequently obtained 105,000 x g supernatant f l u i d .  This technique s o l u b i l i z e d about 30-40%  of the t o t a l enzyme a c t i v i t y i n cardiac c e l l s , and was therefore adopted as the basis f o r further attempts to p u r i f y the enzyme. P u r i f i c a t i o n of the enzyme from the 105,000 x g supernatant f l u i d was also extremely d i f f i c u l t .  Again a number of  c l a s s i c a l techniques of enzyme f r a c t i o n a t i o n were employed. Repeated attempts using high temperatures, ethanol p r e c i p i t a t i o n methods, calcium phosphate gels, Sephadex G-200 columns and zinc-ethanol treatment f a i l e d to give a s a t i s f a c t o r y purification.  However, both ammonium sulfate f r a c t i o n a t i o n  and the i s o e l e c t r i c p r e c i p i t a t i o n methods did provide a small measure of p u r i f i c a t i o n .  The i s o e l e c t r i c p r e c i p i t a t i o n tech-  51.  nique was  adopted as the second step i n the proposed further  p u r i f i c a t i o n of the enzyme. by i s o e l e c t r i c precipitation) sulfate were t r i e d but  Treatment of the extract (obtained with Sephadex G-200 and  the enzyme resisted  A major obstacle to p u r i f i c a t i o n was  further p u r i f i c a t i o n .  the i n s t a b i l i t y of  l i p o l y t i c system i n the i s o e l e c t r i c precipitate p a r t i c u l a r l y to freezing amount of time had  and  ammonium  thawing.  fraction,  Since a considerable  already been expended i n e f f o r t s to  b i l i z e and p u r i f y the enzyme, i t was  decided to use  p a r t i a l l y p u r i f i e d i s o e l e c t r i c precipitate study of the enzyme.  The  the  solu-  the  f r a c t i o n for  the  acid p r e c i p i t a t i o n method gave about  a 3 to 4-fold p u r i f i c a t i o n over the combined supernatant f r a c tion, and  a y i e l d of approximately 457..  however, was  The  overall yield,  only 107. when based on the t o t a l a c t i v i t y of  whole homogenate, owing e s s e n t i a l l y to the extremely nature of the enzyme.  activity.  The  insoluble  I t should be mentioned that the degree  of p u r i f i c a t i o n obtained was or monoolein was  the  about the same when either  Ediol  R  used as substrate for measuring l i p o l y t i c  p a r t i a l p u r i f i c a t i o n obtained i s shown in Table I I .  L i p o l y t i c a c t i v i t y of the i s o e l e c t r i c precipitate d i r e c t l y proportional to enzyme concentration (Fig. 10)  was over  a range of protein which constituted a r e l i a b l e assay. C.  PROPERTIES OF CARDIAC MONOGLYCERIDE-HYDROLYZING ENZYME Using the p a r t i a l l y p u r i f i e d i s o e l e c t r i c  preparation (hereafter referred  precipitate  to as " p a r t i a l l y p u r i f i e d  extract") of r a t heart, a number of experiments were performed  52.  TABLE I I  P a r t i a l p u r i f i c a t i o n of cardiac monoglyceride-splitting enzyme. The a c t i v i t i e s i n the f i r s t and second high speed supernate f r a c t i o n s are included to indicate the increase i n s o l u b i l i z a t i o n obtained with T r i t o n X-100. The standard l i p a s e assay was employed with 15 ueqs monoolein as substrate. One mp u n i t of enzyme a c t i v i t y i s that amount which produced 1.0 mpeqs FFA/60 min/37°. S p e c i f i c a c t i v i t y i s defined as mu units enzyme activity/mg protein/60 min/37°. FRACTION  TOTAL ACTIVITY  SPECIFIC ACTIVITY  105,000 g supernate  220,000  956  (No. 2) " " obtained with T r i t o n X r l O O  646,800  3480  COMBINED 105,000 supernates  766,800  1980  pH 5.2-5.9 i s o e l e c t r i c precipitate  333,333  7000  (No. 1)  53.  VOLUME OF EXTRACT  ADDED (mla)  FIG. 10 FFA production as a function o f enzyme concent r a t i o n with monostearin as substrate. The standard assay was employed, and the enzyme used . was the p a r t i a l l y p u r i f i e d i s o e l e c t r i c preparation*  54.  to study some of the properties of this enzyme.  I t was hoped  that information obtained from such studies would greatly a s s i s t i n the assignment of a possible p h y s i o l o g i c a l r o l e of the enzyme i n cardiac tissues. 1.  Albumin Requirement —  Under p h y s i o l o g i c a l conditions,  long chain free f a t t y acids are normally transported blood as free f a t t y acid-albumin complexes.  i n the  This binding of  free f a t t y acids to albumin e f f e c t i v e l y s o l u b i l i z e s a considerable amount of otherwise insoluble FFA»s which have been released into the c i r c u l a t i o n from adipose t i s s u e s . For the same reason, albumin has frequently been added to l i p o l y t i c reaction mixtures to function as a FFA-acceptor i n the i n v i t r o system.  As may be seen i n F i g . 11, the require-  ment f o r albumin i n the assay mixtures was not absolute, but the presence of more than 12 mg albumin increased the a c t i v i t y about 2-fold.  Routinely, 20 mg of p u r i f i e d bovine serum  albumin was included i n the assay system. 2.  pH-Qptimum —  When monoolein was used as substrate, the  a c t i v i t y of the enzyme extended over a wide pH range (pH 6.09.0) with the maximum a c t i v i t y exhibited near pH 6.5-7.0 (Fig. 12).  This observation  i s consistent with the report of  Bjorntorp and Furman's (13) that a sodium f l u o r i d e - i n h i b i t e d l i p o l y t i c a c t i v i t y having a pH optimum near pH 6.8 existed i n r a t hearts.  The pH optimum of l i p o p r o t e i n l i p a s e i s near 8.5.  Since, i n the experiment, no t r i g l y c e r i d e or diglyceride substrate and no serum was present, and since the reaction was performed i n the presence of 0.5 M NaCl, t h i s enzyme can be  o 100 > cr  a.  < 50  0  4  8 ALBUMIN  12 ADDED  16 (mg)  20  FIG. 11 E f f e c t of concentration o f albumin on l i p o lytic, activity. Incubation was carried out according to the standard assay, using 0.1 ml of 1:19 d i l u t i o n o f E d i o l as substrate, except that the albumin concentration was varied as indicated. The enzyme used was the p a r t i a l l y p u r i f i e d pH 5.2-5.9 preparation. .  56.  pH FIG. 12 Hydrolysis of monoolein by p a r t i a l p u r i f i e d lipase.as a function of pH. The incubation mixture contained 15 peq monoolein, 20 mg p u r i f i e d bovine serum albumin, 0.5 M NaCl, enzyme, 0.08 M T r i s and 0.08 M phosphate buffers at the pH*s indicated. The incubations were performed i n a t o t a l volume of 1.0 ml f o r 60 minutes at 37° on a Dubnoff Metabolic Shaker.  57.  r e a d i l y distinguished from lipoprotein l i p a s e . 3.  Temperature Optimum —  The optimum temperature f o r the  in v i t r o enzymatic hydrolysis of monostearin was near 45° ( F i g . 13).  This optimum i s beyond the normal p h y s i o l o g i c a l  range expected of any enzyme i n b i o l o g i c a l systems.  There-  fore, the significance of t h i s observation can only be extended to the enzymatic hydrolysis of monostearin jLn v i t r o , and i s of academic interest only.  However, i t further empha-  sizes the s i m i l a r i t y between t h i s enzyme and the monoglyceri d e - s p l i t t i n g enzyme from adipose tissue because Vaughan jet _al.(6) has shown that t h i s enzyme has a temperature optimum of 45°. 4. E f f e c t of Physical State of Substrate —  One of the  c r i t e r i a used! f o r distinguishing between true lipases and non-specific esterases i s that the former does not normally attack esters i n aqueous solution (109).  In order to study  the nature of the l i p o l y t i c a c t i v i t y i n the p a r t i a l l y p u r i f i e d extract, the enzyme was added to various concentrations of monoolein.  A s e r i a l d i l u t i o n of monoolein was made such  that at low concentrations, the reaction mixture was o p t i c a l l y c l e a r , presumably because the monoolein was i n true solution. At higher concentrations, the substrate was insoluble and i n the form of a suspension. Careful v i s u a l examination of a series o f reaction tubes p r i o r to addition of enzyme indicated that the t u r b i d i t y of the reaction mixture became apparent at monoolein concentrations i n the region of 1.5 or 2.25 ueq/ml. When the l i p o l y t i c a c t i v i t y was measured i n the usual manner,  58.  25 30 35 40 45 50 55 TEMPE RATURE INCUBATION  FIG. 13 E f f e c t of temperature on cardiac monoglyc.eride« hydrolyzing lipase a c t i v i t y . The standard assay was employed using monostearin as substrate, except that the incubations were performed at the temperatures indicated. Control tubes were also incubated, and the enzyme added a f t e r the reactions were stopped.  59.  i t was observed that a marked increase i n a c t i v i t y near 2.0-4.0 ueq/ml monoolein concentration  occurred  ( F i g . 14). I t  i s r e a d i l y conceded that the v i s u a l estimation of t u r b i d i t y gave only a rough approximation of the actual physical state of the monoolein substrate i n aqueous media.  Nevertheless,  the marked increase i n l i p o l y t i c a c t i v i t y over a r e l a t i v e l y narrow monoglyceride concentration range suggested strongly that the enzyme i s a true l i p a s e , and not a simple non-specific esterase. 5.  E f f e c t of Some Enzyme Inhibitors on Monoglyceride-  hydrolyzing Lipase A c t i v i t y —  Many enzyme i n h i b i t o r s have  been employed i n order to i d e n t i f y the active s i t e s and to study the mechanism of action of enzymes.  I t appears, how-  ever, that the use of i n h i b i t o r s i n the study o f l i p a s e s has been l a r g e l y r e s t r i c t e d to distinguishing one l i p a s e a c t i v i t y from another.  Even when used f o r t h i s purpose, many inconsis-  tencies are noted i n the l i t e r a t u r e with respect to the e f f e c t of i n h i b i t o r s on mammalian tissue l i p a s e s , presumably due to the use of crude enzyme preparations and grossly impure substrates. I t was hoped that the use of some known l i p a s e i n h i b i t o r s i n this study would y i e l d a d d i t i o n a l information as to whether the cardiac monoglyceride-splitting l i p a s e was s i m i l a r to those which have been reported i n t e s t i n a l mucosa.  to e x i s t i n adipose tissue and i n the  The r e s u l t s of these studies are presented  i n Table I I I , including the data obtained by other investigators f o r reference.  60.  1 2 3 4 5 6 7 8 9 10 // 14 MONOOLEIN CONCENTRATION (ueq/ml)  FIG. 14 E f f e c t of monoolein concentration on the a c t i v i t y of the p a r t i a l l y p u r i f i e d cardiac lipase. The reaction mixture contained a l l components of the standard assay, except that the monoolein concentration was varied as indicated and 0.5 M NaCl was included.  TABLE I I I I n h i b i t i o n of Monoglyceride-splitting Lipase by Various Compounds The standard assay was used with 15 ueq monoolein as substrate, except that 0.5 M NaCl was included i n a l l the reaction mixtures. The various i n h i b i t o r s were pre-incubated for 15 minutes at 37° with the p a r t i a l l y purified enzyme preparation prior to addition of substrate. COMPOUND  THIS STUDY cone.  NaF  Kupieki (16)  % inhib. cone. % inhib.  0.15M  75  0.20M  0  DFP  2 x 10""%  70  1 x 10-%  39  EDTA  5 x 10"%  0  5 x 10"%  0  Protamine sulfate  400 ug/ml  0  300 ug/ml  0  N-Ethyl Maleimide  1 x 10"%  25  1 x 10*"%  33  Iodoacetic acid  1 x 10"%  0  1 x 10"%  0  1 x 10"%  79  P-chloromercuribenzoate  mm mm  Strand et al» Pope et al» (15) (597"~ cone. 7o inhib. cone. 7. inhib. 0.20M  45  (0.0 2ml/ml)  Isopropanol  Vaughan et al« (6) cone. % inhib.  (0.025ml/ ml), 5 x 10 %  0 97  84  (0.025ml/ ml)  1 x 10"^  0 mm mm  100 mm mm  1  X  10"%  100  —  *• mm mm  —  mm mm  «m  mmm» tm  mm mm  mm mm  mm mm  mm mm  Wm mm  mm mm  -  —  mm mm  1 x 10"%  0  Substrate  Monoolein  Monoolein  Monostearin  Monoolein  Monoolein  Monoglycerides p l i t t i n g lipase source  Partially p u r i f i e d from r a t heart  Partially p u r i f i e d from r a t adipose tissue  Crude extract adipose tissue  Crude extract adipose tissue  Highly p u r i f i e d from rabbit i n t e s t i n a l mucosa  62.  The most potent i n h i b i t o r s of the monoglyceride l y z i n g a c t i v i t y were NaF and DFP,  and the e f f e c t s  hydro-  observed  were i n good agreement with those seen f o r monoglyceridehydrolyzing lipases of other tissues.  The mechanism of  i n h i b i t i o n by NaF i s not known, but the r e l a t i v e l y high concentrations normally required to show t h i s i n h i b i t i o n r e s t r i c t s the usefulness of this compound, except perhaps to distinguish between monoglyceride-hydrolyzing a c t i v i t y and lipoprotein lipase a c t i v i t y .  On the other hand, DFP  the enzyme at a r e l a t i v e l y low concentration. DFP  inhibited  i s known  to combine i r r e v e r s i b l y with the -OH function of enzymes (e.g. esterases), thus blocking the active s i t e of the enzyme. In t h i s respect, the monoglyceride-hydrolyzing enzyme appears to resemble an esterase. The complete absence of i n h i b i t i o n by protamine  sulfate  (400 ug/ml) was expected since any lipoprotein lipase a c t i v i t y which may have been present i n the extract would have been e f f e c t i v e l y inhibited under the conditions of the assay. The presence of EDTA (5 x lO'^M) did not i n h i b i t the a c t i v i t y , which l i k e l y indicates  that no p o s i t i v e l y changed  metal cations were required f o r a c t i v i t y . The a l k y l a t i n g agents, N-ethylmaleimide  and iodoacetate,  used at the same concentrations would have been expected to give similar r e s u l t s . N-ethylmaleimide, reconcile,  Therefore the i n h i b i t i o n observed with  but not with iodoacetate i s d i f f i c u l t to  although i t must be noted that Kupieki (16) obtained  e s s e n t i a l l y similar r e s u l t s with these compounds.  The  effects  63. of these i n h i b i t o r s on the monoglyceride-splitting l i p a s e a c t i v i t y of heart tissue i s i n good agreement with those observed f o r the monoglyceride-hydrolyzing enzymes studied i n other tissues, and further indicated the s i m i l a r i t y of these a c t i v i t i e s . 6.  Intracellular Localization —  I t has been previously  mentioned that the enzyme was bound to tissue p a r t i c l e s . 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 monoglyceride-splitting lipase was  investigated i n order to provide some evidence as  to i t s possible physiological function i n cardiac tissue. As shown i n Table IV, about 85% of the a c t i v i t y was bound to the p a r t i c u l a t e components of the c e l l , about 50% of the t o t a l a c t i v i t y being located i n the nuclear f r a c t i o n .  In  contrast, when t r i p a l m i t i n was used as substrate, the only c e l l u l a r f r a c t i o n to exhibit any l i p o l y t i c a c t i v i t y was  the  microsomal f r a c t i o n (Table IV), although the a c t i v i t y was low as to make accurate determinations d i f f i c u l t . experiments, 0,5 M NaCl was  so  In these  included i n the assay system i n  order to i n h i b i t lipoprotein lipase a c t i v i t y .  The p a r t i c l e -  bound nature of the monoglyceride-hydrolyzing lipase i n heart tissue i s similar to that reported recently by Pope et: a l . (59) for the p u r i f i e d monoglyceride  lipase of i n t e s t i n a l mucosa,  i n which only about 10% of the t o t a l a c t i v i t y was the soluble f r a c t i o n .  locatedlin  64.  TABLE IV D i s t r i b u t i o n of monoglyceride-splitting lipase and t r i glyceride l i p a s e a c t i v i t i e s i n various fractions of a r a t heart homogenate. Ventricular tissues were pooled from 6 r a t hearts and homogenized i n 12 volumes of 0;25 M sucrose containing 0.05 M T r i s buffer, pH 7.0, for two 1-minute i n t e r v a l s using a S e r v a l l Omnimixer to obtain the whole homogenate. The nuclear f r a c t i o n was obtained by centrifuging the homogenate at 250300 x g for 15 minutes. The l i g h t l y packed sediment was washed with 15 mis 0.25 M sucrose pH 7.0, re-centrifuged and the supernatant f l u i d s combined. The mitochondrial and microsomal fractions were obtained by centrifuging the combined supernatant f l u i d s at 6000 x g x 15 minutes and 105,000 x g x 60 minutes respectively. A l l procedures were c a r r i e d out at 0-4°. Standard assay conditions were employed, using either monoolein or t r i p a l m i t i n as substrates, except that NaCl 0.5 M was also included i n each reaction mixture. The assay for t r i g l y c e r i d e - s p l i t t i n g a c t i v i t y was performed using 10 times more extract than used for the monoglyceride-splitt i n g lipase a c t i v i t y , and incubation was for 120 mins at 37°. Enzyme a c t i v i t y i s as defined i n the text. MONOGLYCERIDE-SPLITTING LIPASE Fraction  Total A c t i v i t y (mp  Whole Homogenate Nuclear Fraction Mitochondrial Fraction Microsomal Fraction 105,000 x g Supernate  units)  %  536,500 287,500 110,000 75,000 80,600  (100%) 53.520.5 14.0 15.0  2590 0 0 2000 0  (100%) 0 0 77.2 0  TRIGLYCERIDE-SPLITTING LIPASE Whole Homogenate Nuclear Fraction Mitochondrial Fraction Microsomal Fraction 105,000 x g Supernate  65.  DISCUSSION The object of the perfusion studies was p r i m a r i l y to investigate the p o s s i b i l i t y that endogenous t r i g l y c e r i d e s were u t i l i z e d by the heart, and to compare t h i s u t i l i z a t i o n with that of glycogen when no exogenous substrates were available.  In general, the changes observed i n the  glycogen  levels of r a t v e n t r i c u l a r tissue during f a s t i n g and during substrate-free perfusions were not unexpected.  The increase  i n f a s t i n g cardiac glycogen levels deserves further comment. Although Cruikshank (110) observed that pancreatectomy caused a s h i f t i n l i v e r glycogen to cardiac glycogen,  the  f i r s t to observe t h i s phenomenon i n f a s t i n g was Evans (8) i n 1934.  Evans found that glycogen content increased i n r a t  hearts from 341 i 15 mg/100 g to 578 ± 14 mg/100 g wet weight a f t e r a 48-hour f a s t .  Then Lackey and co-workers (111^113)  found that cardiac glycogen increased i n the alloxan diabetic state (111), that there was  a d i r e c t r e l a t i o n s h i p between  blood ketone concentrations and cardiac glycogen levels 113).  Next, i t was  (112,  shown by Russell and Bloom (114) that  growth hormone was necessary f o r glycogen to increase i n heart during f a s t i n g .  Lukens (115) suggested that the  increase i n cardiac glycogen was due to the increased amount of FFA reaching the heart, owing to the action of growth hormone on adipose tissue. firmed by Bowman (116).  This suggestion was  l a t e r con-  More recently, Newsholme and Randle  (9) and Garland and co-workers (10) showed that ketone bodies,  66.  FFA,  and pyruvate e f f e c t i v e l y i n h i b i t the g l y c o l y t i c enzyme,  phosphofructokinase i n perfused r a t hearts.  Parmeggiani and  Bowman (11) demonstrated further that the i n h i b i t i o n of phosphofructokinase was  due to the increased tissue l e v e l s  of c i t r a t e . The increase i n cardiac glycogen content during f a s t i n g and i t s immediate decline upon re-feeding was the course of t h i s work.  As may  observed during  be noted i n F i g . 15, cardiac  glycogen increased over 2.5-fold during seven days of f a s t i n g . The immediate depletion of glycogen a f t e r one day of re-feeding to the c o n t r o l l e v e l indicates a very rapid u t i l i z a t i o n and/or mobilization of glycogen to other tissues of the body, probably the l i v e r . The slower rate of glycogen disappearance from perfused (control) hearts from fasted rats as compared with those from the fed animals suggested that i n vivo, the b i o l o g i c a l mechanisms for u t i l i z i n g l i p i d s was r a t hearts.  accelerated i n f a s t i n g  In f a c t , the t r i g l y c e r i d e l e v e l s of the fasted  group i n the "control" series appeared to decrease during perfusion, suggesting perhaps, that endogenous t r i g l y c e r i d e s may  have been u t i l i z e d under these perfusion conditions.  However, i n view of the unexplainable  increases i n t r i g l y -  ceride l e v e l s found under d i f f e r e n t perfusion conditions, i t i s d i f f i c u l t to state c a t e g o r i c a l l y at t h i s time that t r i glycerides were mobilized and oxidized by the working r a t heart.  At best, the data i s interpreted as only  that this might have been the case.  suggestive  3 OOf 27-01  ,24-0 2 1-0 z LU  g 18-0 o ^15-0 o a: 12-0 -  <t _j ZD  9-0 -  UJ  3.0  (I)  o or 6-0 >  2 3 4 DAYS FASTED  FIG. Leve1.  15  E f f e c t o f F a s t i n g on M y o c a r d i a l  Glycogen  R a t s were f a s t e d f o r the d u r a t i o n i n d i c a t e d . A r a t was r e - f e d ad l i b , on t h e s i x t h day and the v e n t r i c u l a r g l y c o g e n c o n t e n t determined a f t e r 24 hours r e - f e e d i n g (dotted l i n e ) . T i s s u e g l y c o g e n c o n t e n t s a r e shown as t h e means " i s t a n d a r d e r r o r o f the mean. The number o f a n i m a l used i s shown i n p a r e n t h e s i s . ~  68.  The increased rate of depletion of glycogen from epinephrineperfused hearts was expected.  I t was calculated that within  about 10 minutes, between 1.8  to 3.0 mg of glycogen a c t u a l l y  disappeared from a heart of a fasted r a t under the influence of epinephrine.  Since i t was d i f f i c u l t to believe that a l l  the glucose derived from glycogenolysis under these conditions was being converted to lactate, or even oxidized by the Krebs cycle, the perfusate was analysed for glucose, but none could be detected by the glucose-oxidase method. The mechanism of action of epinephrine on glycogenolysis i s perhaps the best understood of a l l hormones investigated. B r i e f l y , epinephrine promotes the formation of c y c l i c 3",5»AMP  from ATP by stimulation of an enzyme, adenyl cyclase.  C y c l i c 3',5'-AMP then brings about the conversion of inactive phosphorylase  b to active phosphorylase  phorylase t> kinase.  Phosphorylase  y i e l d i n g glucose-l-phosphate.  a., mediated by phos-  a stimulates glycogeno l y s i s ,  This scheme of glycogenolysis  has been reviewed by Sutherland (12). The data f o r the tissue t r i g l y c e r i d e series i s most d i f f i c u l t to interpret.  I f the increases i n t r i g l y c e r i d e content  of epinephrine and heparin perfused hearts were r e a l , this would be a most i n t e r e s t i n g observation, but the idea that t r i g l y c e r i d e s were a c t u a l l y synthesized during substrate-free perfusions i s most unacceptable to the author.  I t i s more  l i k e l y that some product of glycogenolysis was extracted into the organic layer during the extraction of tissue Chromotropic  lipids.  acid would then l a t e r combine with the compound  69. to give f a l s e l y high values f o r t r i g l y c e r i d e s . The experiments with epinephrine and heparin therefore yielded no u s e f u l information regarding the breakdown of t r i g l y c e r i d e by the heart.  On the other hand, t r i g l y c e r i d e s  may have been hydrolyzed and metabolized series.  i n the "CONTROL"  I t would appear that i n order to demonstrate c l e a r l y  whether t r i g l y c e r i d e s are indeed u t i l i z e d by the heart, perfusion conditions must be altered somehow so that hearts w i l l function normally f o r 60 minutes or more, even under substratefree medium.  Further studies using longer periods of perfusion  (without epinephrine or heparin) and using a larger population of r a t s would most l i k e l y be necessary before the  disappearance  of t r i g l y c e r i d e s i n r a t hearts can be conclusively demonstrated. Evidence has been presented which c l e a r l y establishes that a l i p o l y t i c a c t i v i t y other than lipoprotein lipase e x i s t s i n r a t myocardium.  The properties of the enzyme d i f f e r greatly  from those of l i p o p r o t e i n l i p a s e , so that the two enzymes can be distinguished even i n crude preparations.  In many  respects, the properties of the enzyme are similar to monoglyceride-hydrolyzing lipases reported i n adipose tissue (6, 15, 16) and i n t e s t i n a l mucosa (56, 58, 59), and p a r t i c u l a r l y to the Ediol --hydrolyzing enzyme system i n r a t hearts (13) . 8  The pH optimum of the monoglyceride-hydrolyzing  enzyme of  cardiac tissue agrees with the optimum pH of 6.8 reported by Bjorntorp and Furman (13) f o r an unspecified cardiac enzyme which a c t i v e l y hydrolyzed E d i o l . R  The monoglyceride-hydro-  l y z i n g a c t i v i t y i n adipose tissue described by Strand et a l .  (15) also has a pH optimum within t h i s range (pH 7.0).  On  the other hand, Kupieki (16) and Vaughan and co-workers (6) have reported pH optimums of 7.5 and 8.0,  respectively, f o r  the same monoglyceride-splitting enzyme i n adipose t i s s u e . These inconsistencies may  have arisen from the fact that  d i f f e r e n t buffers and monoglyceride substrates were used i n these studies.  The pH optimum of the cardiac enzyme i s s i g -  n i f i c a n t l y lower than that of the i n t e s t i n a l mucosa l i p a s e which i s reported to have an optimum pH of 7.8 9.0  (59).  (56) and  8.5-  The monoglyceride-splitting enzyme of r a t and  l i v e r has a pH optimum of 8.2  hog  (55).  The optimal temperature (45°)  f o r the jLn v i t r o  lipolytic  a c t i v i t y of the heart l i p a s e enzyme i s consistent with that found by Vaughan and associates (6) f o r the enzyme i n adipose tissue.  No other temperature optima have been reported f o r  further comparison. The r e l a t i v e a c t i v i t y of monoglyceride-hydrolyzing systems against d i - and t r i g l y c e r i d e s have been described for the i n t e s t i n a l mucosa.  Senior and Isselbacher  (56)  observed the comparative rates of t r i - , d i - , and monoglyceride hydrolysis to be 0.3,  1.6,  and 91.9  respectively.  Pope  and associates (59) found the r e l a t i v e rates of hydrolysis of t r i - , d i - , and monoolein to be 0..0, tively.  0.0,  and 1.0  respec-  McPherson jet a l . (58) observed that monoolein was  hydrolyzed  16 times as f a s t as t r i o l e i n .  In adipose tissue,  the comparative rates were 1, 43, 73, and 100 f o r t r i o l e i n , 1,2-diolein, 1,3-diolein, and monoolein respectively as  71.  reported by Strand and co-workers  (15). Again i n adipose  tissue, Kupieki's data (16) indicates the r e l a t i v e rates are 0.19, 0.17, and 1.00 f o r t r i - , d i - , and monostearin.  The  accumulated evidence indicates that whereas the i n t e s t i n a l mucosa enzyme i s highly s p e c i f i c f o r monoglycerides, the adipose tissue enzyme i s r e l a t i v e l y less s p e c i f i c i n this respect. In the present study, no experiments were designed s p e c i f i c a l l y to investigate the a c t i v i t y of the cardiac enzyme with respect to i t s a c t i v i t y against d i - and t r i g l y cerides.  However, from the data collected  from occasional  experiments i n which both t r i p a l m i t i n and monostearin (or monoolein) were used as substrates, i t may be r e a d i l y seen that the a c t i v i t y against monoolein and monostearin were s i g n i f i c a n t l y higher than against t r i p a l m i t i n (Table V). Comparison of t r i p a l m i t i n and monostearin hydrolysis i n one experiment ( F i g . 8) showed that the rate of monostearin hydrol y s i s was as much as 20-fold greater than that of t r i p a l m i t i n hydrolysis.  No d e f i n i t e statement can be made at present  regarding the absolute s p e c i f i c i t y of the cardiac enzyme. There i s no doubt that the enzyme hydrolyzes monoglycerides at an appreciably faster rate than i t does t r i g l y c e r i d e s , but much more data i s required to indicate i t s actual degree of substrate s p e c i f i c i t y .  For example, the hydrolysis rates of  mono-, d i - , and t r i o l e i n as compared with the rates of mono-, d i - , and t r i p a l m i t i n would be of i n t e r e s t .  I f i t i s established  that the enzyme i s highly s p e c i f i c f o r monoglycerides only, then i t s action on the 1- and 2-isomers of monoglycerides,  TABLE V Relative rates of Hydrolysis of Monoolein, Monostearin and T r i p a l m i t i n  SUBSTRATE  ACTIVITY mueq FFA/60 min  RELATIVE ACTIVITY (Tripalmitin-1.0)  EXPT. A  Monoolein Tripalmitin  34.0 2.8  12.1 1.0  EXPT. B  Monostearin Tripalmitin  42.0 11.0  3.8 1.0  EXPT. C  Monostearin Tripalmitin  208.0 38.0  5.2 1.0  In Experiment "A", the microsomal f r a c t i o n of heart tissue was used as the enzyme source. Standard assay conditions were used except that the reaction mixture containing monoolein and t r i o l e i n were incubated i n 0.05 M T r i s buffer, pH 7.0 and 8.5 respectively. In Experiment "B" and "C", the enzyme preparations were the i s o e l e c t r i c p r e c i p i t a t e . f r a c t i o n and whole homogenate (1:10) respectively. Standard assay conditions were employed.  73.  and i t s a c t i v i t y against medium and short chain monoglycerides. must be investigated.  Further studies of t h i s nature, of  course, must await a more extensive p u r i f i c a t i o n of the enzyme than has been obtained  i n this study.  The 757. i n h i b i t i o n of the cardiac l i p o l y t i c enzyme by 0.2 M NaF compares favourably with the s i m i l a r type of l i p o l y t i c a c t i v i t y reported i n adipose tissue (15, 16) and i n t e s t i n a l mucosa (59) where i n h i b i t i o n s of 84%., 45%, and 100% r e s p e c t i v e l y have been reported.  Since NaF does not  i n h i b i t l i p o p r o t e i n l i p a s e , the potent i n h i b i t o r y action of 0.2 M NaF must indicate the presence of another l i p o l y t i c system i n cardiac and other t i s s u e s .  The i n h i b i t i o n by r e l a -  t i v e l y low concentrations of DFP appears to be a common feature of monoglyceride-splitting enzymes. Thus, when discussing the p o s s i b i l i t y that the 300-fold p u r i f i e d monog l y c e r i d e - s p l i t t i n g enzyme i n the i n t e s t i n a l mucosa may be an esterase (rather than a l i p a s e ) , Pope and associates (59) have agreed that t h e i r enzyme was, i n fact, a l i p a s e , owing to the observation that "an increasing s o l u b i l i t y of the substrate i s associated with a decreasing rate of hydrolysis". I t was likewise indicated i n t h i s study that the increased a c t i v i t y of the cardiac enzyme was associated with the increased a v a i l a b i l i t y of insoluble substrates. The p h y s i o l o g i c a l r o l e of l i p o p r o t e i n lipase i n heart i s not completely understood at the present time.  I t s major r o l e  may be to hydrolyze exogenously supplied t r i g l y c e r i d e s to diglycerides, monoglycerides and FFA.  Since the enzyme i s  74.  r a p i d l y eluted from heart and adipose tissue ±n v i t r o by heparin, i t s l o c a t i o n on or near the plasma membrane has been postulated.  A l o u s i and Mallov (17) found the following d i s -  t r i b u t i o n of l i p o p r o t e i n l i p a s e i n cardiac c e l l s : f r a c t i o n , 1.33 ± 0.14;  mitochondrial  microsomal f r a c t i o n , 1.16 ± 0.14; 0.13.  nuclear  f r a c t i o n , 0.83 ± 0.25;  soluble f r a c t i o n , 0.99 ±  The t o t a l enzyme a c t i v i t y was 4.14 i 0.34.  The high  concentrations of the enzyme a c t i v i t y i n the nuclear and microsomal fractions give support to the concept that l i p o protein lipase i s e s s e n t i a l l y a membrane-located enzyme. 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 cardiac monoglyceride-hydrolyzing  lipase (Table IV) also indicated that the  enzyme i s membrane-bound.  Therefore, one might reasonably  speculate that the primary physiological r o l e of the monoglyceride-hydrolyzing  lipase i n cardiac tissue might be that  of completing the f i n a l step i n the hydrolysis of t r i g l y c e r i d e s . A s i m i l a r co-ordinated l i p o l y t i c system i s also thought to e x i s t i n the hydrolysis of t r i g l y c e r i d e s i n the i n t e s t i n a l t r a c t jln vivo.  I t i s known that pancreatic  lipase hydrolyzes  t r i g l y c e r i d e s to monoglycerides, and that i t s action essent i a l l y stops at t h i s stage of the hydrolytic process i n the i n t e s t i n a l lumen.  However, after monoglycerides are absorbed  into the i n t e s t i n a l mucosal c e l l s , they are e i t h e r further hydrolyzed to g l y c e r o l and FFA, or are r e - e s t e r i f i e d to higher glycerides.  I t i s highly a t t r a c t i v e to speculate that perhaps  in the case of cardiac c e l l s as well, the t r i g l y c e r i d e s i n chylomicrons and very low density lipoproteins are f i r s t hydro-  75. lyzed to monoglycerides at the outer surface of the c e l l s (including the lumen of the endoplasmic reticulum), then absorbed, and completely hydrolyzed by the monoglycerides p l i t t i n g l i p a s e , or r e - e s t e r i f i e d to higher glycerides.  How-  ever, this idea i s not supported by isotopic studies which indicated that t r i g l y c e r i d e s are taken up intact by the heart. Therefore one i s l e f t with the alternative idea that the actions of both l i p o p r o t e i n lipase and the monoglycerides p l i t t i n g lipase occur i n t r a c e l l u l a r l y , but on or near the inner aspect of the plasma membrane. Some of the problems encountered during attempts to p u r i f y the monoglyceride-splitting l i p a s e a c t i v i t y from cardiac tissue have already been b r i e f l y mentioned.  However, the d i f f i c u l t i e s  involved i n attempting to s o l u b i l i z e and p u r i f y the enzyme cannot be over-emphasized.  The enzyme was t i g h t l y bound to the  p a r t i c u l a t e materials of the c e l l and was extremely r e s i s t a n t to the usual s o l u b i l i z i n g techniques. and co-workers  I t i s noted that Pope  (59) used 0.37o sodium deoxycholate to rupture  the microsomal p a r t i c l e s and thus s o l u b i l i z e d the enzyme p r i o r to p u r i f i c a t i o n and study of the monoglyceride-hydrol y z i n g enzyme i n the i n t e s t i n a l mucosa.  In our hands, 0.17o  deoxycholate treatment resulted i n some s o l u b i l i z a t i o n , but t h i s technique also resulted i n low recoveries of a c t i v i t y . Although butanol extraction was considered as a possible technique, the reasonably s a t i s f a c t o r y s o l u b i l i z a t i o n obtained with Triton X-100 obviated the necessity f o r i t s use. The degree of p u r i f i c a t i o n of the monoglyceride-hydrol y z i n g enzyme i n heart tissue i s admittedly very small.  76.  Nevertheless,  i t should be born i n mind that with the possible  exception of plasma l i p o p r o t e i n lipase, pancreatic lipase and the most recently reported monoglyceride-hydrolyzing  lipase  of i n t e s t i n a l mucosa, extensive p u r i f i c a t i o n of lipase i n biol o g i c a l systems have been overwhelmingly unsuccessful.  Kupieki  (16) for example, succeeded i n obtaining a modest 4.8-fold p u r i f i c a t i o n of a monoglyceride-hydrolyzing tissue.  lipase from adipose  Nevertheless, when one considers the importance of  l i p i d metabolism i n mammalian systems, i t i s e s s e n t i a l that greater e f f o r t s be directed i n the future toward p u r i f y i n g and studying the lipase i n these organisms. The indiscriminate use of E d i o l  as substrate f o r mea-  suring l i p o l y t i c a c t i v i t y has been c r i t i c i z e d i n this thesis. With the aid of t r i p a l m i t i n - l - C  1 4  incorporated into E d i o l , R  the present study has shown that even when the hydrolysis of triglycerides in Ediol  was completely abolished, s i g n i f i c a n t  amounts of free f a t t y acids were s t i l l being produced during the i n i t i a l 20-25 minutes of the r e a c t i o n .  Since the addition  of 0.2 M NaF caused a further consistent and  significant  (70-757.) decrease of t h i s l i p o l y t i c a c t i v i t y , the source of these FFA*s must have been derived from the monostearin component of E d i o l .  From these observations,  incidentally,  TD  i t was  suggested that the unspecified E d i o l -hydrolyzing  a c t i v i t y i n extracts of r a t hearts which was reported by Bjorntorp and Furman (13) was probably e n t i r e l y due to the hydrolysis of the monoglyceride component. have recently looked upon the use of E d i o l  Other workers R  i n lipase studies  77.  with some suspicion.  Very recently, Kupieki (16) reported  that the l i p o l y t i c a c t i v i t y of an adipose tissue extract prepared as described by Rizack (107) liberated as much free f a t t y acids from monostearin as from E d i o l  R  ( i . e . , 10.80  versus 9.84 ueq/hr/100 mg tissue from monostearin and E d i o l , R  respectively).  Furthermore, Kupieki (16) found that the pH  optima were v i r t u a l l y indistinguishable when either of these substrates were used.  He concluded that ". . . i t appears  (that) when the hydrolysis of E d i o l  R  i s used to follow l i p o -  l y t i c a c t i v i t y , the r e s u l t s can be misleading;  the added TJ  monostearin which serves as an emulsifier i n E d i o l can account f o r a large part of the FFA released by t h i s  substrate".  The f i r s t hint, however, that the monoglyceride component of E d i o l ^ may be r a p i d l y hydrolyzed  was offered by Vaughan e t a l .  (6) who observed that g l y c e r o l production was not proportional to adipose tissue homogenate concentration when E d i o l used as substrate.  was  R  When the amount of g l y c e r o l derivable  from the stated amount of monostearin present i n E d i o l  R  was  subtracted from the amounts of g l y c e r o l produced, the "corrected" g l y c e r o l production was l i n e a r with respect to homogenate concentration and a straight l i n e was obtained through the o r i g i n .  The r e s u l t s of the present study there-  fore not only support the suspicions of Kupieki (16) and Vaughan et: al» (6) but provided stearin component i n E d i o l  d i r e c t evidence that the mono-  i s , i n f a c t , r a p i d l y hydrolyzed  by cardiac enzymes. I t i s safe to conclude from these studies that a lipase  78.  other than l i p o p r o t e i n lipase e x i s t s i n r a t myocardium. of i t s properties have been described.  Some  Much further study  w i l l be required to c l a r i f y i t s true p h y s i o l o g i c a l function i n o v e r a l l cardiac energy metabolism.  The knowledge w i l l  come only with success i n p u r i f y i n g the enzyme.  Whether i t  w i l l be subject to regulation through epinephrine or some other hormone can only be a matter of speculation at the present time.  79.  PART  STUDIES  ON 3 , 5 • - C Y C L I C 1  II  NUCLEOTIDE  PHOSPHODIESTERASE  so.  INTRODUCTION In recent years, the molecular regulation of c e l l u l a r a c t i v i t y has been one of the most intensively studied areas i n biochemistry.  I t i s not our purpose here to review the  mechanisms by which the l i v i n g c e l l regulates i t s metabolic activity.  We wish only to point out that many small molecules,  bearing no necessary s t r u c t u r a l r e l a t i o n to substrate or product can profoundly influence the action of an enzyme i n either a p o s i t i v e or negative d i r e c t i o n .  The physiological  implications of such a l l o s t e r i c e f f e c t s are boundless.  One  such compound which has attracted much attention as an e f f e c t o r or mediator of important enzymatic reactions i s adenosine S ' ^ ' - c y c l i c phosphate  (cyclic  3',5 -AMP). ,  The discovery of c y c l i c 3*,5*-AMP i n mammalian tissues i n 1958 originated from studies on l i v e r phosphorylase.  In  1951, Sutherland and C o r i (119) noted that when l i v e r s l i c e s were incubated with epinephrine and glucagon, the glycogen content decreased (glycogenolysis) and phosphorylase a c t i v i t y increased.  Studies on l i v e r phosphorylase, the enzyme which  degrades glycogen to glucose-1-phosphate, revealed that i t existed i n an active and inactive form.  Epinephrine was shown  to mediate glycogenolysis by s h i f t i n g the balance of l i v e r phosphorylase i n favour of the active enzyme.  The enzyme,  phosphorylase phosphatase, was isolated which catalyzed the inactivation of highly active l i v e r phosphorylase (120, 121). At this time, R a i l and Sutherland (122) also reported on  81.  another enzyme which catalyzed the conversion of i n a c t i v e l i v e r phosphorylase to the active form.  This enzyme, which was  given the name phosphorylase kinase, required Mg"*""^ ions and ATP f o r a c t i v i t y . concluded  From their studies, these investigators  that epinephrine stimulated glycogenolysis not by  acting on phosphorylase,  but by acting i n some way on the  l a t t e r enzyme, phosphorylase kinase. (123) then made the important  R a i l and h i s associates  observation that when p a r t i c u -  late f r a c t i o n s of dog l i v e r homogenates obtained by low speed centrifugation were incubated with epinephrine (or glucagon) together with ATP and M g  ++  ions, a heat-stable  factor was produced which stimulated the a c t i v a t i o n of inactive l i v e r phosphorylase.  I t became c l e a r that the action of  epinephrine was i n d i r e c t , i n that i t stimulated the synthesis of a heat-stable factor i n c e l l p a r t i c l e s which i n turn acted on phosphorylase kinase, with the r e s u l t that phosphorylase was activated, leading to increased glycogenolysis.  This  factor was soon isolated from dog l i v e r and c r y s t a l l i z e d by Sutherland and R a i l (124, 125) i n 1958, and was f i n a l l y characterized (126) as adenosine 3»,5»-cyclic phosphate (Fig. 16). An enzyme system i n l i v e r which catalyzed the formation of c y c l i c St^-AMP was f i r s t reported by R a i l and Sutherland (127) i n 1958.  In the presence of Mg"*"* ions, ATP, epinephrine  and glucagon, 1200 x g p a r t i c l e s of l i v e r (and also of heart, s k e l e t a l muscle and brain) formed s i g n i f i c a n t amounts of c y c l i c 3»,5 -AMP. I  The name "adenyl cyclase" was adopted by  FIG. 16 Structural formula of adenosine-3',5«-phosphate ( c y c l i c 3',5«-AMP).  83.  Sutherland et al.(128) f o r the enzyme.  Adenyl cyclase was  detected i n a l l animal tissues studied (128), except i n dog red blood c e l l s .  The tissues possessing the highest s p e c i f i c  a c t i v i t y were c e r e b r a l cortex and cerebellum of beef, c a l f , sheep and p i g .  Equally high a c t i v i t i e s were found i n l i v e r  flukes (Fasciola hepatica) and i n earthworms (Lumbricus terrestris) .  Intermediate  l e v e l s of c y c l i c 3», 5«-AMP-forming  a c t i v i t y were found i n t e s t i s , uterus, i n t e s t i n a l mucosa of dog, the l i v e r of cat and r a t , s k e l e t a l muscle of rabbit and blood c e l l s of pigeons.  Low i n adenyl cyclase a c t i v i t y were  r a t epididymal f a t pads, f l y larva, and minnow.  The r e l a t i v e  a c t i v i t i e s (on protein basis) i n dog tissues were:  brain  cortex (11.0), spleen (2.0), s k e l e t a l muscle (2.0), heart v e n t r i c l e (2.0), lung (1.5), kidney cortex (1.0), l i v e r (0.5), aorta (1.0), i n t e s t i n a l muscle (1.0), femoral artery (1.5) and adipose tissue (1.0).  Studies on the 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 t h i s enzyme indicated that i t might be derived from plasma membranes of from n u c l e i . Davoren and Sutherland  Later studies by  (129) with pigeon erythrocytes demon-  strated that no adenyl cyclase a c t i v i t y was associated with the n u c l e i .  I t appeared, therefore, that adenyl cyclase was  located on the plasma membrane of c e l l s .  The preparation o f  the enzyme i n a p u r i f i e d form was seriously hampered by i t s association with p a r t i c u l a t e materials of the "nuclear" f r a c tion, by i t s i n s t a b i l i t y , and by i t s close association with T r i t o n X-100 after s o l u b i l i z a t i o n (128).  The p u r i f i c a t i o n of  adenyl cyclase was 2- to 3-fold from brain and about 15-fold from l i v e r .  84.  I t was previously mentioned that epinephrine stimulated the synthesis of c y c l i c 3*,5i-AMP, and i t i s now known that the immediate s i t e of action of epinephrine i s adenyl cyclase. Murad and associates (130) studied the r e l a t i v e potencies of several catecholamines  on the adenyl cyclase system of dog  myocardial and l i v e r p a r t i c l e s .  In the myocardial system,  the r e l a t i v e potencies were as follows: L-isopropylnorepinephrine  (7.8), L-epinephrine (1.0), L-norepinephrine (1.0)  and D-epinephrine potencies were:  (0.12).  In the l i v e r system, the r e l a t i v e  L-isopropylnorepinephrine (4.0), L-epinephrine  (1.0), L-norepinephrine  (1.0).  .Dichloroisopropylnorepine-  phrine (DCI), an adrenergic blocking agent, blocked the stimulating e f f e c t of catecholamines.  The e f f e c t of epine-  phrine on the p a r t i c u l a t e preparations of adenyl cyclase from brain were also invetigated by Klainer ej: a± (131). These workers observed that i n the presence of epinephrine, a 2-fold increase i n c y c l i c 3*,5*-AMP formation by p a r t i c u l a t e preparations from the cerebellum was obtained.  Cyclase  preparations from the cerebral cortex, pons, medulla, and spinal cord were also stimulated by epinephrine. I t has become c l e a r that c y c l i c 3*,5*-AMP plays an important r o l e i n mediating the metabolic e f f e c t s of catecholamines. The p a r t i c i p a t i o n of c y c l i c S^M-AMP i n the glycogenolytic response of the l i v e r to epinephrine i s shown i n F i g . 17. E s s e n t i a l l y , a similar series of enzymatic  reactions occur i n  s k e l e t a l muscle (132) and i n cardiac muscle (133). b o l i c r o l e of c y c l i c 3*,5 -AMP i ,  n  The meta-  glycogenolysis has thus  Epinephrine (or glucagon) ATP  Adenyl Cyclase  »- C y c l i c 3«,5«-AMP Glycogen depho sphopho sphorylase kinase ATP M g + +  dephosphophosphorylase (inactive)  phosphorylase (active)  phosphorylase phosphatase  Glucose-1-P Pi  *—^| (phosphatase)  I  Glucose  FIG. 17 Mediation o f c y c l i c 3*,5*-AMP i n the glycogenolytic response o f l i v e r epinephrine (or glucagon).  86.  been f i r m l y established. Investigations by several workers have indicated that the r o l e of c y c l i c 3*,5,*-AMP i n b i o l o g i c a l  systems extends f a r  beyond i t s p a r t i c i p a t i o n i n the process of glycogenolysis. The a c t i v i t y of a number of enzyme systems (other than the phosphorylase system i n l i v e r , heart and s k e l e t a l muscle) have been shown to be influenced by c y c l i c 31,5*-AMP. Haynes and Berthet (134) found that the addition of adrenocortical hormone (ACTH) to adrenal tissue s l i c e s caused a rapid and s p e c i f i c a c t i v a t i o n of phosphorylase i n these tissues.  Furthermore,  Haynes (135) showed that when ACTH was added to s l i c e s of beef adrenal cortex, c y c l i c 3 5 -AMJ? accumulated 1  t  i  tissues.  i n these  When c y c l i c 3* ,5}-A3XE i t s e l f was added, phosphory-  lase a c t i v i t y increased. Thus, he concluded that the action of ACTH on the adrenal cortex was mediated by c y c l i c 3«,5*-AMP. Another enzyme which i s affected by c y c l i c 3»,5*-AMP i s glycogen synthetase (UDPG-ac-glucan Transglucosylase).  Belo-  copitow (136) observed a decrease i n glycogen synthetase a c t i v i t y i n r a t diaphragms after these tissues were incubated with epinephrine.  In order to investigate further the mecha-  nism of action of epinephrine on glycogen synthetase, he incubated a 4000 x g supernatant of r a t s k e l e t a l muscle homogenates with c y c l i c 3»,5«-AMP and ATP.  When c y c l i c 3»,5«-AMP  was omitted from the system, an increase i n synthetase a c t i v i t y was observed.  When both ATP and c y c l i c 3«,5'-AMP were omitted,  a further increase i n the enzyme a c t i v i t y was observed. observations led Belocopitow (136) to suggest that c y c l i c  These  87.  3»,5»-AMP may have been i n h i b i t i n g the synthetase system. Recently, Rosell-Perez and Larner (137)  showed that the action  of c y c l i c 3*,5*-AMP on the i n h i b i t i o n of glycogen synthetase a c t i v i t y was c l o s e l y associated with enhancing the  conversion  of the " I " (Independent,active) form of the enzyme to the "D" (Dependent,inactive) form.  I t has become evident,  therefore,  that the i n a c t i v a t i o n of glycogen synthetase coupled with the simultaneous a c t i v a t i o n of phosphorylase by c y c l i c 3*,5*-AMP would constitute an important r o l e for the c y c l i c  nucleotide  in the regulation of glycogen breakdown and synthesis. 1960,  In  Mansour and h i s co-workers (138) made an i n t e r e s t i n g  observation r e l a t i n g to the phosphorylase system i n the l i v e r fluke, F a s c i o l a tiepatica.  These workers demonstrated that  the a c t i v a t i o n of phosphorylase i n t h i s organism was obtained not with epinephrine,  but with 5-hydroxytryptamine (serotonin).  Furthermore, their studies showed that serotonin caused a rapid increase i n c y c l i c 3*,5*-AMP l e v e l s , and also that a c t i vation of phosphorylase was mediated by the c y c l i c nucleotide. Mansour (139) also demonstrated that g l y c o l y s i s i n homogenates of l i v e r flukes was regulated by the a c t i v i t y of phosphofructokinase  (PFK), and that stimulation of g l y c o l y s i s by seroto-  n i n resulted i n a marked increase i n PFK a c t i v i t y . and Mansour (140) reported  Mansour  that c y c l i c 3,»,5,»-AMP could a c t i -  vate l i v e r fluke PFK which had been i n h i b i t e d by ATP, and that the c y c l i c nucleotide could also activate an inactive preparation of PFK.  Similar e f f e c t s of c y c l i c 3*,5*-AMP i n  PFK a c t i v i t y have been demonstrated with the enzyme isolated from mammalian cardiac tissue (141, 142) and s k e l e t a l  88.  muscle (143) • The observation  that c y c l i c 3*,5*-AMP mediated the glyco-  genolytic response of the l i v e r to epinephrine  led investiga-  tors to study the p o s s i b i l i t y that c y c l i c 3«,5»-AMP might also mediate the l i p o l y t i c response of adipose tissue to epinephrine.  Indeed, Rizack (144)  showed that an epinephrine-  sensitive l i p a s e i n c e l l - f r e e extracts of adipose tissue could be activated by the addition of c y c l i c 3*,5»-AMP i n the presence of ATP and Mg"*"** ions. (145)  Butcher and h i s co-workers  found that when a derivative of c y c l i c 3«,5«-AMP, N -2t6  O-dibutyryl c y c l i c AMP was incubated with i n t a c t isolated f a t c e l l s , l i p o l y s i s was stimulated  some 10-fold.  Hence, another  r o l e f o r c y c l i c 3*,5*-AMP, that of increasing the mobilizat i o n of free f a t t y acids from f a t depots, has been established. The enzyme, tryptophan pyrrolase, which opens the indole r i n g of tryptophan to y i e l d formylkynurenine, appears to e x i s t i n an active and an inactive form.  Although studies on the  e f f e c t s of c y c l i c 3»,5»-AMP on t h i s enzyme system have not been extensive, there are indications that t h i s enzyme can be converted from the inactive to the active form by c y c l i c 3*,5«AMP (146, 147), although a recent report (148)  indicates that  5*-AMP, guanine, guanosine and 5«-GMP were also highly e f f e c tive. There are several other b i o l o g i c a l processes i n which c y c l i c 3»,5»-AMP has been implicated.  For example, the p o s i -  t i v e inotropic response of the heart to epinephrine  and other  catecholamines has been widely studied and the indications  89.  are that this process may also be mediated by c y c l i c 3»,5*-AMP (149). and  Recent studies had shown that the inotropic response  the a c t i v a t i o n of cardiac phosphorylase were apparently  unrelated  (150, 151). Then Robison and h i s associates (149)  observed that a single dose of epinephrine  caused a 4-fold  increase i n c y c l i c 3*,5»-AMP l e v e l s i n cardiac tissue within 3 seconds a f t e r i n j e c t i o n , while the c o n t r a c t i l e force increased about 1.4-fold at 20 seconds.  These observations  therefore  favour the hypothesis that c y c l i c 3«,5»-AMP may mediate the inotropic response of the heart to catecholamines. I t was mentioned e a r l i e r that the a c t i v a t i o n of adrenoc o r t i c a l phosphorylase by ACTH was shown to be mediated by c y c l i c 3*,5»-AMP.  Since ACTH action on the adrenal  cortex  stimulates the synthesis of c o r t i c o s t e r o i d s , one might expect that c y c l i c 3«,5»-AMP i t s e l f might mimic the action of ACTH. Indeed, Haynes e t al.(152) found that when they added c y c l i c 3»,5«-AMP to fragments of incubating r a t adrenals, the t o t a l c o r t i c o i d production was stimulated almost 5-fold.  Recent  studies have indicated that the stimulation of steroid production by the adrenal cortex was not s o l e l y due to the a c t i v a t i o n of phosphorylase. 154,  Roberts and co-workers (153,  155) found that c y c l i c S'j^-AMP s e l e c t i v e l y stimulated  C-11/& hydroxylase a c t i v i t y i n r a t adrenal homogenates f o r t i f i e d with glucose-6-phosphate and NADP, r e s u l t i n g i n increased formation of corticosterone from exogenous terone or progesterone.  11-deoxycorticos-  They also showed that the conversion  of progesterone to 11/5 -hydroxyprogesterone was increased.  90. These workers concluded that the action of 3»,5*~AMP on the stimulation of steroidogenesis was independent of phosphorylase a c t i v a t i o n , NA13PH generation and the presence of endogenous c o r t i c o s t e r o i d precursors.  Roberts e t alo(156, 157)  showed that c y c l i c 3»,5*-AMP also stimulated the hydroxylation of 11-deoxycorticosterone to  18-hydroxy-ll-deoxycorticosterone,  as well as the conversion of exogenous c h o l e s t e r o l to pregnenolone by isolated r a t adrenal mitochondria.  Karaboyas and  K o r i t z (158) recently observed that c y c l i c 3*,5*-AMP stimulated the incorporation of acetate into corticosterone, and the conversion of c h o l e s t e r o l to corticosterone. Darrington and K i l p a t r i c k (159) AMP stimulated  A report by  indicates that c y c l i c 3»,5«-  the synthesis of two progestational steroids,  4-pregnen-20 oc-ol-3-one and progesterone by ovarian tissues of r a b b i t s . Pryor and Berthet  (160) reported that the incorporation  of leucine into protein of r a t l i v e r s l i c e s was inhibited when these tissues were incubated with c y c l i c 3«,5*-AMP or with glucagon.  Exton and Park (161, 162) have indicated that  the e f f e c t of these hormones on glueoncogenesis appears to be mediated by c y c l i c 3«,5»-AMP.  Furthermore, these authors  have suggested that the d i r e c t s i t e of c y c l i c 3*,5*-AMP action may be the a c t i v a t i o n of phosphopyruvate carboxylase hexose phosphate phosphatases.  and the  Strong evidence also e x i s t s  that c y c l i c 3*,5*-AMP may mediate the secretion of enzymes . from r a t parotid glands.  Bdolah and Schramm (163) have i n d i -  cated that when r a t parotid s l i c e s are incubated with d i b u t y r y l  91.  c y c l i c AMP,  the release of amylase from the glands i s stimu-  lated almost to the same extent as that observed with epinephrine.  O r l o f f and Handler (164) reported that the addition  of c y c l i c 3*,5*-AMP to media i n which toad bladders were immersed, caused a s i g n i f i c a n t increase i n the permeability of the membrane to water.  Since vasopressin ( a n t i d i u r e t i c  hormone) i s also known to e l i c i t the same response, these authors suggested that the a n t i d i u r e t i c action of vasopressin might also be mediated by c y c l i c 3*,f>*-AMP.  Recent studies  on the action of vasopressin on the toad bladder by Strauch and Langdon (165) and by Handler and associates (166) have indicated that the action of t h i s hormone may be a d i r e c t stimulation of adenyl cyclase.  The c e l l u l a r mode of action  of vasopressin has been recently reviewed by O r l o f f and Handler (167).  The decreased incorporation of acetate into  f a t t y acids and c h o l e s t e r o l of l i v e r s l i c e s , and the increased production of ketone bodies by epinephrine, glucagon and by c y c l i c 3»,5«-AMP has been reported by Berthet (168).  Cyclic  3*,5*-AMP has also been implicated i n sugar transport i n the thyroid gland (169), and i n the stimulation of hydrochloric acid secretion by gastric mucosa (170, 171).  The  widespread  interest i n c y c l i c 3*,5"-AMP has led to the appearance of several review a r t i c l e s , the most recent of which are those on the metabolic e f f e c t s of catecholamines by the following authors:  Sutherland and Robison (172), Butcher (173), Krebs  et al.(132), Mansour (174), and Exton and Park (162). The p a r t i c i p a t i o n of c y c l i c S'j^'-AMP i n glycogenolysis,  92.  steroidogenesis, ketogenesis,  l i p o l y s i s , a n t i d i u r e s i s and pos-  s i b l y other p h y s i o l o g i c a l processes  demonstrates c l e a r l y t h e  e x c e e d i n g l y d i v e r s i f i e d and i m p o r t a n t r o l e p l a y e d by t h i s c y c l i c nucleotide i n regulating various b i o l o g i c a l  processes.  I t would f o l l o w t h a t an e q u a l l y i m p o r t a n t p h y s i o l o g i c a l mechanism i s n e c e s s a r y  f o r t h e t e r m i n a t i o n o f the a c t i o n o f c y c l i c  3',5'-AMP i n b i o l o g i c a l systems.  Indeed, an enzyme w h i c h  h y d r o l y z e s c y c l i c 3',5*-AMP t o 5'-AMP i s p r e s e n t tissues.  i n most  The e x i s t e n c e o f such an enzyme a c t i v i t y was f i r s t  suggested by S u t h e r l a n d  and R a i l (124)  i n 1958 w h i l e  a u t h o r s were s t u d y i n g t h e p r o p e r t i e s o f c y c l i c formed by t i s s u e p a r t i c l e s .  3',5'-AMP  The enzyme has been  p u r i f i e d from b e e f h e a r t by B u t c h e r and S u t h e r l a n d r e c e n t l y from dog h e a r t by N a i r (176). Perrott-Yee  these  subsequently (175) and  E a r l i e r , Drummond and  (177) had s t u d i e d t h e d i s t r i b u t i o n o f t h e d i e s -  t e r a s e i n v a r i o u s mammalian t i s s u e s , and had found t h a t nervous t i s s u e s , p a r t i c u l a r l y t h e b r a i n , p o s s e s s e d by f a r the h i g h e s t a c t i v i t y . r a b b i t contained  The k i d n e y , h e a r t , s p l e e n and l i v e r o f  o n l y 10 - 257o o f t h e a c t i v i t y o f t h e b r a i n .  S t u d i e s made on the p r o p e r t i e s o f t h e d i e s t e r a s e from b r a i n r e v e a l e d i t s a b s o l u t e r e q u i r e m e n t f o r magnesium i o n s , and t h a t the p r o d u c t o f h y d r o l y s i s was e x c l u s i v e l y 5'-AMP. Considering the f u n c t i o n o f c y c l i c diverse b i o l o g i c a l processes, must p l a y a n i m p o r t a n t  3',5'-AMP i n so many  i t f o l l o w s that the d i e s t e r a s e  r o l e i n r e g u l a t i n g the a c t i o n o f the  c y c l i c nucleotide i nvarious tissues.  The enzyme i s more  a c t i v e i n b r a i n than i n any o t h e r t i s s u e .  Adenyl cyclase i s  also more active i n brain than elsewhere i n nature.  The  precise p h y s i o l o g i c a l function of c y c l i c 3«,5»-AMP i n nerve tissue i s a problem of paramount importance.  The work des-  cribed i n this part of the thesis constitutes a further study of the properties and p a r t i a l p u r i f i c a t i o n of c y c l i c 3*,5'-nucleotide phosphodiesterase from mammalian brain. Some studies on the d i s t r i b u t i o n of the enzyme throughout the plant and animal kingdom are also reported.  9 4 .  EXPERIMENTAL PROCEDURE  Materials Cyclic  3»,5'-AMP and c a l f i n t e s t i n a l adenosine deaminase  were purchased from Sigma Chemical Company.  C y c l i c 3',5'-GMP  and c y c l i c 3',5»-UMP were prepared by Smith, Drummond and Khorana (178).  Cyclic  3',5«-dAMP, c y c l i c 3«,5«-dGMP, c y c l i c  3',5»-dCMP and c y c l i c 3* ,5*-1MB were prepared by Drummond, Gilgan, Reiner and Smith (179).  Cyclic  2«,3«-AMP was pre-  pared by the method of Smith, Moffatt and Khorana (180). The c y c l i c nucleotides 3»,5«-dAMP, 3«,5«-GMP and 3',5«-dGMP were chromatographically pure.  Crotalus adamanteus venom was  purchased from Ross A l l e n ' s Reptile Institute,  S i l v e r Springs,  F l o r i d a and from Sigma Chemical Company.  Methods Standard Assays - Routinely, c y c l i c 3«,5'-nucleotide phosphodiesterase a c t i v i t y was assayed according to Butcher and Sutherland (175) by measuring the release of inorganic phosphate when Crotalus adamanteus venom was included i n the assay system.  The snake venom contains a potent 5'-nucleoti-  dase which hydrolyzes 5'-nucleotides to give the corresponding nucleoside and inorganic phosphate.  The reaction mixture  contained 0.54 umoles c y c l i c 3',5'-AMP, 0.90 umoles MgS04,  95. 0.1 to 0.4 rag snake venom, 88 umoles T r i s buffer, pH 7.5, with an appropriate d i l u t i o n of the phosphodiesterase sample being assayed, i n a t o t a l volume of 0.9 ml.  Incubations were per-  formed at 30° f o r 30 minutes, and the reaction terminated by the addition of 0.1 ml cold 557. t r i c h l o r o a c e t i c  acid.  The  reaction tubes were centrifuged f o r 10 minutes at 10,000 x g to sediment the denatured proteins, and 0.5 ml aliquots of the supernatant f l u i d taken for inorganic phosphate  analysis  based on the method of Fiske and SubbaRow (181) as modified by Butcher and Sutherland (175).  Thus, to 0.5 ml aliquots of  the supernate were added 0.1 ml 2.57. ammonium molybdate solution i n 5 N I^SO^, 0.35 ml g l a s s - d i s t i l l e d water and 0.05 reducing agent.  ml  Colour was allowed to develop f o r 15 minutes  before reading at 720 mu i n a Beckman Model DU spectrophotometer, using a l i g h t path of 1.0 cm.  The standard curve f o r  inorganic phosphate as measured by this method i s shown i n F i g . 18.  One unit of enzyme a c t i v i t y i s defined as that  amount which caused the l i b e r a t i o n of 1 umole of inorganic phosphate  i n 30 minutes at 30°.  The s p e c i f i c a c t i v i t y of the  enzyme i s defined as the umoles of inorganic phosphate released per mg of enzyme protein i n 30 minutes at 30°. For k i n e t i c experiments, the assay was based on the conversion of c y c l i c SS^'-AMP to inosine i n the presence of brain extract, snake venom and i n t e s t i n a l adenosine deaminase. (One unit of deaminase a c t i v i t y i s defined as the number of umoles of adenosine deaminated per minute at 30° i n 0.1 M c i t r a t e buffer at pH 6.5 at an adenosine concentration of  INORGANIC PHOSPHATE  FIG. 18  Inorganic  ADDED (mjumoles)  phosphate c o n c e n t r a t i o n  curve.  To a s e r i e s o f tubes c o n t a i n i n g 0.2 m l 0.4 M T r i s , pH 7.5, 0.05 m l 18 mM.MgSO^, 0.10 m l 5 5 % t r i c h l o r o a c e t i c a c i d , were added t h e i n d i c a t e d amounts o f i n o r g a n i c phosphate and s u f f i c i e n t g l a s s - d i s t i l l e d water t o make a f i n a l volume o f 1.0 m l . An 0.5 m l a l i q u o t was t a k e n from each tube and assayed f o r i n o r g a n i c phosphate as d e s c r i b e d i n the t e x t .  97.  0.45 x 10~4 M.)  The concentrations of c y c l i c 3,*,5»-AMP were  between 13 to 52 uM.  The reaction mixture also contained 1.0  mM MgSO^ and 88 mM T r i s buffer at pH 7.5 i n a f i n a l volume of 1.5 ml.  A f t e r the addition of c y c l i c 3*,5*-nucleotide phos-  phodiesterase, the decrease i n absorbancy was followed a t 265 mp at 1-minute i n t e r v a l s using a Beckman Model DU spectrophotometer and a l i g h t path of 0.5 cm ( F i g . 19). For c e r t a i n experiments where the i d e n t i f i c a t i o n of the reaction products by paper chromatography was considered advantageous,  the assay system consisted of 0.25 umoles c y c l i c  3»,5»-AMP, 0.8 mM MgS0 , 75-150 mM T r i s buffer, pH 7.5, and 4  enzyme i n a t o t a l volume of 0.2 ml. venom was used.  In t h i s system, no snake  The reaction was stopped a f t e r 15 minutes  incubation a t 30° by the addition of 0.02 ml g l a c i a l acetic acid.  The tubes were centrifuged at 10,000 x g f o r 15 minutes  and 0.02 ml aliquots of the supernate spotted on Whatman No. 1 f i l t e r paper.  The chromatograms were developed by descending  technique with isopropanol-ammonium hydroxide-0.1 M boric acid (7:1:2).  This solvent system e f f e c t i v e l y separates adenine,  adenosine, and the adenosine  nucleotides.  Stock solutions of snake venom (8 mg/ml) used i n the diesterase assay were prepared by d i s s o l v i n g the l y o p h i l i z e d powder i n 0.02 M T r i s , pH 7.5. Centrifugation was occasionally required  to sediment insoluble p a r t i c l e s .  The snake venom  a c t i v i t y was completely stable to repeated freezing and thawing over a period of several months.  Protein was measured  by the biuret method (182) and by the o p t i c a l method of War-  98.  If—.  e L/l  0  1  1  2  3  L 4  1—i  5  1 6  1 T  1  1  8  9  L. 10  M I N U T E S  FIG. 19  Spectrophotometrie Assay of Phosphodiesterase.  The i n c u b a t i o n m i x t u r e c o n t a i n e d 0.03 mM c y c l i c 3',5'-AMP, 1.0' mM MgS0 , 88 mM T r i s , pH 7.5, 0.15 u n i t s o f i n t e s t i n a l adenosine deaminase, 0.32 mg snake venom, and 27 pg r a b b i t b r a i n p h o s p h o d i e s t e r a s e i n a f i n a l volume of. 1.5 m l . The r e a c t i o n was s t a r t e d by the a d d i t i o n o f the d i e s t e r a s e and ~Zb>265 r e c o r d e d a t 1 minute i n t e r v a l s . 4  w  a  s  99.  burg and C h r i s t i a n (183). Enzyme P u r i f i c a t i o n - Mature rabbits were stunned by a blow behind the head and the neck vessels severed immediately. The brains were removed, placed i n i c e and usually frozen before use.  Only the cerebral lobes were used f o r the prepa-  r a t i o n of the extract.  The tissue was homogenized i n 10  volumes of 0.25 M unbuffered sucrose f o r 5 minutes at 0-4° using a glass mortar f i t t e d with a motor-driven t e f l o n p e s t l e . A l l subsequent procedures, unless otherwise noted, were performed at 0-4°.  The homogenate was centrifuged at 105,000 x  g x 30 minutes, and the supernatant f l u i d thus obtained was set aside.  The 105,000 x g sediment was re-homogenized with  5 o r i g i n a l volumes of 0.25 M sucrose containing 0.1% deoxycholate.  The homogenate was centrifuged f o r 30 minutes at  105,000 x g, and the supernatant f l u i d s combined f o r the f o l lowing ammonium sulfate f r a c t i o n a t i o n step.  I t was found  l a t e r that the centrifugation could be more conveniently performed at 37,000 x g x 60 minutes, giving; e s s e n t i a l l y the same degree of p u r i f i c a t i o n . Step 1 - Ammonium Sulfate Fractionation - The supernatant f l u i d was adjusted to 0.3 saturation by the addition of s o l i d enzyme-grade ammonium sulfate with constant s t i r r i n g over a 15-minute period.  The pH was maintained between 6.9 and 7.1  by the dropwise addition of 1.0 N KOH.  A f t e r at l e a s t 15  minutes e q u i l i b r a t i o n , the p r e c i p i t a t e was collected by cent r i f u g i n g f o r 20 minutes at 37,000 x g. The p r e c i p i t a t e was taken up to approximately 15% of the o r i g i n a l combined super-  100.  natant f l u i d volume using 1 mM imidazole,  pH 7.5 containing  1 mM MgSO^.^ The milky extract was dialyzed against 300 v o l umes of the same buffer (pH 7.5) f o r 3 hours i n the cold room. Step-2 - Repeated"Freezing-and Thawing - A f t e r d i a l y z i n g , the extract was centrifuged at 37,000 x g x 15 minutes to remove the heavy f l o c c u l e n t material, and the s l i g h t l y cloudy supernatant f l u i d was stored at -20°.  Upon thawing the extract,  more f l o c c u l e n t material always appeared, which was r e a d i l y removed by centrifugation.  The supernate was re-frozen,  thawed and centrifuged once more before taking the preparat i o n to the next p u r i f i c a t i o n step, or was stored at -20°. About 5% of the enzyme a c t i v i t y was l o s t with the sediment, but no attempt was made to recover t h i s a c t i v i t y .  The repeated  freezing and thawing of the f i r s t ammonium sulfate f r a c t i o n usually gave a 6- to 10-fold p u r i f i c a t i o n of the enzyme, and an o v e r a l l y i e l d of about 15 to 207.. Step 3 - Heat Benaturation and Acid P r e c i p i t a t i o n - A f t e r the repeated freezing and thawing procedure, 0.11 volumes of 0.5 M imidazole,  pH 7.5, and 0.02 volumes of. 0.5 M glycine, pH 10,  was added to the c l e a r supernate.  The pH was taken to 10 by  the addition of 1 N KOH, and the temperature of the solution brought quickly to 45°.  A f t e r maintaining t h i s temperature  for about 15-16 minutes, the solution was immediately c h i l l e d by immersion i n an ice-bath.  The solution was then slowly  taken to pH 5.8 with 0.3 N acetic acid and s t i r r e d f o r at l e a s t 15 minutes before centrifuging at 37,000 x g x 45 min-  101* utes.  The p r e c i p i t a t e was discarded, and the pH of the super-  nate brought back to 7.5 with 0.5 N KOH.  The solution thus  obtained was dialyzed against 300 volumes ofl.jmM imidazole, pH 7.5 containing 1 mM MgSO^ pH 7.5, with constant for  stirring  at least 6 hours. Although  the f i r s t ammonium sulfate step and the freezing  and thawing gave reasonably consistent degrees of p u r i f i c a t i o n , the alkaline-heat, a c i d - p r e c i p i t a t i o n step gave r e s u l t s which varied from one preparation to another.  Unless otherwise  indi-  cated, t h i s preparation was used f o r studying the properties of the brain phosphodiesterase.  The o v e r a l l y i e l d at t h i s  step was about 5 to 10%, and the p u r i f i c a t i o n obtained ranged from 8- to 16-fold.  The enzyme became exceedingly unstable  with increasing p u r i f i c a t i o n , probably owing to the d i l u t i o n of  the enzyme during and a f t e r the alkaline-heat step.  Con-  centration of the 6-hour dialysate obtained from the a l k a l i n e heat step was accomplished  by immersion of the d i a l y s i s bag  into 1 l i t r e of a 60% solution of sucrose.  This technique  resulted i n a 90%. decrease i n volume of the dialysate within a few hours.  The concentrated enzyme was  stored at -20° for  one week with no appreciable loss of a c t i v i t y . Further p u r i f i c a t i o n of the enzyme could be obtained by taking a second (0.3 to 0.6  saturation) ammonium sulfate f r a c -  tion after the alkaline-heat step.  However, despite the 15  to 25-fold p u r i f i c a t i o n obtained by t h i s method, the f i n a l y i e l d of enzyme a c t i v i t y was as a routine procedure was  low;  hence the use of this step  impractical. Nevertheless,  this  102.  preparation was occasionally used i n some of the experiments where the use of a more highly p u r i f i e d preparation was i n d l cated.  103.  RESULTS 1.  Preliminary - Before p u r i f i c a t i o n o f the c y c l i c 3',5'-  nucleotide phosphodiesterase from brain was attempted, the enzymatic components of the diesterase assay system were examined.  The snake venom used i n the diesterase assay con-  tains a potent 5*-nucleotidase which hydrolyzes 5 -AMP to !  adenosine and phosphate.  I t was therefore necessary to deter-  mine the minimum quantity of snake venom required to hydrolyze a l l of the 5*-AMP produced by the diesterase under standard assay conditions.  As may be seen i n F i g . 20, 0.36 umoles of  5 -AMP was almost completely hydrolyzed by 30 ug snake venom ,  at 30° i n 10 minutes.  When the standard assay system was  subsequently developed which contained 0.54 umoles c y c l i c 3*,5»-AMP, an excess (100-400 ug) of snake venom was routinely used i n the incubation mixture i n order to eliminate any poss i b i l i t y of the 5*-nucleotidase l i m i t i n g the o v e r a l l rate.  reaction  When 100 pg of snake venom was used, no c y c l i c S ^ 1  1  nucleotide phosphodiesterase could be detected i n the venom preparation.  However, i n some l a t e r experiments, larger  amounts of snake venom were used i n the assay system.  Analy-  s i s f o r the presence o f c y c l i c 3*,5*-AMP hydrolyzing a c t i v i t y at these higher concentrations o f venom indicated that a trace of diesterase a c t i v i t y was present, as shown i n Table VI. Although the presence of diesterase a c t i v i t y i n the snake venom had i n s i g n i f i c a n t e f f e c t on the results o f most experiments where brain diesterase a c t i v i t y being measured was high, data from those few experiments where the a c t i v i t y was low  104.  O a.  o < CO  0  10 20 30 40 50 60 70 SNAKE VENOM ADDED  80 90 100 (He)  FIG. 20 H y d r o l y s i s o f 5'-AMP by snake venom adamanteus).  (Crotalus  The i n c u b a t i o n m i x t u r e c o n t a i n e d 0.36 umoles 5'-AMP, 20 mM MgSO^, 40 mM T r i s b u f f e r , pH 7.5, i n a f i n a l volume o f 1.0 m l . The i n d i c a t e d amounts o f snake venom were added t o i n i t i a t e t h e r e a c t i o n . The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n o f 0.1 m l 55% t r i c h l o r o a c e t i c a c i d a f t e r 10 minutes- i n c u b a t i o n a t 30°, Phosphate was a n a l y z e d a s described i n the t e x t .  105.  TABLE VI Relative A c t i v i t i e s of 5'-Nucleotidase and C y c l i c 3',5'Nucleotide Phosphodiesterase i n Snake Venom. The incubation mixture contained either 0.72 umoles c y c l i c 3',5'-AMP or 5'-AMP, 18 mM MgS0 , 0.04 M T r i s , pH 7.5, the indicated amounts of Crotalus adamanteus venom (Sigma) and s u f f i c i e n t water to make 0.9 mT~. The reaction was stopped by the addition of 0.1 ml 55% t r i c h l o r o a c e t i c acid after 30 minutes incubation at 30°. Inorganic phosphate was assayed according to the standard procedure as described i n the text. 4  Amount of Snake Venom added (ug)  jamoles P i released from 5'-AMP  & from cyclic 3',5'-AMP  0.43  0.01  0  0.86  0.03  0  2.15  0.09  0  4.3  0.24  0  8.6  0.45  0  21.5  0.75  .002  43.0  0.75  .004  129.0  0.77  215.0  0,74  .010  106.  was corrected f o r the inherent c y c l i c 3 ,5'-nucleotide diess  terase i n the venom.  Experiments indicated that 400 ug snake  venom was capable of hydrolyzing 0.02  umoles c y c l i c 3*,5«-AMP  i n 30 minutes at 30°, and therefore this correction factor was  used. Preliminary experiments designed to test the v a l i d i t y of  the coupled enzyme assay indicated that the rate of c y c l i c 3«,5«-AMP hydrolysis was  d i r e c t l y proportional to the amount  of diesterase used ( F i g . 21). f o r these experiments was  The enzyme preparation used  an extract of an acetone powder  which had been prepared 2 years previously and stored at -20°. The experiments therefore indicated, i n addition, that the brain diesterase was  quite stable to storage when prepared i n  acetone powder form. 2.  P a r t i a l - P u r i f i c a t i o n of Brain Phosphodiesterase - Drummond  and Perrott-Yee  (177) reported  rabbit brain diesterase was  that a p a r t i a l p u r i f i c a t i o n of  r e a d i l y obtained  by taking a  20,000 x g supernatant f r a c t i o n of the whole homogenate to 0.4  saturation with ammonium s u l f a t e .  recovered  from the p r e c i p i t a t e .  The enzyme a c t i v i t y  was  The present study also found  most of the a c t i v i t y associated with the 0.4  saturated ammo-  nium sulfate f r a c t i o n , although the highest s p e c i f i c a c t i v i t y was  recovered  i n the 0.40-0.45 saturated f r a c t i o n .  I t was  observed, however, that when 0.17. sodium deoxycholate  was  included i n the 0.25 M sucrose solution used f o r re-homogeni z i n g the i n i t i a l 105,000 x g sediment, the diesterase precipitated at lower ammonium sulfate concentrations.  was  107.  0  0-22  0-44  PROTEIN  0-66  0-88  I'TO  ADDED (mg)  FIG. 21 P h o s p h o d i e s t e r a s e A c t i v i t y as a L i n e a r of Protein Concentration.  Function  The r e a c t i o n m i x t u r e c o n t a i n e d 0.36 pinoles c y c l i c 3',5 AMP, 20 mM MgSO^, 40 mM T r i s , pH 7.5, 60 p g snake venom, t h e i n d i c a t e d amounts o f p r o t e i n and s u f f i c i e n t water t o make a f i n a l volume o f 0 . 9 m l . The r e a c t i o n was stopped by t h e a d d i t i o n o f 0.1 m l t r i c h l o r o a c e t i c a c i d a f t e r 30 m i n u t e s i n c u b a t i o n a t 30°. The d i e s t e r a s e p r e p a r a t i o n used was an acetone powder e x t r a c t o f b e e f b r a i n . P i was assayed as described i n the t e x t . 1  108. Furthermore, repeated freezing and thawing of the dialyzed extract obtained from the 0.3 saturated ammonium sulfate f r a c t i o n c o n s i s t e n t l y resulted i n a 6-10-fold p u r i f i c a t i o n .  The  alkaline-heat treatment followed by the acid p r e c i p i t a t i o n frequently gave an a d d i t i o n a l 2-fold p u r i f i c a t i o n .  An example  of the p u r i f i c a t i o n of the diesterase from rabbit brain i s shown i n Table VII.  The s p e c i f i c a c t i v i t y of the f i n a l prepa-  r a t i o n from brain was about h a l f of that reported by Butcher and Sutherland  (175) who p u r i f i e d the enzyme from beef heart.  While t h i s work was i n progress, Nair (176) also reported p u r i f y i n g the diesterase from dog heart.  The s p e c i f i c a c t i -  v i t y of the p u r i f i e d enzyme from dog heart was around 27.5, which i s i n the same range as that obtained f o r the beef heart enzyme.  Attempts to p u r i f y the brain enzyme by the use of  Sephadex G-200 columns, or by adsorption on calcium phosphate gels under various conditions were unsuccessful.  Several  attempts to p u r i f y the enzyme on DEAE-cellulose columns were also unsuccessful, owing to the i n s t a b i l i t y of the enzyme i n d i l u t e solutions. 3.  Properties of the P a r t i a l l y P u r i f i e d Phosphodiesterase (a)  -  The i n i t i a l studies on brain diesterase by Drummond  and Perrott-Yee  (177) indicated that the r a b b i t brain dies-  terase had an absolute requirement f o r Mg"*"" ions and was com1  p l e t e l y inhibited by EDTA (1.0 mM). f u l l y confirmed (b)  i n the present  These observations were  study.  The e f f e c t of 0.06 M imidazole on brain diesterase  a c t i v i t y was investigated. As may be seen i n F i g . 22, imi-  109.  TABLE VII P a r t i a l P u r i f i c a t i o n and Y i e l d of C y c l i c 3*,5'-nucleotide Phosphodiesterase from Rabbit Brain. The cerebral cortex from a rabbit brain was fractionated as described i n the text. A c t i v i t i e s are also defined i n the text.  Fraction  Total Activity  Specific Activity  7o Yie°ld  Purification  Homogenate  728  0.9  (100)  1.0  Combined Supernate (37,000 x g)  660  2.3  90  2.4  0.3 Ammonium sulfate  159  4.3  22  4.6  Fro zen- Thawed twic e  156  9.0  21  9.2  20  14.0  2.7  14.8  Alkaline-Heat and Acid p r e c i p i t a t i o n  110.  pH  F I G . 22 pH Curve o f B r a i n C y c l i c 3«,5'-Nucleotide P h o s p h o d i e s t e r a s e and E f f e c t o f I m i d a z o l e . The r e a c t i o n m i x t u r e c o n t a i n e d 0.8 mM c y c l i c S'j^'-AMP, 1.3 mM MgSOA, 4 ug enzyme p r o t e i n o f s p e c i f i c a c t i v i t y 19 u n i t s , and 0.06 M T r i s p l u s i m i d a z o l e o r 0.12 M T r i s . A f t e r a 20-minute i n c u b a t i o n a t 30°, t h e r e a c t i o n was t e r m i n a t e d by h e a t i n g t h e tubes i n b o i l i n g w a t e r f o r 1 m i n u t e . The pH o f t h e r e a c t i o n m i x t u r e s were a d j u s t e d t o n e u t r a l i t y , snake venom (400 ug) was added, and t h e tubes i n c u b a t e d f o r 10 minutes a t 30°. The r e a c t i o n xvas stopped by the a d d i t i o n o f 0.1 ml c o l d t r i c h l o r o a c e t i c a c i d . I n o r g a n i c phosphate was assayed as d e s c r i b e d i n t h e t e x t .  111.  dazole caused a s i g n i f i c a n t  increase i n diesterase a c t i v i t y  over the entire pH range examined.  Peak enzyme a c t i v i t y  near pH 7.0, whether imidazole was present or not.  was  Stimula-  tion of the beef heart enzyme by imidazole has also been reported by Butcher and Sutherland (175), although their data indicated l i t t l e stimulation at pH (c)  Inhibition  8.5.  by Theophylline i n v i t r o - The methyl  xanthines, p a r t i c u l a r l y  theophylline, are known to i n h i b i t  c y c l i c 3*,5*-nucleotide phosphodiesterase.  The e f f e c t of  z x 10*"% theophylline on the p a r t i a l l y p u r i f i e d terase a c t i v i t y was i n F i g . 23.  brain dies-  investigated, and these r e s u l t s  are shown  The i n h i b i t i o n of brain diesterase by theophylline  appears to be competitive i n nature.  The  of the enzyme  was about 0.8 x 10*"M with c y c l i c 3*,5»-AMP as substrate. 4  The  value obtained indicated a marked s i m i l a r i t y to those  values reported f o r the beef heart enzyme. ever, has reported K  m  Nair (176), how-  values near 4.9 x 1 0 " %  for the dog  heart diesterase. (d)  Cyclic  3t,5«-dAMP/cyclic 3«,5«-AMP a c t i v i t y  in successive fractions obtained during p u r i f i c a t i o n  ratios - I t was  reported (177) that the brain diesterase hydrolyzed c y c l i c  3*,  5«-dAMP at about 50% of the rate at which c y c l i c 3«,5«-AMP was hydrolyzed.  The present studies showed that the c y c l i c  3*,5»-dAMP/cyclic 3«,5»-AMP a c t i v i t y r a t i o s were about  0.45.  In order to determine whether c y c l i c 3*,5*-dAMP and c y c l i c 3*,5*-AMP might be hydrolyzed by the same or d i f f e r e n t enzyme, the c y c l i c 3* ,5*-dAMP/cyclic  3»,5*-AMP a c t i v i t y r a t i o s were  112.  FIG. 23. Nature o f B r a i n C y c l i c 3 • , 5 • - N u c l e o t i d e P h o s p h o d i e s t e r a s e I n h i b i t i o n by T h e o p h y l l i n e . The p a r t i a l l y p u r i f i e d b r a i n d i e s t e r a s e p r e p a r a t i o n ( s p e c i f i c a c t i v i t y , 9.1) used i n these e x p e r i m e n t s was s t o r e d a t -20° i n a c o n c e n t r a t e d s u c r o s e s o l u t i o n . A f t e r t h a w i n g and making the a p p r o p r i a t e d i l u t i o n , the enzyme p r e p a r a t i o n was i n c u b a t e d i n the presence o f 2 x 10"^M t h e o p h y l l i n e as d e s c r i b e d i n the t e x t f o r the s p e c t r o p h o t o m e t r i e assay.  \ \  113.  determined i n the various fractions obtained during p u r i f i cation.  These r e s u l t s are shown i n Table VIII.  there was a s i g n i f i c a n t  Although  difference between the r a t i o s obtained  for the whole homogenate and the combined supernatant f r a c t i o n s , the succeeding fractions showed i n s i g n i f i c a n t  differences.  I t was also observed that when equimolar concentrations of c y c l i c S^S'-dAMP were included with c y c l i c 3',5*-AMP i n the standard phosphodiesterase assays, a 15-197. i n h i b i t i o n diesterase a c t i v i t y was consistently obtained.  of  These obser-  vations strongly indicate that both c y c l i c 3»,5»-AMP and c y c l i c 3 ,5 -dAMP are hydrolyzed by the same enzyme. ,  ,  However,  further investigations are necessary i n order to show more conclusively whether these compounds are indeed hydrolyzed by the same diesterase. (e)  Hydrolysis Rates of other Purine and Pyrimidine  c y c l i c 3*,5«-nucleotides - The rate of hydrolysis of other c y c l i c 3 ,5^-nucleotides were investigated i n order to further 1  determine the s p e c i f i c i t y of the brain phosphodiesterase. r e l a t i v e hydrolysis rates of a l l the c y c l i c  The  3*,5'-nucleotides  which were investigated are l i s t e d i n Table IX.  Included f o r  comparison are the rates obtained f o r the brain and heart enzyme extracts by other investigators.  The data c l e a r l y  demonstrate:;, the high s p e c i f i c i t y of the enzyme f o r purine c y c l i c 3*,5*-nucleotides.  C y c l i c 3»,5»-UMP was  the only  pyrimidine c y c l i c nucleotide which was hydrolyzed appreciably by the brain diesterase preparation, although the a c t i v i t y was only about 137. of c y c l i c 3*,5*-AMP hydrolysis rates.  114.  TABLE VIII Cyclic  3S5«-dArtP/cyclic 3«,5»-AMP a c t i v i t y  ratios.  The c y c l i c 3*,5*-nucleotide phosphodiesterase was part i a l l y p u r i f i e d as described i n the text. The s p e c i f i c a c t i v i t y of the f i n a l preparation was 15.0. Assays were performed by the standard method as described i n the text, except that c y c l i c 3»,5»-dAMP was used at a concentration of 0.50 umoles/ml.  Fraction  c y c l i c 3',5'-dAMP c y c l i c 3«,5»-AMP  Whole Homogenate  .76  Combined 105,000 x g supernatant  .53  0.3 Ammonium sulfate Frozen-Thawed x2  .52  Alkaline-heat, Acid treatment  .45  Activity Ratio  TABLE I X R e l a t i v e H y d r o l y s i s R a t e s o f P u r i n e and P y r i m i d i n e C y c l i c 3«,5«-Nucleotides. D i e s t e r a s e a c t i v i t i e s were assayed by t h e standard p r o c e d u r e , e x c e p t t h a t the o t h e r c y c l i c 3»,5"-nucleotides as i n d i c a t e d were s u b s t i t u t e d f o r c y c l i c 3»,5«AMP i n t h e a s s a y i n e q u i m o l a r c o n c e n t r a t i o n s . THIS STUDY (Rabbit brain)  DRUMMOND (Rabbit brain)  BUTCHER & SUTHERLAND (175) (Beef h e a r t )  3*,5»-AMP  1,00  1.00  1.00  3»,5«-dAMP  0.45  3»,5»-GMP  0.50  3»,5«-dGMP  0.48  3»,5»-CMP  0.0  3«,5«-dCMP  <0.04  3»,5»-UMP  0.13  3«,5«-TMP  <0.07  COMPOUND  (X77)  0.33  NAIR (176) (Dog, heart)  DRUMMOND et al(189) TBeef brain)  1.00  1.00  1.30  0.50  0.33 0.44  0.0  0 0.10  0.11  0.17  0.12-0.15 0.10  116.  Hardman and Sutherland  (184) have recently reported p u r i f y i n g  a c y c l i c 3*,5*-nucleotide phosphodiesterase from beef heart that hydrolyzed i t hydrolyzed  c y c l i c S^^-IMP at a much faster rate than  c y c l i c 3»,5*-AMP.  Their findings suggest the  p o s s i b i l i t y that the appreciable hydrolysis o f c y c l i c 3 ,5»l  UMP by the p a r t i a l l y p u r i f i e d  fraction  o f rabbit brain may be  due to the presence of a separate enzyme for hydrolyzing c y c l i c 3»,5«-UMP. The hydrolysis of c y c l i c 3»,5*-AMP, c y c l i c 3«,5*-GMP and their deoxy analogues by the brain diesterase  preparation  were followed by paper chromatography (Figs. 24-27). one hydrolysis product could be detected c y c l i c nucleotides.  Only  for each of the  I t should be mentioned here that the  isopropanol-ammonium hydroxide-0.1 M boric acid (7:1:2) s o l vent system e f f e c t i v e l y  separates 3*- from  5'-nucleotides.  Although no reference standards are shown f o r these compounds on these chromatograms, i t was noted that the product o f c y c l i c 3 , 5'-AMP hydrolysis was indistinguishable from authens  t i c 5'-AMP. Perrott-Yee  Furthermore, e a r l i e r studies by Drummond and (177) have shown that the hydrolysis products o f  c y c l i c 3*,5*-GMP and c y c l i c 3«,5«-AMP were their corresponding 5"-nucleotides.  Therefore,  i t i s reasonable to suggest that  the products of c y c l i c 3*,5*-deoxy-nucleotides were also  their  corre spond ing 5 *-deoxynucleo tide s• (f)  Further studies on S p e c i f i c i t y of Brain Diesterase -  The presence of 5"-nucleotidase purified  a c t i v i t y i n the p a r t i a l l y  preparation of brain diesterase was investigated.  117.  . '7  >! i T  m  FIG. 24 H y d r o l y s i s o f C y c l i c 3«,5»-AMP by P a r t i a l l y P u r i fied Brain Diesterase. The r e a c t i o n m i x t u r e c o n t a i n e d 0.25 umoles c y c l i c 3*,5*-AMP, 0 . 9 mM MgSO-4, 150 mM T r i s , pH 7.5, 45 ug p a r t i a l l y p u r i f i e d enzyme p r o t e i n , i n a t o t a l volume, o f 0.11 m l . I n c u b a t i o n a t 30° was stopped a t 15 minutes by t h e a d d i t i o n o f 0.02 m l g l a c i a l a c e t i c a c i d . An a l i q u o t (0.02 m l ) was s p o t t e d on Whatman No. 1 f i l t e r paper and t h e chromatogram developed w i t h i s o p r o p a n o l ammonium h y d r o x i d e - 0 . 1 M b o r i c a c i d ( 7 : 1 : 2 ) . The "0" and "15" i n d i c a t e d u r a t i o n o f i n c u b a t i o n (rain). The s o l v e n t f r o n t extended 16 cm from t h e o r i g i n . 5»-nucleot i d e s always remain n e a r the o r i g i n under t h e c o n d i t i o n s u s e d .  118.  FIG. 25  H y d r o l y s i s o f C y c l i c 3«,5«-dAMP.  The enzyme i n c u b a t i o n c o n d i t i o n s and c h r o m a t o g r a p h i c p r o c e d u r e s were as d e s c r i b e d under F i g . 24, e x c e p t t h a t t h e subs t r a t e was 3«,5*-dAMP. The s o l v e n t f r o n t extended 16 cm from t h e o r i g i n .  119.  -v '• V^..\..\v.v>'  F I G . 26.  Hydrolysis of Cyclic  3»,5*-GMP.  The en2yme i n c u b a t i o n c o n d i t i o n s and c h r o m a t o g r a p h i c p r o c e d u r e s were as d e s c r i b e d tinder F i g . 24, e x c e p t t h a t t h e s u b s t r a t e was c y c l i c 3*,5»-GMP. The s o l v e n t f r o n t was 16 cm from t h e o r i g i n .  120.  •K,  •i;s  JHBHBMHFHHFm  FIG. 27 H y d r o l y s i s o f C y c l i c  3»,5»-dGMP.  The enzyme i n c u b a t i o n c o n d i t i o n s and c h r o m a t o g r a p h i c p r o c e d u r e s were a s d e s c r i b e d under F i g . 24, e x c e p t t h a t t h e s u b s t r a t e was c y c l i c 3»,5*-dGMP. The s o l v e n t f r o n t was 14.5 cm from t h e o r i g i n .  121.  As shown i n F i g . 28, no 5'-nucleotidase a c t i v i t y was  present  in the preparation. The presence of, a c y c l i c 2«,3»-nucle0tide phosphodiesterase a c t i v i t y was reported by Drummond and Perrott-Yee  (177)  i n t h e i r ammonium sulfate preparation from rabbit brain.  The  p a r t i a l l y p u r i f i e d preparation obtained as described i n the present investigation also contained a considerable amount of c y c l i c 2*,3'-nucleotide  phosphodiesterase  activity.  However,  when the f r a c t i o n obtained by the alkaline-heat, acid p r e c i p i t a t i o n method was  further fractionated with ammonium sulfate  (0.3-0.6 saturation), the c y c l i c 2«,3"-nucleotide phosphodiesterase a c t i v i t y was completely eliminated ( F i g . 29). Furthermore, the 0.3-0.6 saturated ammonium sulfate f r a c t i o n yielded up to a 2-fold increase i n p u r i f i c a t i o n of the 3',5*nucleotide phosphodiesterase v i t i e s i n the range 15-25.  a c t i v i t y having s p e c i f i c a c t i However, the f i n a l ammonium sulfate  step gave extremely low y i e l d of enzyme and was not s u f f i c i e n t l y reproducible to merit consideration as a routine technique f o r p u r i f y i n g the brain diesterase. 4.  C e l l u l a r D i s t r i b u t i o n of C y c l i c 3,»,5'-nucleotide Phospho-  diesterase - Fractionation of rabbit brain into several c e l l u l a r components was performed as described by De Robertis et a l (185).  The r e s u l t s (Table X) indicated that about 50%  of the diesterase a c t i v i t y was supernatant  fraction.  located i n the 105,000 x g  The microsomal and mitochondrial f r a c -  tions contained considerable amounts of diesterase a c t i v i t y , but l i t t l e a c t i v i t y was  located i n the nuclear f r a c t i o n .  TABLE X C e l l u l a r D i s t r i b u t i o n of C y c l i c 3»,5*-Nucleotide Phosphodiesterase i n Rabbit Brain. The cerebral cortex from a rabbit was homogenized f o r 5 minutes i n 8 volumes of 0.33 M sucrose (unbuffered) with the aid of a glass homogenizer f i t t e d with a t e f l o n pestle and centrifuged f o r 10 minutes at 900 x g. The sediment was washed twice with 0.33 M sucrose, and after r e - c e n t r i f u gation at 900 x g, the supernates were combined with the o r i g i n a l 900 x g supernate. The mitochondrial f r a c t i o n was obtained by centrifuging the combined supernates f o r 20 minutes at 11,500 x g. The sediment was again washed twice with 0.33 M sucrose. The microsomal f r a c t i o n was obtained by centrifuging the combined 11,500 x g supernates f o r 30 minutes at 105,000 x g. The microsomal f r a c t i o n thus obtained was washed once only. A l l procedures were carried out at 0-4°. Enzyme a c t i v i t y i s defined i n the text. The standard assay was used to determine diesterase a c t i v i t y .  Fraction Whole homogenate  Total A c t i v i t y (units)  % Total Activity  780  (100%)  Nuclear  22  2i8~  Mitochondrial  63  8.1  Microsomal  103  13.2  105,000 x g supernate  394  50.5  582  74.9  Recovery  FIG. 28. Absence o f 5 ' - N u c l e o t i d a s e A c t i v i t y i n P a r t i a l l y Purified Brain Diesterase Fraction. The r e a c t i o n m i x t u r e c o n t a i n e d 1.8 umoles 5»-AMP, 0.9 mM MgSO^ 80 mM T r i s , pH 7.5, p a r t i a l l y p u r i f i e d b r a i n d i e s t e r a s e ( s p e c i f i c a c t i v i t y , 14) and s u f f i c i e n t w a t e r t o make 0.2 m l . I n c u b a t i o n was f o r 30 minutes a t 30°, and the e n t i r e c o n t e n t s o f t h e r e a c t i o n m i x t u r e were s p o t t e d on Whatman No. 1 f i l t e r paper and developed as d e s c r i b e d i n t h e t e x t . From the l e f t , s p o t 1 - r e f e r e n c e 5»-AMP; s p o t 2 - r e f erence adenosine; s p o t 3 and 4, s u b s t r a t e 5 -AMP i n c u b a t e d i n t h e absence and presence o f 13.5 ug enzyme p r o t e i n respectively. The s o l v e n t f r o n t was 17 cm from t h e o r i g i n . 1  124  F I G , 29 Absence o f C y c l i c 2«,3*-AMP H y d r o l y t i c A c t i v i t y i n t h e F i n a l (Ammonium s u l f a t e 0.3-0.6 s a t u r a t i o n ) P r e p a r a t i o n of Brain Diesterase. The r e a c t i o n m i x t u r e c o n t a i n e d 0.24 umoles c y c l i c 2»,3»AMP, 0.9 mM MgSO,, 80 mM T r i s , pH 7.5, 6 ug b r a i n d i e s t e r a s e p r e p a r a t i o n , i n a t o t a l volume o f 0.2 m l . I n c u b a t i o n was f o r 15 m i n u t e s a t 30°. A l i q u o t s o f 0.02 m l were t a k e n , s p o t t e d on paper and developed a s d e s c r i b e d i n t h e text. S p e c i f i c a c t i v i t y o f t h e d i e s t e r a s e p r e p a r a t i o n was 27. The s o l v e n t f r o n t was 14.5 cm from t h e o r i g i n .  125.  While this work was  i n progress, Cheung and Salganicoff  reported that 40% of the t o t a l a c t i v i t y was mitochondrial  f r a c t i o n of r a t brain.  Drummond and Perrott-Yee  (186)  located i n the  On the other hand,  (177) reported  that the a c t i v i t y  was  l o c a l i z e d e n t i r e l y i n the 100,000 x g supernatant f r a c t i o n . I t i s therefore d i f f i c u l t to reconcile the differences observed between the r e s u l t s of the present study and those of the other authors.  De Robertis e_t a l . (185) have indicated that  the f i t t i n g of the t e f l o n plunger had to be s p e c i a l l y determined i n order to produce a minimal breakage of nerve terminals.  I t i s therefore l i k e l y that the differences i n the  r e s u l t s reported on the c e l l u l a r d i s t r i b u t i o n of brain diesterase a r i s e e s s e n t i a l l y from the extent to which c e l l u l a r components are disrupted during homogenization. The present study indicated l i t t l e diesterase a c t i v i t y i n the nuclear f r a c t i o n (which also contains fragments of plasma membrane).  These findings are i n good agreement with  those of Cheung and Salganicoff (186) who the a c t i v i t y i n t h i s f r a c t i o n .  found only 77. of  Since the adenyl cyclase sys-  tem which synthesizes c y c l i c 3*,5*-AMP i s located on the plasma membrane, i t would appear that the adenyl cyclase system and the phosphodiesterase a c t i v i t i e s are s p a t i a l l y separated the c e l l .  I t seems that such a s p a t i a l arrangement may  in per-  haps be b i o l o g i c a l l y important, since c y c l i c 3*,5»-AMP must be given time to act before i t s destruction by the diesterase. 5.  D i s t r i b u t i o n of C y c l i c 3',5 -Nucleotide Phosphodiesterase 1  i n Various Areas of the Central Nervous System and i n Lower  126.  Organisms - As described e a r l i e r , c y c l i c 3*,5*-AMP i s involved in the regulation of several important b i o l o g i c a l  processes.  The enzyme, c y c l i c 3»,5*-nucleotide phosphodiesterase, which terminates the action of c y c l i c SS^-AMP has been shown to e x i s t i n most of the higher organisms which have been examined for i t s a c t i v i t y .  Because the diesterase must play a r o l e i n  the regulation of i n t r a c e l l u l a r c y c l i c 3*,5*-AMP l e v e l s , the distribution  of the enzyme was investigated i n several tissues;  namely, the human brain, the nervous system of the dog, i n marine organisms and i n the plant kingdom.  As may be observed  i n Table XI, the diesterase a c t i v i t y was highest i n the cereb r a l cortex of the human brain.  The survey of the d i s t r i b u t i o n  of diesterase a c t i v i t y i n various areas of the dog nervous system (Table XII) also indicated that the enzyme a c t i v i t y was highest i n the cerebral cortex.  The high content of diesterase  a c t i v i t y found i n the cerebral cortex i s consistent with that observed by other investigators.  The d i s t r i b u t i o n  of the  enzyme i n several available marine organisms which were examined i s shown i n Table XIII.  The survey of diesterase a c t i -  v i t y i n various areas of the human brain, dog nervous system and i n marine organisms were performed by Mr. Lorne K. Massey, a medical student working i n this laboratory. Several available plants were also investigated f o r diesterase a c t i v i t y .  As may be seen i n Table XIV, no d i e s t e r -  ase a c t i v i t y was detected were examined.  i n any of the plant specimens which  Several techniques were used to ensure complete  disintegration of yeast c e l l s , but no a c t i v i t y was detected.  127.  TABLE XI D i s t r i b u t i o n of c y c l i c 3»,5*-Nucleotide Phosphodiesterase A c t i v i t y i n Human Brain. Specific  a c t i v i t y i s as described i n the text.  Area of Brain Cerebral cortex - grey  - white  Specific  Activity  1.6 1.4  Cerebellar cortex  1.0  Pons  0.4  Corpus Callosum  1.0  Thalamus  1.0  Caudate nucleus  1.0  Vermis  0.8  Hypothalamus  0.4  128.  TABLE XII C y c l i c 3*,5*-Nucleotide Phosphodiesterase A c t i v i t i e s i n Various Areas of Dog Nervous System. Specific a c t i v i t y i s as described i n the text.  Nervous Tissue  Specific  Cerebral cortex  3.2  Cerebellum  0.4  Basal ganglion and internal capsule  2.0  Medulla  1.0  Pons  0.3  Activity  Spinal Cord - c e r v i c a l - upper thoracic  0.4  - lower thoracic  0.6  - lumbar  0.2  Midbrain  0.5  Hypothalumus  2.1  Caudate nucleus  2.9  S t e l l a t e ganglion  0.4  Thoracic sympathetic ganglion  0.05  Phrenic nerve  0.3  S c i a t i c nerve  0.2  Thoracic sympathetic axons  0.5  Cervical  sympathetic axons  0.1  Cervical  superior ganglion  0.1  Nodose ganglion  0.2  Vagus nerve  0.5  Thalamus  3.1  129.  TABLE XIII D i s t r i b u t i o n of C y c l i c 3 * , 5 " - n u c l e o t i d e Phosphodiesterase A c t i v i t y i n Several Marine Organisms. S p e c i f i c a c t i v i t y i s as defined i n the text.  Organism Steelhead  Trout  Genus Salmo  it  Tissue  Specific Activity  brain  0.21  s k e l e t a l muscle  0.07  Salmon  Oncorhynkus  heart  0.34  Sea anemone  Metridium  musele  0.54  Tubeworm  Nereis  (whole organism)  0.48  Sea urchin  Stronglyocentrotus  gonads  0.26  intestine  0.85  ii  n  II  Oyster  Crosostrea  adductor muscle  0.0  Clam  Mytilus  adductor muscle  0.0  Snail  Thais  (whole organism)  0.45  Hermit Crab  Pagarus  (whole organism)  0.0  Sea cucumber  Stichopus  longitudinal muscle  0.25  Crab  Cancer  gill  0.0  liver  0.0  pancreas  0.0  heart  0.0  aorta  0.0  it  II  it  n  it  it  it  ti  130.  TABLE XIV D i s t r i b u t i o n of Phosphodiesterase A c t i v i t y i n Plants and Micro-organisms. Plant tissues were washed with 0.15 M KC1, frozen i n l i q u i d nitrogen, ground to a powder i n a c h i l l e d mortar, and further homogenized i n suitable volumes of 0.5 M T r i s , pH 7.5. Whole homogenates were used f o r assaying diesterase a c t i v i t i e s , unless indicated otherwise. The assays were performed as described i n the text f o r the chromatographic method of detecting diesterase a c t i v i t y . A l l negative r e s u l t s were re-examined by using larger volumes of tissue homogenates or extracts and increasing the incubation time to 2 hours. JB. ferrooxidans and E_. c o l i c e l l s were disrupted by sonication at y kc/sec f o r 30 and 10 minutes, respectively. Specific a c t i v i t y as described i n the text.  Organism  Specific A c t i v i t y  Plants Higher plant leaf - (Genus Tradescantia)  0  Moss (Liverwort) - (Genus Lunularia)  0  Fungus (mycelium) - Coprinus macrorhizus  0*  Yeast - Saccharomyces cerevisiae  0*  Algae (Red) - Gymnogongrus norvegicus  0*  Algae (Green) - Spongomorpha coalita  0*  Algae (Blue) - Phaeostrophion irregulare -  0*  Micro-organisms Bacteria - B a c i l l u s ferrooxidans  0*  Bacteria - Escherichia c o l i  0.27*  * indicates 37,000 x g supernate and sediment were examined for a c t i v i t y .  131.  On the other hand, Cheung (187) has reported recently that extremely low diesterase a c t i v i t y was Saccharomyces carlsbergensis.  Two  present  i n the yeast,  available microorganisms  were also examined for diesterase a c t i v i t y .  I t was  noted that  _E. c o l i possessed an appreciable l e v e l of phosphodiesterase activity.  In t h i s organism, the e n t i r e a c t i v i t y was  in the 37,000 x g supernate. a c t i v i t y i n E_. c o l i was  located  A f t e r the presence of diesterase  demonstrated i n the present  Brana and C h y t i l (188) reported  study,  s i m i l a r observations.  authors also noted that the diesterase a c t i v i t y was in the supernatant f r a c t i o n obtained  These  located  a f t e r centrifugation of  sonicated E. c o l i c e l l s at 20,000 x g.  132.  DISCUSSION The e l u c i d a t i o n of the r o l e s of c y c l i c 3*,5*-AMP i s curr e n t l y under intense study i n many laboratories.  Experiments  with the adenyl cyclase system has led Sutherland  (190)  to  propose a general picture of a two-messenger system f o r the expression of hormonal c o n t r o l i n b i o l o g i c a l systems.  He  suggests that a hormone ( f i r s t messenger) interacts with s p e c i f i c e f f e c t o r c e l l s at the plasma membrane.  This i n t e r -  action r e s u l t s i n the formation of a second messenger within the c e l l to modify i n t r a c e l l u l a r enzyme a c t i v i t y . s p e c i f i c example, Sutherland cyclase by epinephrine  As a  c i t e s the stimulation of adenyl  ( f i r s t messenger) which r e s u l t s i n  the increased biosynthesis of c y c l i c 3»,5*-AMP (second messenger) and consequently, the several p h y s i o l o g i c a l e f f e c t s of epinephrine Sutherland  which are observed.  (190)  The scheme proposed by  i s i l l u s t r a t e d i n F i g . 30.  In such a system,  the a c t i v i t y of the phosphodiesterase must be equally important as that of adenyl cyclase i n maintaining  the required  i n t r a c e l l u l a r l e v e l s of c y c l i c 3*,5*-AMP. As yet, no hormonal mechanism for c o n t r o l l i n g the a c t i v i t y of the phosphodiesterase has been detected. The r o l e of c y c l i c 3»,5*-nucleotide phosphodiesterase in the brain i s being a c t i v e l y investigated. ganicoff (186) have reported was  Cheung and S a l -  that the diesterase a c t i v i t y  located mainly i n the cholinergic nerve endings and i n  the soluble synaptic neuroplasm. diesterase i n brain was  They suggested that the  probably more c l o s e l y associated with  133.  (EXTRACELLULAR)  (INTRACELLULAR) I PHYSIOLOGICAL I RESPONSES. J  Glycogenolysis \  HORMONE • (FIRST MESSENGER)  ATP  Glycolysis Lipolysis  V  Cardiac inotropic response  DIESTERASE c y c l i c 3*,5»-AMP— — >-5«-AMP (SECOND MESSENGER)  FIG. 30 The Two-Messenger Concept f o r the Expression of Hormonal Control as Modified from Sutherland(190).  134.  the regulation o f glucose metabolism than i n synaptic  trans-  mission. Studies of several properties of the p a r t i a l l y  purified  c y c l i c 3*,5'-nucleotide phosphodiesterase from rabbit brain revealed that the properties of the brain enzyme are very s i m i l a r to the diesterase which has been p u r i f i e d tissue.  from cardiac  As reported e a r l i e r by Drummond and Perrott-Yee  (177),  brain diesterase catalyzed the conversion of c y c l i c 3»,5»-AMP s p e c i f i c a l l y to 5*-AMP.  No other product was formed.  I t was  also confirmed that the enzyme requires Mg"*"* ions f o r a c t i v i t y and was completely inactive i n the presence o f 1 mM EDTA. E a r l i e r studies on brain and heart diesterases suggested that the a c t i v i t y o f the enzyme was more selective f o r the c y c l i c 3»,5'-nucleotide which contain purine bases than those with pyrimidine bases.  Indeed, the present studies have shown  that the enzyme has v i r t u a l l y no a c t i v i t y against  pyrimidine  c y c l i c 3» ,5*-nucleotides, with the exception of c y c l i c 3»,5»UMP.  However, Hardman and Sutherland  that a phosphodiesterase was present  (184) recently reported i n heart that  hydrolyzed  c y c l i c 3',5*-UMP a t a much faster rate than c y c l i c 3«,5'-AMP and other available c y c l i c 3',5«-nucleotides.  Their obser-  vation therefore suggests that the s l i g h t but s i g n i f i c a n t a c t i v i t y against c y c l i c 3',5«-UMP found i n the p a r t i a l l y p u r i fied brain preparation may have been due to another diesterase which was s p e c i f i c  f o r c y c l i c 3*,5'-UMP.  I t was noted that  the hydrolysis of c y c l i c 3',5'-AMP was c o n s i s t e n t l y inhibited by the presence of c y c l i c 3»,5«-.dlMP. Preliminary experiments  135.  also indicated the i n h i b i t i o n of c y c l i c 3»,5'-AMP hydrolysis by c y c l i c 3«,5'-GMP and c y c l i c 3»,5«-dGMP.  The data strongly  indicates that c y c l i c 3',5'-nucleotides possessing bases are hydrolyzed ATP  purine  by the same enzyme.  (0.125 mM and 1.25 mM) did not appear to i n h i b i t the  brain diesterase.  This i s i n contrast to a recent report by  Cheung (191) who indicated that brain diesterase was inhibited by ATP and pyrophosphate, and have attributed to ATP a regul a t o r y r o l e on the enzyme. The s p e c i f i c i t y of brain diesterase for the c y c l i c 3«,5»diester linkage was unequivocally demonstrated when the f i n a l ammonium s u l f a t e f r a c t i o n a t i o n (0.3-0.6 saturation) was performed.  This f i n a l step e f f e c t i v e l y removed the c y c l i c 2»,3«-  nucleotide phosphodiesterase a c t i v i t y by p r e c i p i t a t i n g the enzyme into the 0-0.3 saturated ammonium sulfate f r a c t i o n , leaving a highly p u r i f i e d preparation of c y c l i c 3*,5»-nucleotide phosphodiesterase i n the higher ammonium sulfate f r a c t i o n . The present study also demonstrated that the brain diesterase i s stimulated by imidazole.  The quantitative response to  imidazole i s i n agreement with that observed for.heart diesterase.  The  KJJJ. value  of brain diesterase f o r c y c l i c 3*,5»-  AMP was about 0.8 x 10" M, which i s s i m i l a r to that observed 4  for beef heart diesterase,.but  lower than that observed by  Nair (176) f o r dog heart diesterase.  The i n v i t r o i n h i b i t i o n  of brain diesterase by theophylline observed i n the present investigation i s also a common feature possessed by the brain and heart enzymes.  The nature of theophylline i n h i b i t i o n  136.  appears to be competitive  i n both instances.  As one might  expect, caffeine and theobromine also i n h i b i t s diesterase a c t i v i t y , although i t has been reported  (175)  that these  compounds were only about 167 as potent as theophylline i n 0  this respect.  The a b i l i t y of methyl xanthines to i n h i b i t  diesterase a c t i v i t y i s most l i k e l y due  to the s i m i l a r i t y i n  the structures of the methyl xanthines to c y c l i c 3 ,5 -AMP, ,  as indicated i n F i g . 31.  One might even speculate that the  lower potency of caffeine.and presence or  ,  theobromine may  be due to the  -methyl groups on these compounds, which may  interfere with their binding to the diesterase. Theophylline has been known for many years to produce a number of pharmacological e f f e c t s .  The compound  stimulates  the c e n t r a l nervous system, p a r t i c u l a r l y the cerebral cortex, although to a lesser extent than c a f f e i n e .  However, theo-  p h y l l i n e i s the most potent of a l l the methyl xanthines i n i t s d i u r e t i c action on the kidney, i t s stimulation of cardiac and  s k e l e t a l muscle, and i t s e f f e c t on the relaxation of  smooth muscle.  These pharmacological responses to  theophyl-  l i n e administration i s l i k e l y due to the i n h i b i t i o n of phosphodiesterase i n vivo, which i n turn would r e s u l t i n increased l e v e l s of c y c l i c 3',5*-AMP.  For example, i t i s  known that when s k e l e t a l muscle i s exposed to methyl xanthines (e.g., c a f f e i n e ) , large quantities of l a c t i c acids are produced.  These observations  indicate that glycogenolysis  and  g l y c o l y s i s were stimulated owing to increased c y c l i c 3*,5»AMP  l e v e l s i n the tissues.  S i m i l a r l y , theophylline has been  137  CH  CH  3  Theophylline (1,3-Dimethyl xanthine)  3  Theobromine (3,7-Dimethyl xanthine)  0 •  0. \  C y c l i c 3«,5»-AMP  FIG. 31? Structural Formulae of Methyl Xanthines  138.  reported to potentiate the cardiac inotropic response to norepinephrine  (192), increase l i p o l y t i c a c t i v i t y i n adipose  tissue (193), stimulate steroidogenesis (159), increase amylase secretion from r a t parotid gland  (163) and increase the  permeability of toad bladders to water (166).  I t would be  of p a r t i c u l a r interest to investigate the p o s s i b i l i t y that the observed pharmacological  e f f e c t s of c a f f e i n e and theo-  phylline upon the c e n t r a l nervous system might arise from the i n vivo i n h i b i t i o n of brain phosphodiesterase,  resulting i n  higher l e v e l s of c y c l i c 3',5'-AMP i n the brain. The method described i n t h i s study for the p a r t i a l p u r i f i c a t i o n of c y c l i c 3 ,5'-nucleotide 1  phosphodiesterase  from  brain i s considerably more rapid and t e c h n i c a l l y easier than that described f o r the p u r i f i c a t i o n of the enzyme from beef and dog hearts.  However, t h i s advantage i s o f f s e t by the  fact that the enzyme preparation generally has a s p e c i f i c a c t i v i t y equal to about one-half that reported f o r the heart preparations. The use of p u r i f i e d diesterase preparations has aided i n the measurement of c y c l i c 3',5'-AMP i n b i o l o g i c a l materials.  For example, Butcher and Sutherland  (175) have  used the diesterase as a b i o l o g i c a l t o o l i n order to destroy c y c l i c 3',5'-AMP i n extracts of urine, thus obtaining "tissue blanks" i n the assay for the c y c l i c nucleotide i n urine. The universal d i s t r i b u t i o n of c y c l i c phosphodiesterase  3',5'-nucleotide  i n higher organisms of the animal kingdom  contrasts sharply with the absence of diesterase a c t i v i t y i n  139.  any of the plant specimens examined i n t h i s study.  Suther-  land (194) reported that no adenyl cyclase a c t i v i t y could be detected  i n plants.  Therefore,  these observations  suggest  that the b i o l o g i c a l importance of c y c l i c 3',5'-AMP may be confined  l a r g e l y to those organisms within the animal kingdom.  The detection of diesterase a c t i v i t y i n marine organisms indicates that c y c l i c 3',5'-AMP i s also widespread i n lower animal organisms.  The b i o l o g i c a l importance of c y c l i c 3',5'-  AMP i n these organisms i s of no less i n t e r e s t than i t s r o l e in higher organisms. 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