ME filSCOVERY AND PARTIAL CHARMYEREZATION 0F GARMTSNE ACETYLTMNSFERASE ACTM'W FROM RA? LEVER PEROXISGMES; ANS) MECRO’SQMES Dissertation far. ens-Degree' of 9m D. MECHEW sun: umvsasm » MARY ANN mums mama 2974 - ’ This is to certify that the thesis entitled The Discovery and Partial Characterization of Carnitine Acetyltransferase Activity from Rat Liver Peroxisomes and Microsomes presented by Mary Ann K. Markwell has been accepted towards fulfillment of the requirements for Ph.D. Jesteein Biochemistry ‘ $flm llaknrproflessor Date December 20, 1971+ 0-7839 \ ‘2‘: amomo If ‘ HUAB & SUNS' BM WHY INS. LiBRARY BINDERS ' I‘ ‘gfii w_~\_"))ll ' m » n3 Vt L. e. a 0 fl. h“ —\u .H» “‘1. e 1‘ fi ‘l. 1 Al .6 #s .‘l “I ’§ r. .1. .1; -3 \‘~ u; .r. C 3. Wu 7‘ i. .“ $.S 01‘ S P. :. 1. .r5 «. «L - eL liq ‘c .‘ c [N “Pt ‘1‘ ~\. 9" .1 u. .d .?L «(J D! fir. ‘K.w .2 ‘i ABSTRACT THE DISCOVERY AND PARTIAL CHARACTERIZATION OF CARNITINE ACETYLTRANSFERASE ACTIVITY FROM RAT LIVER PEROXISOMES AND MICROSOMES By Mary Ann Kelling Markwell Subcellular localization studies using isopycnic sucrose density gradients showed carnitine palmitoyltransferase (CPT) to be exclu— sively a mitochondrial enzyme in liver and kidney from rat and pig. Little or no carnitine acetyltransferase (CAT) activity was found outside the mitochondrion in kidney. In liver, however, 3 distinct peaks of CAT and carnitine octanoyltransferase (COT) were obtained on zonal gradients. Approximately half of the CAT activity was mitochondrial; the remainder was divided between peroxisomes and a lipid-rich membranous region. Three separate assay systems con- firmed the presence of extramitochondrial CAT. The specific activity of CAT in rat liver peroxisomes was 2— to 3—fold greater than in mitochondria or in the membranous region. Most (>80%) of peroxisomal CAT activity was latent. Maximum activity was obtained only when the peroxisomal membrane had been disrupted using aging, Triton X-100, or freeze-thaw procedures. Fractionation of peroxisomes into core, membrane, and soluble (matrix) fractions indicated CAT was a matrix enzyme. CAT activity (>9OZ) could be released 1 about the skewed the activitie; additive. Hepa CAT and C of COT wa transfera COA'S as .ran additive at about IEgiOn-.C further 5 ilcrosome Mary Ann Kelling Markwell released from the peroxisome by freeze-thaw. COT was present at about the same specific activity as CAT in hepatic peroxisomes and showed the same solubility by freeze-thaw. Peroxisomal transferase activities using acetyl- and octanoyl-CoA's as substrates were additive. Hepatic mitochondria contained all 3 transferases. Mitochondrial CAT and CPT had similar specific activities, but the specific activity of COT was up to 6-fold greater than the other two. Mitochondrial transferase activities using acety1-, octanoy1-, and palmitoyl- CoA's as substrates were additive. Transferase activities with acetyl- and octanoyl-CoA were also additive in the third zonal region. Here CAT and COT were present at about the same specific activity. When components of this region--Golgi membranes, plasma membranes, and microsomes were further separated, CAT and COT activities fractionated with the microsomes. Microsomes isolated by 3 different methods--zona1 centrifugation, high-speed differential centrifugation, and aggre- gation with Ca++ followed by lowbspeed centrifugation--all contained CAT and COT activities. CAT and COT were equally distributed between rough and smooth microsomes. CAT activity could be solubilized from the microsomal membrane by treatment with 0.4 M KCl, 1% Triton X-100, or a combination of both. Microsomal CAT activity was very labile (all activity lost within a few hours of isolation) in low ionic strength or phosphate-buffered isolation media, but could be stored for days without loss of activity in 0.4 M KCl. 1 . . WM'-fil~ Peron fold usin; heZ par on cellul the same CAItesuT man CK activity refocusi transfer tion (59 L-carni: C05 rat] 0f acyl. )4. CA Sueciny in the live}: I from PI in mic Mary Ann Kelling Markwell, Peroxisomal and microsomal CAT activities were purified 200- fold using DEAR—cellulose and cellulose phosphate chromatography. The 2 partially purified activities had the same elution profiles on cellulose phosphate (pH 6.0 and 7.5) and Sepharose hydrazide and the same pH optima (7.2-8.0). Isoelectric focusing of peroxisomal CAT resulted in a single peak at pH 8.3. The major peak of micro- somal CAT also focused at pH 8.3, but a minor peak (20% of the recovered activity) appeared at pH 5.3. After exposure to 0.4 M KCl and refocusing only the pH 8.3 peak remained. The partially purified transferases had the same apparent molecular weight by gel filtra- tion (59,000) and the same apparent Km's for acetyl-CoA (69 uM) and L—carnitine (143-150 uM). Maximum activity occurred using propionyl- CoA rather than acetyl-CoA as a substrate. Activity with a series of acyl-CoA's dropped off rapidly as the carbon chain length became >4. CAT had activity for malonyl- and acetoacetyl-CoA but not for succinyl- and B-hydroxy-B-methylglutaryl-CoA. Several differences in the above properties were observed between CAT activity from rat liver peroxisomes and microsomes and a commercial preparation of CAT from pigeon breast muscle. The results strongly indicated that COT in microsomes and peroxisomes is a different enzyme from CAT. THE DISCOVERY AND PARTIAL CHARACTERIZATION OF CARNITINE ACETYLTRANSFERASE ACTIVITY FROM RAT LIVER PEROXISOMES AND MICROSOMES By Mary Ann Kelling Markwell A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1974 © Copyright by MARY ANN KELLING MARKWELL 1975 DEDICATION This dissertation is dedicated to the one who is my colleague, friend, and spouse - to you, John- 11 A d of the u people i and most wish to Doctors guidance fessor, Enthus 1.; Pas time ACKNOWLEDGMENTS A dissertation comes into being primarily through the efforts of the writer but is shaped by many people. I thank all those people in the biochemistry department who lent equipment, suggestions, and most of all sympathetic ears to this work as it progressed. I wish to express my gratitude to the members of my thesis committee—- Doctors Helmrath, Luecke, Ronzio, and Wells--for their continuing guidance and interest. Finally I would like to thank my major pro- fessor, Dr. Loran Bieber, for his professional example, untiring enthusiasm, and his wisdom in knowing when to offer help and when it was time for me to fly it alone. iii Life itself is infinitely stranger than anything which the mind of man could invent. -Sherlock Holmes iv LIST 0 LIST 0 LIST 0 STAT D! LIIERA Elm: TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . vii LIST OF FIGURES O O O O O O O O C O O O O O O O O O O O O O O Viii LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . .x STATEMENT OF THE PROBLEM. . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW OF THE CARNITINE ACYLTRANSFERASES . . . . . 3 Carnitine Acetyltransferase. . . . . . . . . . . . 4 Discovery and Tissue Distribution . . . . . . 4 Subcellular Localization. . . . . . . . . . . . 5 Question of Multiple Forms. . . . . . . . . . . 6 Partial Purification. . . . . . . . . . . . . . 7 Sources of Substrate L-carnitine. . . . . . . . 7 Kinetic Properties. . . . . . . . . . . . . . 8 Possible Functions. . . . . . . . . . . . . . . 8 Carnitine Palmitoyltransferase . . . . . . . . . . . . 10 Carnitine Octanoyltransferase. . . . . . . . . . . . . 14 Changes in Transferase Levels. . . . . . . . . . . . . 14 Changes with Development, Differentiation and Sex. . . . . . . . . . . . . . . . . . . 14 Changes with Increased Fatty Acid Supply. . . . 16 EXPERIMENTAL PROCEDURES AND RESULTS . . . . . . . . . . . . . 19 Discovery of Extramitochondrial CAT Activity and Localization of Peroxisomal CAT . . .,. . . . . 20 Materials and Methods . . . . . . . . . . . . . 20 Results . . . . . . . . . . . . . . . . . . . . 23 Distribution of CA , COT, an CPT Activities in Liver . . . . . . . . . 23 Distribution of CAT and CPT in Kidney. . 33 Distribution of CAT and CPT in Plants. . 33 Latency and Localization of Peroxi- somal CAT . . . . . . . . . . . . . . 38 Stability of CAT . . . . . . . . . . . . 42 Localization and Solubilization of MicroSomal CAT. . . 43 Materials and Methods . . . . . . . . . . . . . 43 Results . . . . . . . . . . . . . . . . . . . . 45 Localization of CAT in Microsomes. . . . 45 Studies on the Solubilization and Sta- bilization of Microsomal CAT. . . . . 50 V Page Effect of Media on CAT Activity in High-Speed Microsomes . . . . . . . . 55 Partial Purification and Characterization of Peroxisomal and Microsomal CAT Activities . . . . . 58 Materials and Methods . . . . . . . . . . . . . 58 Preparation of Dephospho CoA— Affinity Column . . . . . . . . . . . 58 Procedure for Isoelectric Focusing . . . 59 Results . . . . . . . . . . . . . . . . . . . . 60 Initial Attempts at Purification . . . . 60 Final Purification Scheme. . . . . . . . 63 Partial Characterization and Comparison of PrOperties of Microsomal, Peroxisomal and Commercial CAT. . . . 65 DISCUSSION. 0 O O O O O O O O O O O O O O O O O I O 0 O O O O 90 APPENDIX A. O O O O O O O O O O O O O O O O O O O O O O O O O 100 LIST OF REFERENCES. . . . . . . . . . . . . . . . . . . . . . 102 vi Table 10 ll Table 10 11 LIST OF TABLES Specific activities of carnitine acyltransferases from organelles of rat liver with different acyl-CoA substrates. . . . . . . . . . . . . . . Distribution of carnitine acetyltransferase in particulate fractions from rat liver and kidney gradients. . . . . . . . . . . . . . . . . . Enzymatic characterization of membrane fractions from rat liver 0 O O O O O I O O O O O O O O I O O O 0 Distribution of carnitine acyltransferase activity in smooth and rough microsomal fractions . . . . . . Solubilization of microsomal carnitine acetyl- transferase activity . . . . . . . . . . Recovery of microsomal carnitine acetyltransferase activity in different salt solutions . . . . . . . . . Purification procedure for peroxisomal and micro- somal carnitine acetyltransferase activity . . . . . Proteins used in gel-filtration experiments. . . .-. . Effect of different chloride salts on microsomal, peroxisomal, and commercial carnitine acetyltrans- feraseSo O O O O O O O C O O O I O O I O O O O O . Substrate specificity of peroxisomal and microsomal carnitine acetyltransferases for acyl-CoA's. . . . . . Substrate Specificity of zonal peroxisomes and partially purified peroxisomal carnitine acetyl- tranSferase for aCYI—COA'S o o o o o o o o o o o o o 0 vii Page 28 38 47 50 53 54 64 76 83 87 89 vrj . inur- .L...C I‘d L\ ”\J KL? 10 11 Figure 10 11 LIST OF FIGURES Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase among the organelles Of rat liver 0 C C O I O C O O O O O O O O O O O 0 Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase between peroxisomes and mitochondria from pig liver. . . . . . . . . . Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase among organelles of rat kidney 0 O O O O O O O O O O O O O O O O I O 0 Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase between peroxisomes and mitochondria from pig kidney . . . . . . . . . Fractionation of rat liver peroxisomes . . . . . . Effect of media on microsomal carnitine acetyl- transferase actiVity I O O O O O O O O O O O O O O Cellulose phosphate elution profiles at pH 7.5 of microsomal, peroxisomal, and commercial carni— tine acetyltransferases. . . . . . . . . . . . . . Cellulose phosphate elution profiles at pH 6.0 of microsomal, peroxisomal, and commercial carnitine acetyltransferases . . . . . . . . . . . . . . . . Sepharose hydrazide elution profiles of microsomal, peroxisomal, and commercial carnitine acetyl- transferases . . . . . . . . . . . . . . . . . . . Estimation of molecular weights of microsomal, peroxisomal, and commercial carnitine acetyltrans— ferases by gel filtration. . . . . . . . . . . . . Isoelectric focusing of microsomal, peroxisomal, and commercial carnitine acetyltransferases. . . . viii Page 26 31 35 37 41 57 68 70 73 75 79 I3 Figure 12 13 Page pH profiles of microsomal, peroxisomal, and com- mercial carnitine acetyltransferases . . . . . . . . . 81 Determination of Km values of peroxisomal and microsomal carnitine acetyltransferases for L— earnitine and aCEtYI-COA o o o o o o o o o o o o o o o 85 ix [0.1. “\J wlu \ DI} BSA CAT CM- CoA COT CPT DCCD dCoA DEAE- DTNB NADPH RNA TNBS Tris LIST OF ABBREVIATIONS Bovine serum albumin Carnitine acetyltransferase Carboxymethyl- Coenzyme A Carnitine octanoyltransferase Carnitine palmitoyltransferase N—N'-dicyclohexylcarbodimide 3'-dephospho Coenzyme A Diethylaminoethyl- 5,5'-dithiobis (Z-nitrobenzoate) Michaelis constant Nicotinamide-adenine dinucleotide phosphate (reduced form) Ribonucleic acid 2,4,6-trinitrobenzenesulfonate Tris(hydroxymethyl)aminomethane Uniformly labelled with radioactivity Uridine 5'-diphosphate Maximum velocity I Cagree n b ,. 0;: Lh: met. Car Iai 33: STATEMENT OF THE PROBLEM Chance has put in our way a most singular and whimsical problem and its solution is its own reward. - Sherlock Holmes The problem I chose for research toward a Doctor of Philosophy degree was an offshoot from the ongoing research in our laboratory on the role of carnitine in lipid metabolism. The enzymes which use carnitine as a substrate, the carnitine acyltransferases, had previously been reported to be exclusively associated with mito- chondria, and so their cellular function was explained in terms of this organelle. My initial task was to survey subcellular fractions of rat liver to determine if the assignment of the carnitine acyl- transferases to the mitochondrion was valid. This led to the dis- covery of extramitochondrial carnitine acyltransferase activity and to a reconsideration of the role of carnitine in intermediary metabolism. In the next phase of the project peroxisomal and microsomal carnitine acetyltransferase activities were partially purified from rat liver and characterized to determine if they were forms of the same protein and if their properties differed greatly from the pigeon breast muscle enzyme, the only carnitine acetyltransferase which has been crystallized. A particularly intriguing aspect of the problem was the physical state of these activities. Did the l trans: port a 2 transferases have to be located in a membrane to facilitate trans- port across it? 1’".- ('1 n! LITERATURE REVIEW OF THE CARNITINE ACYLTRANSFERASES It immensely adds to the zest of an investigation...when one is in conscious sympathy with the historical atmosphere of one's surroundings. - Sherlock Holmes This review is intended to cite the major works on the carnitine acyltransferases. Because of the differing physiologies of the experimental animals (insects, birds, ruminants, primates, and rodents) used in these references, discussion of the functions of the transferases will focus on mammalian liver, particularly that of the rat. The carnitine acyltransferases are a family of enzymes which catalyze the following reversible reaction: acyl-CoA + L-carnitine Z CoA + L—acylcarnitine. The exact number, substrate specificity, and function of each remains to be established, but in general they are thought to facilitate the transport of activated fatty acids in the form of acylcarnitines across membranes impermeable to acyl—CoA's. In certain tissues they may also maintain levels of acyl-CoA's and through this regulate certain other key enzymes of intermediary metabolism. This will be discussed in more detail in subsequent sections. Discover Car O-acetyl to be di carnitir by an e: experimt tissues sunably Su by seve aCtivit Specifi epidid} Vere Cc aCtiViI part 01 by Spe: I Klinge chondr oxidat differ insth lEvelS 4 Carnitine Acetyltransferase Discovery and Tissue Distribution Carnitine acetyltransferase (CAT) (acetyl-CoA:L-carnitine O-acetyltransferase, EC 2.3.1.7) was the first of the transferases to be discovered. In 1955 Friedman and Fraenkel demonstrated that carnitine is reversibly acetylated in the presence of acetyl-CoA by an enzyme in soluble extracts of liver.1 Bremer reproduced these experiments using mitochondria prepared from several different tissues and noted that only one optical isomer of carnitine, pre- sumably the L form, participated in the reaction.2 Subsequently a more quantitative investigation was undertaken by several research groups. Marquis and Fritz compared transferase activities in mitochondria from different rat tissues.3’€ High specific activities were found in heart, skeletal muscle, kidney, epididymis, and testes, whereas those of brain and liver mitochondria were considerably lower. Spermatozoa had the highest specific activity of transferase observed in any of the tissues. At least part of the enzyme found in testes and epididymis was contributed by sperm. In their survey of transferase activity Beenakkers and Klingenberg5 noted the correlation between levels of CAT in mito- chondria of various tissues and the carnitine-dependent fatty acid oxidation capacity of that tissue. Particularly striking was the difference between transferase levels in the flight muscle of 2 insects. The locust, which burns fatty acids in flight, had high levels of CAT; the bee, which uses carbohydrates as its energy source have The pred acti Sucr mitc equi Pres or j fol] EEt} assC cou] phOs freE enz; 5 in flight, had no detectable CAT activity.5 The association of CAT activity with fatty acid oxidation did not, however, prove true in all cases. Childress et aZ.6 demonstrated that although blowfly flight muscle does not possess the ability to oxidize fatty acids and thus depends upon a glycolytic source of energy for early flight, it does have high CAT activity. Subcellular Localization... Using the method of differential centrifugation investigators have studied the subcellular distribution of CAT in various tissues. The majority concurred that by this method the enzyme appeared to be predominantly in mitochondrial fractions with lesser amounts of activity in the Particle-free supernatant.2’3’7'-10 However, using a method with greater resolution, centrifugation through a steep sucrose gradient, the distribution of CAT was not identical to the mitochondrial marker enzyme but significantly skewed toward lower equilibrium density fractions.11 These data suggest that CAT was present in a population of mitochondria different than the marker or in an additional organelle. Unfortunately, there has been no follow-up experiment on these results. Subfractionation of rat liver mitochondria by the digitonin method revealed that at least 75% of transferase activity was loosely associated with the inner mitochondrial membrane12 from which it could be easily extracted with sonication or deoxycholate in phosphate buffer.3 Some investigators have reported that neither freeze-thaw, osmotic shock, nor ultrasonic vibration solubilizes the 7,11,13 enzyme from mitochondrial preparations. Differences in the seIe dist was 1005 p001 Cute be 1 P00 YE} SHE Ca] 6 isolation buffer, type of tissue, or age of animal used may account for the discrepancy. CAT can be extracted from pigeon breast muscle, which is used commercially as the source of the enzyme, by homogenization of the frozen tissue in 100 mM phOSphate buffer pH 7.2.14 Question of Multiple Forms The results of studies on the localization of CAT,12 on respira- tion rates using acetate derivatives as substrates,8 and inhibitors selective for CAT15 implied that the mitochondrion contained 2 distinct noninterchangeable pools of CAT activity. The outer pool was thought to lie between the 2 mitochondrial membranes or to be loosely associated with the outer face of the inner membrane. This pool could be easily lost in the isolation of the organelle. The outer transferase could readily use exogenous acetyl-CoA and could be inhibited by low concentrations of bromoacetyl CoA. The inner pool was thought to be firmly bound to the matrix side of the inner membrane. This inner enzyme was not readily accessible to exogenous acetyl- or bromoacetyl-GOA but could be reached by acetyl or bromoacetyl derivatives of carnitine. The question therefore arose as to whether the 2 forms of CAT activity, inner and outer, were expressions of the same enzyme. Recent studies have shown these 2 forms to be freely intercon- vertible and to have similar kinetic properties.14 The results suggest the existence of only a single type of mitochondrial carnitine acetyltransferase. ac: an; int r01 rev but in?.; no, Partial Purification Fritz et al.16 were the first to partially purify carnitine acetyltransferase activity to a state permitting study of some of its general properties. CAT was found to catalyze the reversible transfer of short-chain acyl groups from CoA to L-carnitine with an apparent equilibrium constant of 0.6 at pH 7.0 and 35°C. The pH profile of the enzyme's activity showed a broad optimum between 7.1 and 8.2 and was not affected by the buffer used. The rate of transfer was approximately equal with acetyl-, propionyl- and butyryl-CoA but dropped off rapidly with longer acyl chain lengths. The final preparation of the enzyme from pig heart had little or no fatty acyl-CoA hydrolase or acetylcarnitine esterase activity. Substrate studies showed that only norcarnitine (B—hydroxy-y- dimethylaminobutyrate) and B-hydroxy-Y-aminobutyrate could substi- tute for L(-)-carnitinel6’17 and acetyl-3'-dephospho-CoA for acetyl-CoA,18 but at much higher concentrations. Other carnitine analogs including the unnatural D(+)—isomer served as competitive inhibitors or were inactive in the system. Sources of Substrate L-carnitine The early investigations on the structure, distribution, and role in fatty acid metabolism of carnitine have already been reviewed.19’20 Briefly, L-carnitine (B-hydroxy-Y-trimethylamino- butyrate, vitamin BT) seems to be widely distributed in micro- organisms, plants, and animals but is particularly concentrated in 19,20 4 invertebrate and vertebrate muscle and epididymis. In addi- tion to the nutritional source of carnitine mammals have the ca; 38: x1 enz; tne of c the acet 8 capability of synthesizing at least part of their carnitine require- ment from lysine.21 Kinetic Properties The partially purified CAT activity from pig heart was found to be sensitive to sulfhydryl reagents. Prior incubation of the enzyme with its substrate acetyl-CoA protected against such inhi- bition.17 The Km for L-carnitine calculated from data using the same enzyme preparation was 3.1 x 10..4 M, for acetyl-CoA was 4.1 x 10“5 M. Chase at al.22 crystallized the enzyme from pigeon breast muscle. (This tissue had been shown to be a better source of the enzyme.5) From their data it appears that the substrates bind to tne enzyme in a random order to form a ternary complex, the binding of one substrate to its site having no effect on the affinity of the enzyme for the other substrate.23 The Michaelis constant for acetyl-CoA was given as 3.4 x 10-5 M, for L-carnitine 1.20 x 10-4 M. The pH optimum18 (7.2-8.2) and substrate specificity24 were similar to those previously reported for the preparation from pig heart by Fritz's group.16’17 Possible Functions The precise function(s) of CAT in intermediary metabolism is not clear. In general it is thought to facilitate the transport of short-chain acyl groups as carnitine derivatives across membranes not readily permeable to acyl-CoA's. In rat liver short-chain fatty acids are activated both in the cytoplasm and in the mitochondrial aci is CHI] inn. €112: fan car: agai 8C5 acet earn aCet C0u1 Sist 9 matrix.25 Those generated outside the matrix must be transported across the mitochondrial inner membrane to be oxidized. To facili- tate this transport is one of the most likely functions suggested for mitochondrial CAT.26 The correlation between levels of CAT and the carnitine-dependent fatty acid oxidation capability of the tissue mentioned earlier in this review supports this hypothesis.5 The high activity of CAT in tissues which do not oxidize fatty acids,6 however, suggests that CAT has additional functions. In regard to this Bressler and Katz27 demonstrated that there is a direct pathway via CAT for the transport of acetate in the opposite direction. Thus the product of B-oxidation acetyl-CoA could be transported out of the mitochondrial matrix as acetyl- carnitine by the action of the inner pool of CAT. Once across the inner membrane it could be reconverted to acetyl-CoA by the outer enzyme and used as a substrate for biological acetylations28 or fatty acid and cholesterol synthesis.27 In addition, this conversion of activated acetate to the acetyl- carnitine form could act as a buffer system to protect the cell against rapid changes in acetyl-CoA levels.29 In times of increased 'acetyl pressure', i.e., during increased fatty acid oxidation, the acetyl groups could be removed from the mitochondrion as acetyl- carnitine thereby relieving the pressure on the CoA system. When acetyl-CoA levels start to decrease, cytoplasmic acetylcarnitine could be used to restore levels. For CAT to function in a buffer system the reactants and products should be at or near equilibrium t1 re re 31 th 10 concentrations. This is the case for perfused rat hearts29 and for sheep muscle in vivo.10 In the same manner CAT could provide an important mechanism for the regulation of ketogenesis by maintaining levels of acetyl? CoA in the inner mitochondrial compartment where ketogenesis occurs. Hahn and Skala have speculated that in brown fat CAT could be involved in the oxidation of ketone bodies for heat production. The basis for this is their observation that the postnatal develop- ment of CAT activity follows the rise and fall in blood ketone levels.30 The exact role, if any, of CAT in the production or oxida- tion of ketones needs further clarification. Thus far the investigations into the function of CAT have focused on the uses of acetyl-CoA. However, the enzyme has a broad specificity and can use as substrates such diverse CoA esters as propionyl, butyryl,16 B-methyl-crotonyl,31 isobutyryl, isovaleryl, and 2-methy1butyryl.32 The possibility therefore exists that CAT may have a role in amino acid metabolism or in steroid synthesis from intermediates more complex than acetyl-CoA. Carnitine Palmitqyltransferase Substrate specificity studies of the purified short-chain transferase had shown that palmitoyl-CoA acted as a potent inhibitor rather than as a substrate for that enzyme.24 But the freely reversible transfer of palmitoyl groups between carnitine and CoA 33’34 in a manner similar to also occurs in isolated mitochondria, the reaction catalyzed by CAT. The existence of a carnitine palmi- toyltransferase (CPT) (palmitoyl CoA:L-carnitine 0-palmitoyltransferase, _trif for a and 3 head as 3- local of th aCtiv Cellu USe 0 aCtiv haVe Only Pools 11 EC 2.3.1.21) distinct from the acetyltransferase was first shown. by Norum,35 who separated the 2 activities by ammonium sulfate- precipitation. With the addition of ion exchange chromatography he obtained a 22-fold purified preparation of CPT from calf liver with little or no palmitoyl-CoA hydrolase and CAT activity.36 The enzyme exhibited maximum activity between pH 7.0 and 8.2. The activity of the enzyme increased with increasing chain length to a maximum with 16 carbons. The Michaelis constant for palmitoyl-CoA was 3.1 x 10.5 M, for L-carnitine 2.1 x 10-3 M. Only the L-isomer of carnitine and its acyl esters were enzymatically active. The purified long-chain transferase showed a broad spectrum of activity for acyl derivatives of unsaturated fatty acids such as linoleate and stearate,37 for prostaglandins,38 dicarboxylic acids such as hexadecanedioic,39 and B-oxidation derivatives of fatty acids such as B-hydroxypalmitate.40’41 Like the short-chain transferase, CPT is thought to be mainly localized in mitochondria and associated with the inner membrane 11’42-44 A few investigators have reported CPT 33,45 of that organelle. activity in mitochondrial and microsomal fractions. The sub- cellular distribution of CPT at this point still lay open to question. From respiratory, inhibitor, subfractionation studies and the use of antibodies specific for CPT, 2 pools of mitochondrial CPT activity (an outer and an inner one separated by the inner membrane) have been shown to occur.4.6-51 Each of these 2 pools has access only to the CoA or acyl-CoA on its side of the membrane, but both pools have access to external carnitine or acylcarnitine. Again easi Bent solu 12 as in the case of CAT, the outer pool of CPT seems to be more easily solubilized than the inner. The question again arises whether these 2 separate pools of transferase are composed of 2 different proteins. Some investigators have found no difference in the kinetic properties of the 2 trans- ferase activities.37’42’52 The differences observed by others between the 2 activities within the mitochondrion, such as the apparent immunity of the inner pool to inhibition by bromoacyl derivatives of CoA or carnitine,47’48 can be explained either by intrinsic differences in protein structure or a different environ- ment within an anisotropic membrane. Some investigators using solubilized CPT activity have claimed that 2 distinct carnitine palmitoyltransferases can be distinguished by differences in solu- bility, substrate specificity, and inhibition by 2-bromopalmitoyl derivatives of CoA or carnitine.53’54 These studies are complicated by the possible existence of medium-chain acyltransferases with some activity for palmitoyl-CoA and the problem of allotopy55 (changes in the properties of a membrane-bound enzyme upon solu— bilization). Allotopic effects have been demonstrated with CPT. The membrane-bound CPT is inhibited by D-palmitoylcarnitine and effected by the ionic strength of the assay medium, but there is no inhibitory or ionic effect on the same activity when it is solu- 53,56,57 bilized with detergent. Also, 2 forms of CPT have been iso- lated with different activity toward myristoyl-CoA, but the one form can be transformed into the other by treatment with urea or guanidine.58 eryt? that acros aCI‘OS pr0P0: COQCeI Valem 13 In rat liver long-chain acyl-CoA's, unlike their short-chain counterparts, are activated entirely outside of the mitochondrial matrix in the outer mitochondrial membrane and in endoplasmic reticulumfm’59 The primary function of CPT is to shuttle these long—chain acyl groups from their activation sites to the intra— mitochondrial compartment where they are oxidized. In addition to its role in the mitochondria, CPT may possibly play a more general role in fatty acid transport. The presence of CPT in mature erythrocyte membranes60 and term placenta61 raises the possibility that carnitine may function in translocating long-chain fatty acids across cell surface membranes and in maternal-fetal transport across the placenta. Inhibition of various enzyme activities by long-chain fatty acids and their acyl-CoA esters is well known, and there has been considerable speculation as to whether these may have regulatory roles in vivo. Unspecific inhibition which occurs because of the detergent properties of these compounds is unlikely to have a physio— logical role.62 Examples of specific inhibition that are readily reversible and not due to surfactant properties are rare but may be functional in viva. One such example is the competitive inhibi- tion of adenine nucleotide translocase by long-chain fatty acyl- CoA's.63 Lastly, a somewhat more speculative function for CPT has been proposed. Long-chain fatty acids and their CoA thioesters at low concentrations increase the permeability of mitochondria to mono- valent alkali metal cations in a manner similar to typical ionophore spec: of en chond but 5 ECHO V chond trans feras relat found fEren Garbo tTans SEVer enqu Conta Exact Carni l4 antibiotics.64 The mode of action seems to be a combination of specific as well as nonspecific detergent effects. The small amount of endogenous fatty acids and/or acyl-CoA normally present in.mito—' chondria may act as the natural ionophore responsible for the low but significant permeability the inner membrane normally has for monovalent cations. Carnitine Octanoyltransferase In the different extraction procedures used to purify mito- chondrial CPT several investigators obtained preparations with high 53,54 transferase activity for octanoylcarnitine. The known trans- ferases CAT and CPT also transfer medium-chain acyl groups but at 16’24’36 At about the same time Solberg65 relatively low rates. found in a commercial preparation of CAT a foreign activity dif- ferent from CAT and specific for fatty acids containing 6 to 9 carbon atoms. The pr0posed enzyme designated carnitine octanoyl- transferase (COT) was shown to be present in mitochondria of several different rat tissues. Although a purification of this enzyme was attempted in calf liver,66 the final preparation was still contaminated by short- and long-chain transferase activity. The exact number, substrate specificity, and function of the mitochondrial carnitine acyltransferases remains a problem for further research. Changes in Transferase Levels Changes with Development, Differentiation and Sex The responses of the different carnitine acyltransferases to genetic and environmental stress give further proof of their plurality in p0 15 as well as clues to the function of each. In development the mammalian. fetus is highly dependent on transplacentally derived glucose. After birth mammals, e.g., the rat, shift from glucose metabolism and begin to utilize the fatty acids from milk. In contrast the bird embryo, e.g., the chick, is dependent on the lipid-filled yolk. This difference in energy sources used for fetal growth is reflected in the different developmental patterns for CPT and CAT in the heart and liver of developing rat and chick. In the rat CAT and CPT activi- ties were 1ow in fetal material but rose rapidly during the first week after birth,30’67’68 concomitant with increases in fatty acid oxidation, ketogenesis, and gluconeogenesis.69 In the chick, peak activities of CPT were observed between 13 and 16 days of develop- ment.67 Activity then decreased before hatching on day 21 and increased again to peak level by 2 weeks posthatching. A similar postnatal developmental pattern for CAT was observed.70 CATand CPT do not always show the same developmental pattern in tissues. Levels of CAT in testes of neonatal rats are only 5% of adult levels. A very rapid increase in testicular activity occurred 24-32 days after birth as primary spermatocytes matured. Although there is more than 7-fold increase in CAT activity during this period, other mitochondrial enzymes such as CPT, glutamic dehydrogenase, and cytochrome oxidase did not increase.7 Even in the same tissue at the same stage of development dif- ferences due to hormonal effects may be observed. One laboratory has observed that liver homogenates from female rats consistently gave lower values (30 to 50%) of CAT than those of male rats, bra the Che and aci in fas 3-f day nif epic act: and grot kidt espe Naek effe 16 although there was no significant difference between the sexes in brain or skeletal muscle activities.7 Corresponding CPT levels in these tissues were not reported. Changes with Increased Fatty Acid Supply Because conditions such as fasting, fat-feeding, cold exposure and alloxan-induced diabetes are associated with increased fatty acid oxidation, the levels of CAT and CPT have been investigated in these conditions. The total activity of CPT in livers from fasted, fat-fed, and alloxan—induced diabetic rats increased 2- to 3-fold compared to normal rat liver.72-75 Insulin treatment for 2-4 days reversed the increase of CPT in the diabetic rat.75 No sig— nificant changes were detected in CPT activity in heart muscle or epididymal fatunder the same conditions.74 The increased hepatic activity is probably due to activation of preformed CPT rather than de nova synthesis since it is not prevented by protein synthesis inhibitors.72 There is some controversy as to the effect of fasting upon CAT activity. An increase in CAT levels in pigeon liver homogenates28 and rat liver mitochondria72 upon fasting has been reported. Another group, however, has observed no change in CAT levels in rat liver, kidneys or interscapular brown fat upon fasting.76 Cold exposure causes a marked increase in lipid degradation esPecially in adipose tissue. Exposure to cold for more than 2 weeks doubled CAT activity in brown adipose tissue76 but had no effect on CPT.3O An increase in hepatic CAT activity in animals adapt of er fibr the wee} to 1 how« rep thy Pro ti: 17 adapted to low temperatures was also noted,76 probably the result of enhanced metabolism in the liver. These conditions in which increases in CAT or CPT activity are found are associated with elevation of free fatty acid levels in.the plasma, enhanced flux of fatty acids to the liver, and increased level of activated fatty acids in the tissues. Investigators have therefore tried to link the increase in CAT or CPT with an increase in fatty acid metabolism. The plasma-lipid lowering agent clo- fibrate (p-chlorophenoxyisobutyrate)77 has been shown to increase the activity of all 3 of the known carnitine acyltransferases (CAT, COT, and CPT) but not to the same extent. In rat liver CPT showed the least change, a 2- to 3-fold increase over a period of l to 2 weeks of clofibrate feeding.39’78’79 The activity of COT rose 3- to 4-fold during the same period.78 The most dramatic change, however, occurred with CAT. Elevations of 5- to 13-fold have been reported.50’78’80 Some effects of clofibrate may be mediated by thyroxine,81 which is displaced from its binding sites in plasma proteins by clofibrate.82 Thyroxine treatment, however, does not affect CAT activity80 and therefore its increase with clofibrate is not likely to be mediated by thyroxine. The question remains whether the increase in carnitine acyltransferase activities is a causative factor in the hypolipidemic effect of clofibrate or merely a side effect. The correlation between the carnitine acyltransferases and lipid metabolism has been further strengthened by the investiga- tion of enzymatic levels in certain diseases characterized by EXCB the I seve tori EUS( l8 excessive fat accumulation. In one such disease,‘muscularrdystrophy, the overall activity of CAT in dystrophic muscle was 35% lower than' that in normal muscle.83 In another myopathy characterized by the appearance of numerous lipid-filled vacuoles between muscle fibers, CPT levels were normal but levels of its substrate carnitine were less than 20% of controls.84 In a third myopathy there was no accumu- lation of lipid, but both CAT and CPT levels were below normal, the former decreased 40% and the latter 80% or more.85 Respiratory functions with pyruvate and succinate were normal but with palmi- toylcarnitine were 38% lower than controls. If lipid metabolism is severely impaired by the decrease in CAT and CPT levels, it is curious that there was no abnormal accumulation of lipid in the muscle as in the 2 previous myopathies. 3c of SUI: hat} thi aSS EXPERIMENTAL PROCEDURES AND RESULTS It is a capital mistake to theorize before one has data. Insensibly one begins to twist facts to suit theories, instead of theories to suit facts. — Sherlock Holmes The results section of this dissertation has been divided into 3 chapters, each with its own Materials and Methods. Because many of the techniques were used only in one phase of the project or were modified as research progressed, this format was found to be the one most convenient for future referral. Discussion of results is presented in a completely separate section to offer a unified over- view of the dissertation work. As is often the case, my discovery of new peroxisomal and microsomal enzymes raises more questions than it answers. Some of the more immediate or intriguing ones, along with possible approaches to answering them, are included in the discussion section. As an offshoot of my research I investigated the optimal conditions for the microbial production of Coenzyme A. Because this is not an essential part of my dissertation, but still will be of interest to those using CoA-requiring enzymes, I have summarized these results in Appendix A. Some of the data herein have been published in another form.86 I wish to acknowledge at this point that the zonal gradients were prepared and zonal fractions assayed for marker enzymes by Dr. Tolbert's group, who generously l9 allo CPI SECC "C I Y I a h r n Bio< car fer cer fez 0t] as: 0f ho: in: We ti 20 allowed me to use these fractions. The assays for CAT, COT, and CPT on these gradients as well as the remainder of the results section are entirely my own work. Discovery of Extramitochondrial CAT Activity and Localization of Peroxisomal CAT Materials and Methods Acyl-CoA's were purchased from Sigma Chemical Company, P. L. Biochemicals, Incorporated, and Worthington Biochemical Corporation. The commercial preparation of carnitine acetyltransferase from pigeon breast muscle was obtained from Sigma. ll'C-methyl-labeled carnitine was purchased from International Chemical and Nuclear Corporation. The D and L forms of carnitine and acetylcarnitine were gifts of the Otsuka Pharmaceutical Factory, Naruto, Tokushima, Japan. Livers and kidneys from pigs or from 200-224 g Sprague-Dawley female rats starved for 2 to 3 days were used for most of the zonal centrifugation work. Preliminary experiments using livers of adult female chickens (not starved) were also done. Details of the particle preparation and characterization are described elsewhere.87 (Unless otherwise stated, all operations were carried out at 0-4°C and all assays unless otherwise referenced at 25°C.)' The procedure consisted of homogenization by one pass with a loose fitting Potter-Elvehjem homogenizer, filtration through cheesecloth, and a slow speed centri- fugation to remove cell debris. For rat tissue, the total homogenate was then subjected to isopycnic sucrose density gradient centrifuga- tion in an IEC B-30 zonal rotor. For homogenates of pig liver or and be c SOUE The grac rest to 1 dis tra: dis of - tha act lit 8031 hit 21 kidney or chicken liver, the particles were first concentrated by differential centrifugation for 20 min at 7000 x g, resuspended, and applied to the gradient. Gradients were developed for 3 or 4 hours and then lO-ml fractions were collected. Mitochondria were located by chtochrome c oxidase or succinate dehydrogenase, or both. Peroxi- somes were marked by catalase. Lysosomal distribution was indicated by acid phosphatase activity (data not shown). Protein was determined by the Lowry procedure using bovine serum albumin as a standard. The distribution of the activity of these markers on the sucrose gradients is shown in the upper sections of Figures 1 to 4. The rest of the data (middle and lower sections of Figures 1 to 4) is to be superimposed upon the gradient markers and consists of the distribution of carnitine acetyltransferase and carnitine palmitoyl- transferase total and specific activities. Coincidence of enzyme distribution between CAT or CPT and a marker enzyme is indicative of the organelle location. Analysis of these gradients indicated that in the peak peroxisomal fraction the cytochrome c oxidase specific activity was about 1% of that of the mitochondrial band and, thus, little mitochondrial contamination existed in the whole peroxisomes. Somewhat more (2 to 5%) peroxisomal contamination occurred in the mitochondrial area of the gradient. Microsomes were also isolated by differential centrifugation as described elsewhere88 except that the homogenate was centrifuged twice at 20,000 x g for 20 min rather than once at 10,000 x g. The washed 105,000 x g pellet was resuspended in 150 mM Tris chloride buffer, pH 8.0, and assayed immediately for carnitine acyltransferase activity. photo carni CoA a withc of Cc assa} in t1 SUCTt Frac Brad SFSt and 22 Carnitine acyltransferase activity was routinely assayed in' gradient fractions by following the release of CoA from acyl-CoA using the general thiol reagent DTNB (5,5'-dithiobis[2-nitrobenzoate]) as previously described.86’89 The reaction was initiated by addition of enzyme and the rate followed at 412 nm on a Gilford 2400 spectro- ' photometer. The difference between the rates with and without L- carnitine gave the carnitine-dependent rate for the formation of CoA and was equated to carnitine acyltransferase activity. The rate without carnitine is referred to as the carnitine-independent release of CoA, background, deacylase, or hydrolase activity. The DTNB assay system was not affected by the high levels of sucrose present in the aliquots from the gradient. Particulate fractions from sucrose gradients were assayed for CAT, CPT and in a few cases COT. Fractions with peak activities were assayed at least in duplicate. CAT activity in peak peroxisomal, mitochondrial, and microsomal gradient fractions was further established by using 2 other assay systems. The first follows the formation of acetyl-CoA from CoA and L-acetylcarnitine at 232 nm. The second was based on the forma- tion of a radioactive product [14C]-acetylcarnitine. Gradient frac- tions or a commercial preparation of CAT were incubated with acetyl- CoA and DL-[14C]-carnitine containing L-carnitine in a Tris-buffered system at pH 8.0. DTNB was added after 10 min to pull the otherwise reversible reaction toward synthesis of acetylcarnitine. The samples were immediately frozen and lyophilized. A 95% ethanol extract of each was spotted on Whatman No. 3MM chromatography paper with internal standards of carnitine and acetylcarnitine and chro: (4:1 scan had data hep; the loc. gre fer tio org enz to stL Sm SUI r') o .4 “a CF to 23 chromatographed in a l-butanol-water-glacial acetic acid system (4:1:1). The 14C product was detected with a radiochromatogram. scanner and the standards with Dragendorf spray reagent. Carnitine had an R of 0.24 and acetylcarnitine 0.41 in this system. Only f data using the DTNB assay are presented in the figures. Results Distribution of CAT, COT, and CPT Activities in Liver Because of conflicting reports in the literature as to whether hepatic CAT, COT, and CPT were exclusively mitochondrial activities, the first phase of my research was to determine the subcellular location of these activities in liver, specifically rat liver. The great majority of the previous studies had used the method of dif- ferential centrifugation in attempts to separate subcellular frac- tions. This method suffers from 2 limitations: a) not all the organelles of the cell can be separated in this fashion, and b) some enzymes may be inactivated during the procedure. Therefore I chose to supplement and clarify the results of differential centrifugation studies by using a method with greater resolving power, isopycnic sucrose density centrifugation. A shallow nonlinear gradient of sucrose can separate peroxisomes, lysosomes (primary and secondary), mitochondria, lipid-rich membranes (Golgi, microsomal and plasma) and cytosol into discrete bands upon centrifugation. Each of these particulate fractions was assayed for CAT and CPT activities and in a few cases COT in addition to marker enzymes to indicate the location of various organelles. Each of the in it thi} Witt the 24 transferase assays was corrected for carnitine-independent release of CoA. The amount of CAT, COT, and CPT in the original homogenates or in soluble fractions of the gradient could not be accurately estimated, because of the high level of carnitine-independent activity. Data are shown for one gradient from liver and one gradient from kidney of both rat and pig, but the results of each of these experiments were verified with at least 3 additional gradients. Distribution of CPT activity on sucrose gradients from rat and pig liver homogenates paralleled the activity distribution of the mitochondrial markers, succinate dehydrogenase and cytochrome c oxidase (Figures 1 and 2). Since particulate CPT was present only in mitochondrial areas of the gradient, it was therefore concluded it was exclusively a mitochondrial enzyme. This conclusion was further substantiated by the fact that no detectable CPT activity was found in microsomes isolated by differential centrifugation , (Table 3 in chapter 2). In contrast CAT activity was located in 3 distinct particulate regions of the gradient from rat liver (Figure 1). Only half of the total CAT activity coincided with the mitochondrial marker, cytochrome c oxidase. The rest of the activity was divided between the peroxisomal region and a third region with a sucrose density of about 1.10 to 1.18 g x cc-l. The specific activity of CAT in the third region was approximately the same as in the mitochondria. The specific activity in the first peak, the one that coincided with the peroxisomal marker catalase, was 2- to 3-fold greater than the other 2. Activity for acid phosphatase, the lysosomal marker, 25 Figure 1. Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase among the organelles of rat liver. The gradient was characterized by its sucrose density profile G——-—-, D100) and protein distribution (- - -) (top). Catalase (O--O) and cytochrome c oxidase (A A), respectively, mark the peroxisomal and mitochondrial peaks (top). The activities per milliliter (0—-—-O) and specific activities (0————0) of carni- tine acetyltransferase (middle) and carnitine palmitoyltransferase (bottom) are shown corrected for the carnitine-independent release of CoA (k———523 _ u 1 1.22 a .w .7... x 7...... x «30...... .7... x 7...... x 3.0:... ...-.:xoz.z.w5¢.. w s w a n a a m u u s 4 m u m u m w a o 4 2 . . T. . . . q . . . . m. . . . a . . . . . u. a. m '4 W m. a m. u lotion: 1 M t D C T ‘ ' “\ R e... m at- E m u ,. W m m A II L O . W w o. N 0‘. O T c H a 1|.“ A 9 u u “n. In. , .1 “'3‘... ........... o. 0 O O ‘ I. A o m. o E a... O m \a‘ oo 0 A ,. 1 w u n a ..................... av . . . . u a ..I ...u 1 1 MI . 2 W . 8 I . 4 3 2 1 7.... x 7...... x «30.! I 7.! x 7...... x 3.9.... I .7... x 7...... x 3.02:. 353.3 I _ _ _ _ _ _ _ _ |L|$+$IIIIJWI zap domflllll m a s. 2. m 1. 1. a e of 7 72.... x no .9! 010 .55.... 72. x 72.: x 3.5:... I .2355. 70: x 7...... x 8.0:... 8533 olo - — — n F — C .0 4 2 1 :IJS x 7.2.! x mug wan-X0 0 $1005 I (co ".0 gnu: nines-memo! 3:29.23 I (00 no gum: Emozwamoz. 3.3.5:: I IN GRADIENT VOLUME (ML) Figure l di 5 ya so th CA tit of the or how Car: 27 did not coincide with any of the peaks of CAT activity. Some of the lysosomal activity peaked between the peroxisomes and mito— chondria, while most of it resided in less dense sucrose just above the mitochondria. The absence of CAT in lysosomes agrees.with the results of Norum and Bremer,11 who separated mitochondria and lyso- somes on a sucrose equilibrium density gradient after treatment of the rat with Triton WR-1339. These data are convincing evidence for CAT activity in both respiratory organelles, the peroxisomes and mitochondria, plus at least one other subcellular site in the rat hepatocyte. The diffuse and variable specific activity of the third peak of CAT suggests that it contains more than one organelle or a popula- tion of particles of varying density. From the low specific density of the third peak, it is postulated that CAT is a component of some lipid-rich cell membrane fraction. This region is known to contain microsomes plus components of the Golgi apparatus and plasma membrane. A similar fraction roughly termed 'microsomal' was isolated by dif- ferential centrifugation from rat liver and assayed for CAT activity. These microsomal preparations were contaminated by less than 3% of the total catalase and cytochrome c oxidase and contained very little or no detectable CPT activity. Significant amounts of CAT activity, however, were found in these fractions with a specific activity of the same magnitude or slightly greater than in mitochondrial fractions from the same liver. While these studies on CAT and CPT were in progress, a third carnitine acyltransferase COT was discovered53’6S and partially purl the COT sho reg tio oct hos the or Pe Mi f‘h 28 purified.66 All of the particulate fractions of one gradient plus the peak fractions of several subsequent gradients were assayed for COT. Like CAT, 3 distinct peaks of COT were observed (data not shown) coinciding with the peroxisomal, mitochondrial, and microsomal regions of the gradient. Hepatic peroxisomal and microsomal frac- tions showed about the same or slightly greater activity for octanoyl-CoA as for acetyl-CoA (Table 1). In rat mitochondria, however, COT activity varied from about the same to 6-fold greater than CAT. The latter is in good agreement with reported values for mitochondria.65 TABLE 1 Specific activities of carnitine acyltransferases from organelles of rat liver with different acyl-CoA substrates Substrates Acetyl-CoA Octanoyl-CoA PalmitoyeroA Organelle nmoles x min-1 x mg-l protein Peroxisomes 15.4 19.7 0 Mitochondria 5.1 15.8 4.2 Microsomes 6.2 8.5 0 The occurrence of extramitochondrial CAT in liver is not unique to the rat. In pig liver preparations, the particulate fraction was first enriched by differential centrifugation between 1000 x g and 7000 x g for 20 min, and then the resuspended particles were further separated by sucrose density gradient centrifugation as shown in Figt $0711 the 2 U fer; grac dria shat. latt aCtI aoiz Spe: m3 f1‘0:- aCt; Pig bec. rel! EVE 1Y8: 29 Figure 2. As a result, most of the lighter fractions, i.e., micro- somes, were discarded in the 7000 x g supernatant. Two peaks of CAT activity, a peroxisomal and a mitochondrial one, were seen on the gradient. Again the specific activity of peroxisomal CAT was 2 to 3 times as high as in mitochondria. The third peak of trans- ferase activity in the microsomal region was not detected on this gradient. If the entire pig liver homogenate was applied to the gradient, however, 3 peaks of CAT activity (peroxisomal, mitochon- drial, and microsomal) appeared after zonal centrifugation (data not shown). As with rat liver, a microsomal fraction of pig liver iso- lated by differential centrifugation contained significant CAT activity. Not only mammals such as the rat and pig but another class of animals, birds, have extramitochondrial CAT activity. The highest specific activities on any gradient (77 and 83 nmoles x min.1 x mg.1 protein) were found in the peroxisomal peak of 2 gradients from chicken liver. The corresponding mitochondrial specific activities were 15 and 25 nmoles x min-1 x mg-1 protein. As with pig liver, the microsomal region did not appear on the gradient because a 7000 x g pellet not the total homogenate was applied. On all of the gradients (Figures 1 to 4) carnitine-independent release of CoA or hydrolase activity was widely distributed. Although every fraction had some of this activity, it occurred primarily in lysosomal and soluble regions paralleling the soluble lysosomal marker acid phosphatase (data not shown). On most of the gradients hydrolase activity was equal to or greater than carnitine 30 Figure 2. Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase between peroxisomes and mito- chondria from pig liver. The particles from the liver homogenate of a 2-week-old pig were pelleted by centrifugation for 20 min at 7000 x g, resuspended, and applied to the gradient. Symbols are the same as in Figure 1. H cvrocunonn c axiom (NE. a: nu-' x on"! m m m m. m m m..-8 x o. :3qu .7... x 72... x .3012. .7... x 72.: x 3.9.2. _ _ _ _ d _ . <8 so 34wa 53233:. .2:sz I <8 .6 343... 532232. 22:25". I .7... x or. .55.: u u u u .... a. m n n m w n a n u n I 1 _ d Le _ _ _ _ cm a m — _ d — _ — _ _ _ _ I E t . ... w. u .1. - - . - .. s m m a r ., , _ . . ._ M .1 .- m _ . 1... C / r n a I U L .11. . w m on E . _ W w v M I 0 IA. HxH no» N Oo\. P o» 1 fl _ . E a m u... m m m o u \o\ N m t . n a . 31 PIG LIVER w u u CVTWHOOI‘ C OXIDASE _ u .7... x 7:... x a w a . a a 1. ... s 7.: x 7...... x .39! I ‘ I .‘whg .li x rlg x Dug Etta 0'0 2.2.0.: 79. x 7:... x 8.5.! 010 1w l H 1mm .7... x 7:... x 2 (mg 2x0 0 £381 0 k 0 GRAD!!!" VOL” (”U 1 .l 1 I ‘35; plg x nil-I I a; 0'10 uldl x pl; K a; I Figure 2 acyltra region low bu1 5] to gra: 4 grad nmoles chondr Specif than i organs Althou and mi were a from 1 (n subj ec hydro} in the be det carni‘ glutal all 3 acety] a Subs incorp 32 acyltransferase activity. A notable exception was the peroxisomal region of the gradient from liver in which hydrolase activity was low but CAT activity high (middle section, Figures 1 and 2). Specific activities for CAT, COT, and CPT varied from gradient to gradient and in proportion to each other. For example, data from 4 gradients using 5 rat livers per gradient show a range of 3 to 18 nmoles x min-l x mg.1 protein for peroxisomal CAT, 3 to 7 for mito- chondrial CAT and 4 to 7 for microsomal CAT. In each gradient the specific activity of peroxisomal CAT was always 2 to 3 times greater than in the other 2 organelles. The ratio of CAT to COT in each organelle seemed independent of the ratio in the other organelles. Although the CAT to COT ratio was usually about unity in peroxisomes and microsomes, it varied from 0.5 to 2.0. In mitochondria COT levels were always found to be greater than CAT levels but the factor ranged from 1.5- to lO-fold. Since the DTNB assay for CAT is a difference assay and therefore subject to error especially in samples containing high levels of hydrolase activity, additional evidence was sought for CAT activity in the various organelles. Using the 232 nm assay for CAT, it could be demonstrated that all 3 zonal peaks of CAT activity used Leacetyl- carnitine but not the D isomer as a substrate and none could use glutathione in place of CoA. In the radioactiVe assay, samples of all 3 peaks formed [14C]—acetylcarnitine from DL-[14C]-carnitine and acetyl-CoA. Again, probably because the D form could not be used as a substrate, up to but never more than 502 of the radioactivity was incorporated into the product. p'i_s_t As 1' only in t The dist] rat kidne chondria for pig 1 were not liver, Cl fractions region (7 between 1 the totaj little 0: 13$ Sin liver, a glfv'OXyso be Prese and to p FraCtiOn bean cot Cot‘fledo; Neither ( 33 Distribution of CAT and CPT in Kidney As in the liver, CPT activity in rat and pig kidney was located only in the mitochondrial region of the gradient (Figures 3 and 4). The distribution of CAT activity among the organelle fractions from rat kidney indicated that about 90% of it was associated with mito- chondria (middle section, Figure 3). Similar results were obtained for pig kidney (middle section, Figure 4). Note that microsomes were not applied to the gradient from Figure 4. In contrast to the liver, CAT activity was not detected at all in kidney peroxisomal fractions and only a trace of it could be found in the microsomal region (Table 2). Table 2 dramatically points out the difference between the 2 organs in the distribution of CAT. In liver half of the total particulate CAT is extramitochondrial; in kidney there is little or none found outside the mitochondrion. Distribution of CAT and CPT in Plants Since CAT activity was found in peroxisomes from mammalian liver, an exploration of various plant tissues containing peroxisomes, glyoxysomes, or both was conducted. Carnitine has been reported to be present in some of the higher plants particularly fatty seeds90 and to participate in the mitochondrial oxidation of fatty acids.91 Fractions of isopycnic sucrose gradients of spinach leaves and castor bean cotyledons as well as homogenates of wheat germ, sunflower cotyledons, and avocado mesencarp were assayed for CAT and CPT. Neither of these activities was found in any of the fractions or homogenates using the DTNB assay. 34 Figure 3. Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase among organelles of rat kidney. The gradient was characterized by its sucrose density profile 6---, D100) and protein distribution (- - -) (top). Catalase (O—-—-O) and succinate dehydrogenase (A——-—A), respectively, mark the peroxisomal and mitochondrial fractions (top). The activities per milliliter (O—--O) and specific activities (0——-—O) of carnitine acetyltransferase (middle) and carnitine palmitoyl- transferase (bottom) are shown corrected for the carnitine- independent release of CoA (k--A) (middle and bottom). ! i H “CONAV' DENVOIMINA‘E (Mth I ”I" I “L I 35 m m u u. m m m .78 x 2 >528 . . $ 4 . _ . .74: x 72.: x 8.68.... :5: x 7...... x 3.0:... <8 3 853.. 53223.... 22:25.“. I <8 3 8433. 53.3.3... 2.52:5 I .7... x 0:. 2.2.0.: .3. u u o a. J a. a m a u a u m c u I 4 I I 4 2 \4 .oxq I:Il11 . . .4 q . . . . . m. . a. . q . . . . - . .u m m \ O E O E I I ‘ A TIT , o o m m I I 0,. M n . m 0 v. M m m E N . T o .h m 3 N. E ‘ N L U s N OI . m O R V I T E m . E O h u m t M \ o m o m M - \. \ . H W o‘ a. . L V. m V o M .0 M m L m. m .. a \ o w c O » -m - .. ........ nm .1 films ......... m T A . no .- ‘Mlc M m , .o ./ m o 4... ,. n... :::::::: m p _ _ . _ m— W“ m M n M u - O 4 4 WF 2 1 €82.08; plg x «I: X Gui ghfla CIIO Lwl swirl .748 x 73.: x Mug 848803228 33.803 I 7.... x 7...: x 33...... I 1 3.8.: 79.. x 7...... x 36.! 0'0 |+|+I|r 4m .T 2.2.0.... 708 x 7...... x 3.0.! Oulo Figure 3 36 Figure 4. Distribution of carnitine acetyltransferase and carnitine palmitoyltransferase between peroxisomes and mitochondria from pig kidney. The 7000 x g resuspended pellet of kidney homogenate from an adult pig was applied to the gradient as in Figure 3. Symbols are the same as used in Figure l. 37 I PIG KILDNEY .7... x 7...... x 8.9.... hiklh. manna: mewuwenm O i“ .° «u 1 p 5 .71.: x u... x 3.9.... 84.33 I - r ... 3.3.5.! 79. x 7:... x 3.08... 3...: .7... x 7:... x 3.2.... 83:8 9 39.38:... I 7... x 7...... x 3.0.! I II+|+|JT||TII 83kg pt: X 7.2.8 I an; I CARNITINE PALIITUYLWEIASE .7... x 7:... x 3.9.... (8 .6 33.2. 535.3... 33.25:. I <8 3 33.... 53233.... u... p.253 I 7... x 7:... x 3.082 I .llll. 3.39: 7.... x 7...... x 3.9.... I GRADIENT VOLWE (MU Figure 4 38 TABLE 2 Distribution of carnitine acetyltransferase in particulate fractions from rat liver and kidney gradients Z of total particulate carnitine acetyltransferase activity in: Organ Peroxisomes Mitochondria Third peak Liver Gradient 1 (Figure l) 11 57 32 Gradient 2 (not shown) 17 48 35 Kidney Gradient 1 (Figure 3) -- 94 6 Carnitine acetyltransferase activity in the gradient fractions was summed to give the total from which the percentage in each par- ticulate peak was.calculated. Because there was some variation in the sucrose density in which peroxisomes band, the total peroxisomal activity included the peak fraction of catalase activity plus 5 fractions on each side. The fractions between the peroxisomal frac- tions and a sucrose density of 1.180 g x cc"1 were taken as the mito- chondrial peak. This included at least 95% of the mitochondrial markers, cytochrome c.oxidase, and.succinate dehydrogenase. The third peak occurred between sucrose densities 1.180 to 1.100 g x cc‘l. Latency and Localisation of Peroxisomal CAT Previously a requirement for the detergent Triton X-100 was demonstrated in the DTNB assay for CPT in mitochondrial fractions in order to obtain reproducible results.89 A similar requirement for mitochondrial CAT measurements was encountered, so 0.1% Triton X-100 was routinely included in the assay media. When peroxisomal fractions were assayed for CAT within 2 hours after separation on the sucrose gradient, there was a S-fold increase in detectable CAT activity upon the addition of Triton X-100 to the assay. After storing the gradient 10 days at 4°C the total CAT activity remained 39 unchanged but Triton X-100 was no longer needed for maximum activity. It is presumed that this phenomenon was due to an alteration in the permeability of the peroxisomal membrane as it aged during storage. The small amount of carnitine-independent deacylase present in these fractions was not stimulated by Triton X-100, nor was a commercial preparation of CAT in the soluble form. Freeze-thaw procedures also disrupted the peroxisome to reveal latent CAT activity. These results suggested that CAT activity in the peroxisome as in the mitochondrion was located inside the organelle and not readily available to exogenous acetyl—CoA. An attempt was subsequently made to localize the enzyme within the organelle. Sucrose gradient fractions of peroxisomes from rat liver were pooled, diluted 1:1 with 0.01 M pyrophosphate at pH 8.5, and stored overnight at 4°C in order to rupture the peroxisomes.92 Untreated controls consisted of a 1:1 dilution with water. These peroxisomal samples were then subjected to equilibrium density sucrose gradient centrifugation (Figure 5) designed to fractionate peroxisomes into soluble, mem- branous, and dense core material.93 In the gradient containing pyrophosphate-treated peroxisomes (Figure 5, C and D), urate oxidase sedimented into the densest sucrose, typical of its known property as a component of the stable peroxisomal core. The membranous fraction, identified by cytochrome c reductase, sedimented into the gradient to an isopycnic density between that of the core and the soluble fractions. Catalase, a soluble matrix enzyme in rat liver peroxisomes, remained at the top of the gradient. The entire complement of CAT activity after rupture of the peroxisomes was 40 Figure 5. Fractionation of rat liver peroxisomes. Untreated (A and B) peroxisomes obtained from the sucrose gradient were diluted 1:1 with water and stored overnight at 4°C and frac- tionated on a sucrose density gradient characterized by D10°. Urate oxidase (O————O), a marker for the peroxisomal core; cyto- chrome c reductase (k————A), a marker for the peroxisome membrane; and catalase (F—O), a marker for the peroxisome matrix, are shown in A. The location of carnitine acetyltransferase activity (O——-—O) is shown in B. .Pyrophosphate-treated (C and D) peroxi- somes were diluted 1:1 with 0.01 M pyrophosphate at pH 8.5, stored overnight at 4°C, and then fractionated by centrifugation on a sucrose step gradient. Symbols are the same as in A and B. 41 n u v l. a O m m - 8. a I. a. A ) 3 D x or 1. m :. a .W x Op. a. P x a ... 2+ ZOZD<¢u h2w5<¢0 fl V LI «3. ?n ghw04 w2.h.2¢(u mghgawc U inCZOOgu , whm . wm(4(h(0 20:0(5. .PZwR—(cu up 2 m wm<¢wum2(¢h._>hwu< w2.h.2¢u Owh._._>_.5< ._<..:z_ 3 .23 «we all- TIME (HOURS) Figure 6 58 Partial Purification and Characterization of Peroxisomal and.Microsomal.CAT Activities Materials and Methods Adipic acid dihydrazide was purchased from Eastman Organic Chemicals and fatty acid poor bovine serum albumin (BSA) from Miles Laboratories. Sephadex G-100 was from Pharmacia Fine Chemicals. Cellulose phosphate (medium mesh, Lot #23C-l450), Sepharose 4B, CoA, and CAT (specific activity 80-100 units/mg) were obtained from Sigma Chemical Company. Acyl-CoA's were from Sigma and P-L Biochemicals. DEAR-cellulose DE52 and cellulose phosphate P11 were purchased from Whatman. The latter proved unsatisfactory for use in CAT purification. Wheat seedling 3'-nucleotidase was generously provided by Dr. James Fairley and warren Kroeker. Protein concentrations were quantitated by absorbance at 210 nm using BSA as a standard.113 CAT, COT, and CPT were routinely assayed by the DTNB method (Methods, first chapter). The 232 nm assay (Methods, first chapter) was used in determining the pH profile of partially purified CAT activity. Preparation of Dephospho CoA—Affinitngolumn The 3'-dephospho CoA (dCoA) affinity column was synthesized by the general procedure for coupling nucleotides to agarose by hydra- zone formation.114 CoA (50 mg) was dephosphorylated at the 3'- 115 116 position by wheat seedling nuclease as referenced. By means of the Bartlett assay117 it was shown that 0.99 moles of inorganic phosphate were released per mole of CoA added. The final product had an adenine:ribose:phosphate ratio of l.07:1:2.18 where adenine 59 was estimated by UV absorption at 260 nm based on a millimolar~ absorption coefficient of 16,000.116 Ribose was determined by the orcinol method118 and phosphate by the Bartlett method.117 The arsenolysis testllg’120 showed no detectable CoA present in the dCoA product. The dCoA ligand was to be attached through its ribose ring to activated Sepharose by means of a hydrazide arm as follows. Cyanogen bromide (4 g) was added to Sepharose 4B (30 m1) and the activation of Sepharose carried out as referenced.121 Adipic acid dihydrazide (2.7 g) was added to the activated agarose gel to produce Sepharose hydrazide. The deep red of the 2,4,6-trinitrobenzene-sulfonate (TNBS) test indicated a high degree of coupling. The ligand dCoA was oxidized with metaperiodate to the dialydehyde form by the method of Gilham.122 Sepharose hydrazide (10 ml) was added to the dialdehyde. After 3 h of reaction the TNBS test indicated that most of the hydrazide arms had reacted with dCoA. From the 260 nm absorbance of the wash it was estimated that 50% or about 30 nmoles of the dialdehyde remained covalently coupled to the column. This efficiency of coupling (3 nmoles/m1 Sepharose) is comparable to that achieved for other nucleotides by this method.114 Procedure for Isoelectric Focusing A 10-ml column of 1% Ampholine in a 10-50% sucrose gradient was used for isoelectric focusing. A 3% ethanolamine solution was used at the cathode, 3% H2804 at the anode. ‘Samples of CAT activity were dialyzed against 1% glycine overnight, combined with the carrier ampholytes of pH 3-10 or pH 7-10, and sucrose added to give a 10% 60 and 50% solution as described.123 A gradient mixer was used to form the 10-50% sucrose gradient. Samples were electrofocused 36 h at 200 v. Results Initial Attempts at Purification Having determined the subcellular location of CAT activity in the rat hepatocyte, the third phase of my research was to solubilize and purify the extramitochondrial CAT activities to a state which would permit comparison of their general properties. To character- ize these CAT activities the final preparations would have to be relatively free (<5% contamination) of other carnitine acyltrans- ferase activities and acyl-CoA deacylases. A combination of well-established conventional procedures and newer methods such as ammonium sulfate gradient solubilization, ion filtration, and affinity chromatography was the approach I decided on. Three considerations indicated that the purifications could be rather difficult: a) microsomal CAT was a membrane-bound enzyme, b) under most conditions CAT activity solubilized from the micro- somal membrane or the disrupted peroxisome was unstable, and c) the specific activity of CAT in rat liver is low (CAT makes up <1% of the microsomal membrane). In the initial purification attempts only the peak zonal frac- tion with the highest specific activity for CAT of the peroxisomal or microsomal region was used. CAT activity was solubilized by rupturing the peroxisome with pyrophosphate (Chapter 1) and treating 61 the microsomal membrane overnight with 0.4 M KCl (Chapter 2). The solubilized activity was dialyzed overnight against 150 mM Tris HCl, 1.5% Triton X—100 at pH 8.0. Ammonium sulfate fractionation of the solubilized samples resulted in about two-thirds of CAT in the 60% saturated pellet and the remainder in the 80% pellet. No separation from COT was achieved and recovery was low (515%). The inclusion of a protamine sulfate step (at 0.04%) did not increase the fold purification nor significantly alter the 280/260 ratio which ranged from 1.2-1.4 for both samples. Because an ammonium sulfate precipitation is a necessary prerequisite for ammonium sulfate solubilization124 another method was tried next. DEAE-Sephadex A-25 and CM-Sephadex C-25 have been successfully employed for ion filtration chromatography, a procedure that combines ion exchange and gel filtration chromatography.125 Neither peroxi- somal nor microsomal CAT was adsorbed onto DEAE-Sephadex at pH 8.0 or 9.5. A distinct blue band of protein formed on the column with the peroxisomal sample but not the microsomal one. Samples of CAT were absorbed onto CM-Sephadex at pH 7.0 or 8.0 but the enzyme was unstable at the low ionic strength needed for binding. CAT activity in the microsomes was also unstable at acidic pH's (Chapter 2) so these could not be used on the CM-Sephadex column. Because of prob- lems of instability of CAT and poor flow rates on these columns and the small sample volumes needed for resolution ion filtration could not be used with these crude samples of CAT. Affinity chromatography using the newly synthesized dCoA column (Methods) was next tried with samples of commercial CAT. 62 The enzyme (0.125 mg) could be totally absorbed onto the column if allowed to incubate with it 1 h, but BSA (2.50 mg) under the same conditions washed through. The inclusion of 1 mM L-carnitine or L-acetylcarnitine in the applied sample or eluting buffer did not change the behavior of CAT, nor did the reduction of the dCoA groups with dithiothreitol. A clue to the mystery of why an unusually long equilibration time was needed for the enzyme to bind to the dCoA column was the finding that the enzyme would adsorb onto Sepharose hydrazide without any equilibration period and was eluted by the same ionic strength (0.20 M KCl) from Sepharose hydrazide as from the dCoA column. Commercial CAT did not interact with Sepharose 4B alone. The COT impurity in the commercial sample also adsorbed onto Sepharose hydrazide and eluted at the same ionic strength as CAT. Again the low ionic strength needed for adsorption of CAT combined with the small size of the column dictated that Sepharose hydrazide be an intermediate or final rather than initial step in the purification process, because of probable inactivation of the enzyme in the time required to apply large samples. Because CAT would adsorb onto CM-Sephadex a stronger cation exchanger cellulose phosphate was tested in batch and column pro- cedures. Whatman cellulose phosphate P11 completely destroyed microsomal and peroxisomal CAT activities whether added batchwise or used as a column. The Sigma product, however, in identical pro- cedures adsorbed CAT and all of the original activity could be recovered. Substrate elution with 1 mM L-carnitine did not change the point of elution of CAT from the cellulose phosphate column. 63 Final Purification Scheme Peroxisomal and microsomal regions from 5 zonal gradients. stored at -20°C were thawed overnight at 4°C, pooled, and.made.0.4 M in KCl. This process solubilized CAT from the microsomal membrane, disrupted the peroxisomal membrane to free CAT in the peroxisomal matrix, and stabilized both CAT activities. The zonal samples con— : tained approximately equal amounts of CAT and COT but no detectable CPT activity. The solubilization process proceeded 12 h after which time the samples were diluted 4-fold and ethanolamine added to yield solutions of CAT in 0.1 M KCl, 0.01 M ethanolamine at pH 9.5.- The solutions were applied to a DEAR-cellulose column (4.1 x 35 cm). The pH 9.5 flow-through was concentrated by pressure dialysis using a P—lO membrane. This first step achieved a 3- to ‘4-fold purification without substantial loss of CAT activity (Table 7). At the same time a 6-fold decrease in COT was observed in the flow-through but no detectable COT activity could be eluted from the DEAE-cellulose column by a 0.1-1.0 M KCl gradient at pH 9.5 The dialysis chamber and membrane were washed 3 times with 0.015 M phosphate buffer at pH 7.5 or 6.0 and the washings combined with the concentrated solution to yield a volume of about 100 ml. The microsomal sample was brought to a pH of 7.5 because at this stage microsomal CAT was more stable at this pH than at pH 6.0 which was found to be optimal for the purification of the peroxi- somal enzyme. Microsomal and peroxisomal samples were applied to a Sigma cellulose phosphate column (0.6 x 12 cm) and eluted with linear KCl gradients of 0.1-0.5 M (see Figure 7, section A, for 64 TABLE 7 Purification procedure for peroxisomal and microsomal carnitine acetyltransferase activity CAT Specific Purifi- Purification activity Recovery Protein activity cation step (units)* (%) (mg) (units*/mg) (fold) Zonal microsomes 1029 100 776 1.33 1.00 DEAE-cellulose 1016 98.7 210 4.83 3.64 Cellulose phosphate (pH 7.5) Center fractions 450 43.7 1.40 321 242 (27-33) of peak Remainder of (21- 548 53.3 5.03 109 82.1 26, 34-39) peak 998 97.0 Zonal peroxisomes 843 100 224 3.76 1.00 DEAR-cellulose 776 92.1 69.8 12.2 3.25 Cellulose phosphate (pH 6.0) Center fractions 218 25.9 0.26 825 . 219 (28-32) of peak Remainder of (24- 582 69.0 3.04 192 50.9 27, 33-36) peak 800 94.9 CAT activities were assayed using the DTNB method and protein calcu- lated from the 210 nm absorbance as referenced in Methods. * A unit of CAT activity produces 1 nmole of CoA and L-acetyl- carnitine from acetyl CoA and L-carnitine per min per m1 of reaction mix at 25°C and pH 8.0 in the DTNB assay method. 65 elution profile of microsomal CAT) or 0.2—0.6 M (see Figure 8, section B, for elution profile of peroxisomal CAT). Little or no COT activity was found associated with the peak of CAT activity. Furthermore no detectable COT activity was found in the flow— through, in other fractions of the gradient, or in a l M KCl wash of the column after the gradient. CAT activity eluted from cellu— lose phosphate in a single symmetrical peak without substantial loss of activity. The center peak fractions of CAT activity from the cellulose phosphate columns were more than ZOO-fold purified over the starting material (Table 7) and contained less than 5% contamination by COT or acyl-CoA deacylase. These fractions were used for gel filtration, isoelectric focusing, and the kinetic studies of CAT. The remaining fractions in the peak which were more than 50-fold purified (Table 8) and again contained <5% con- tamination by COT and acyl—CoA deacylases were used for cellulose phosphate, Sepharose hydrazide, pH profile, and chloride salt studies. Although microsomal and peroxisomal CAT activities were substantially purified by these procedures, it was estimated that a further 100-fold purification would be needed to bring them to the specific activity of the commercial enzyme which still contained contamination by COT if not by other proteins. Partial Characterization and Comparison of Properties of Microsomal, Peroxisomal and Commercial CAT Some of the general properties of the 2 partially purified extramitochondrial CAT activities were examined in an attempt to ascertain whether they were expressions of the same protein. It was 66 recognized that solubilization of the membrane-bound microsomal CAT could produce allotopic effects that could lead to false differ- ences observed between it and its peroxisomal counterpart. These CAT activities from rat liver were in turn compared to a commercial preparation of the pigeon breast muscle enzyme, the only CAT which has been crystallized. In the purification procedure neither peroxisomal nor micro- somal CAT was adsorbed onto DEAE-cellulose when applied in 0.1 M KCl, 0.01M ethanolamine at pH 9.5. The commercial enzyme behaved simi- larly when applied under the same conditions. Because of insta- bility of the crude microsomal preparation the purification step using cellulose phosphate was run at 2 different pH's for the 2 different samples. The interaction of peroxisomal and microsomal CAT plus that of the commercial enzyme was investigated at these 2 pH's. At pH 7.5 (Figure 7) all 3 enzymes eluted at about the same ionic strength; microsomal and peroxisomal CAT (sections A and B) at 0.34 110.02 M KCl, and commercial CAT (section C) at 0.37 t 0.02 M KCl. Activities eluted in single symmetrical peaks and were stable for at least 24 h when stored at 4°C. Similar elution pat- terns and stabilities were observed at pH 6.0 (Figure 8). Micro- somal and peroxisomal CAT (sections A and B) again eluted at the same ionic strength (0.42 1:0.02 M KCl and 0.43 i;0.02 M KCl) but commercial CAT (section C) required a significantly higher ionic strength (0.58 1:0.02 M KCl). It had been previously shown that commercial CAT could be totally adsorbed onto a Sepharose hydrazide column. This same 67 Figure 7. Cellulose phosphate elution profiles at pH 7.5 of microsomal, peroxisomal, and commercial carnitine acetyltransferases. A 0.6 x 12 cm column of Sigma cellulose phosphate was equilibrated with 0.015 M potassium phosphate buffer at pH.7.5- CAT activity was eluted with.a linear 50 ml gradient of 0.1-0.5 M KCl. .One milliliter fractions were collected and CAT activities located by the DTNB assay (Methods). 68 AMUCROSOMAL 4o " 0.34—f ,0" 20 ' o’ ‘ B) PEROXISOMAL ‘L ' , " 2 45' 'éo ' x 0.34» " q.- u- ' ,v E v""’ z 2 P " 'o' X o" (D III-I _I o C)COMMERCIAL E g z ,o’ 5 o 0.37—t'a 25 l- ',o' " r’ “ l J 2O 3O VOLUME (ml) Figure 7 0.4 0.2 40 50 KCI (M) 69 Figure 8. Cellulose phosphate elution profiles at pH 6.0 of microsomal, peroxisomal, and commercial carnitine acetyltransferases. A 0.6 x 12 cm column of Sigma cellulose phosphate was equilibrated in 0.015 M potassium phosphate buffer at pH 6.0. Microsomal and peroxisomal CAT activities were eluted with a 50 ml linear gradient of 0.2-0.6 M KCl. A.similar gradient of 0.3-0.7 M KCl was used to elute commercial CAT activity. One milliliter fractions were collected and CAT activities located by the DTNB assay (Methods). NMOLES x MIN'1 x ML" 70 MMICROSOMAL “o’ I- "”O‘ 4,. 0.42—5— a" . ,¢" ’v v” al- 8) PEROXISOMAL "¢’ I- """ ,0 0.43—5 ¢" 4”‘ 'v '4 IO ’ clconmencm. ,.r 75' o"’ 0058—:' o"‘ o"’ o 50' "v" a 25- 0 IO 20 so 2"- VOLUME (ml) Figure 8 5‘ 0.2 71 column was now used for the 2 partially purified activities. All 3 activities eluted at 0.20 i 0.01 M KCl (Figure 9, sections A,. B and C). The peroxisomal and microsomal samples lost more than 90% of their activity on this column and were not stabilized by the addition of 2 mg/ml BSA either to the applied samples or the eluted fractions. The commercial enzyme showed no such instability. A very crude estimate of the apparent molecular weight of peroxisomal CAT on P-100 had shown it to be in the range of the BSA monomer but lower. A more refined estimate was attempted on a G-lOO column (2.6 x 78 cm) using the proteins listed in Table 8 as molecular weight markers. Attempts to use the column at room temperature and/or at low ionic strength failed because of the instability of the 2 partially purified enzymes. No activity could be detected on these columns. If the column was run, however, at 4°C and in 0.4 M KCl at pH 7.5 sufficient activity (onv 0cm meaonm 000 no mafia: OHN on m I > GOHuomsm sflannam asumm oaHvawGMleo Armawmuomuorv mmamzouwc< ooo ¢s oNH mo soaumvfixo scuwownuuoz mommaxoumm mocmumwmm uzwfioa umanomHoE monuma >mmm< umfiaeanm cflmuoum mo mumafiumm new refionauomoa musmsflumexo coaumuuaamlamw ca 0mm: w mqm