V%.fi.? . Mm“? e- or.“ This is to certify that the dissertation entitled The Metaboiic Role of Carnitine in The Yeast, Toruiogsis bovina presented by Ronald K. Emaus has been accepted towards fulfillment of the requirements for Doctorate degfienlBiochemistrx 7/0/ng M ajor professor Dam June 30, 1982 MSUirnn 4n- .- A ~ I- lnrr ‘1 r r. .' 0-12771 MSU LIBRARIES \- RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. THE METABOLIC ROLE OF CARNITINE IN THE YEAST, TORULOPSIS BOVINA By Ronald K. Emaus A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1982 C‘ l \ ".\C_";.._ i ABSTRACT THE METABOLIC ROLE OF CARNITINE IN THE YEAST, TORULOPSIS BOVINA By Ronald K. Emaus This study shows that carnitine participates in the biosynthesis of amino acids in the carnitine-requiring yeast, Torulopsis bovina ATCC 26014, a finding which is both new and novel and provides a basis of the metabolic role of carnitine in this yeast. When 0.5-5 uM L-carnitine is added to the media, the growth rate of ‘1.‘bgvigg is doubled for both aerobic and anaerobic cultures. The stimulation occurs about 18 minutes after carnitine is added. High concentrations of glutamate stimulate growth without altering the carnitine content of the cells. Cells grown without added carnitine contain 0.4 nmol/g wet weight of carnitine while cells grown with 5 uM carnitine accumulate 1400 nmol/g wet weight by the end of exponential growth. Very high levels of carnitine acetyltransferase (CAT) activity are present in this yeast, the levels being unaffected by anaerobiosis or chloramphenicol but decreasing nearly 50% in yeast grown with carnitine. The substrate specificity and kinetic parameters of the enzyme in cell-free extracts were determined. The transferase is most active with acetyl-, propionyl-, and isobutyrleoA. When provided with carnitine, acetylcarnitine is the only acylcarnitine formed_ig vivo. Adding [1-14C]acetylcarnitine to cultures 0f.I°.QQ!iflé doubles the growth rate with much of the radioactivity becoming cell associated. The majority of the 14C is incorporated into cell protein although some 14C is recovered in the fatty acid fraction of saponified cells. Analysis of the amino acids derived from radiolabeled protein revealed that acetylcarnitine contributes carbons to the synthesis of glutamate, arginine, proline, leucine, and lysine. In contrast, [1-14C]acetate only labels leucine and lysine. The labeling pattern with [14C]acetate plus carnitine is the same as that with [14C]acetylcarnitine. Isopycnic density gradient analysis of lysed spheroplast preparations revealed that CAT is mostly associated with mitochondria while acetleoA synthetase is in the cytosol. Catalase was not detected indicating the absence of peroxisomes. Less than 15% of the CAT activity is soluble in yeast preparations in which the mitochondria are ruptured. Thus the subcellular localization of CAT is consistent with carnitine facilitating the transfer of acetyl groups from the cytosol into the mitochondria where 2-oxoglutarate is synthesized. TABLE OF List of Tables . . . . . . . . . . . List of Figures. . . . . . . . . . . List of Abbreviations. . . . . . . . I. Introduction . . . . . . . . . A. Thesis Statement . . . . . B. Literature Review. . . . . 1. Biochemistry of carnitine, CONTENTS a. Carnitine as a growth factor . . . . . (i) Tenebrio . . . . . . . . (ii) Torulopsis. . . . . . . (iii) Other organisms. . . . b. Discovery of CAT . . . . . . c. CAT involvement in fatty acid oxidation. 0 d. CAT purification and pr0perties. . . . . . e. Distribution of CAT. . . . . . . . . . . . f. Intracellular localization of CAT. 9. CAT isozymes . . . . . . . . . . h. Acetylcarnitine metabolism . . . . . i. Involvement of aceytlcarnitine in fatty acid synthesis and biological 2. Yeast metabolism . . . 3. Restatement of the problem . 11. Experimental Procedures and Results. A. Materials and Methods. . . 1. Materials. . . . . . . 2. Methods. . . . . . . . a. Organism . . . . . b. Growth media . . . c. Maintenance and growth 0 O O acetylations. . . . acetylcarnitine, and CAT. conditions. d. Uptake of radioactive carnitine. ...... e. Carnitine analysis . . . . . . . . . f. Ferment analysis . . . . . . . . . g. Radioactive tracer studies . . ii vi viii h. (i) Lipid extraction . . . ...... . (ii) Phenol extraction . . . . . . . (iii) Protein hydrolysis . . . . . . (iv) Amino acid analysis . . . . . . (v) Radioactivity measurements . . . CAT studies. . . . . . . . . . . . . . (i) Preparation of mechanically disrupted cell-free extracts . . . . . . . . . . . . . (ii) Preparation of spheroplasts and isolation of mitochondria . . . . . . . . . . . . . . (iii) Enzyme Assays. . . . . . . . . . . . . . . (iv) Protein determinations. . . . . . . . . . . B. RESUItSO O O O O O O O O O O O O O O O O O O O O O O O O 1. GrOWth StUd1es 0 O O O O O O O O O O O O O O O O O O a. b. C. d. e. f. 20 l. a. b. c. 3. Carnitine and acetylcarnitine metabolism . . . . . a. b. c. d. e. f. 4. Intracellular localization of CAT. . . . . . . . . a. b. c. d. Carnitine stimulation of growth. . . . . . . . Effect of air and anaerobiosis on carnitine stimulated growth. . . . . . . . . . . . . . . . Response time of cells to carnitine addition/ depletion. . . . . . . . . . . . . . . . . . . . Uptake of carnitine by growth arrested cells . . Carnitine levels in I. bovina. . . . . . . . . . Other growth promoters and potential carnitine precursors . . . . . . . . . . . . . . . . . . . bovina CAT. . . . . . . . . . . . . . . . . . . . Extraction, assay conditions and partial purifications. . . . . . . . . . . . . . . . . CAT production in I. bovina. . . . . . . . . . Substrate specificities and kinetic properties Identification of acetylcarnitine as the major acylcarnitine. . . . . . . . . . . . . . . . . . Acetylcarnitine and the synthesis of N-acetyl glutamate. . .4 . . . . . . . . . . . . . . . . . Addition of [14C]acetylcarnitine to growing yeast. . . . . . . . . . . . . . . . . . . Distribution of [14C] among cellular components. Acetylcarnitine and protein acetylation. . . . . Identification of radioactive labeled amino acids in protein hydrolysates. . . . . . . . . Evidence for membrane bound (particulate) CAT. Isopycnic density gradient analysis of CAT . . Association of CAT with mitochondria . . . . . Utilization of Acetylcarnitine by Isolated Mitochondria . . . . . . . . . . . . . . . . . . O O O O C III. DiSCUSSion O O O O O O O O O O 0 O O O O O 0 O O O O O O O 0 IV. References V. Appendix I 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 O O O O O O O O O O O O O O O O O O O O O O O O 0 111 116 118 128 128 132 135 143 147 162 174 Table LIST OF TABLES Composition of Stock Solutions for I. bovina Media Preparation 0 O O 0 O O O I O O O O O 0 O O O O 000000 Carnitine Recoveries in Boiled Media Solutions. . . . . . . The Effect of L-carnitine on Yeast Growth Rate ....... Effect of Carnitine on Cell Yield . . . . . . . . . . . . . Effect of Different Carbon Sources on Growth. . . . . . . . Growth Rates with Carnitine Added at Various Times after InCUbation. O O O O O O O O O O O O O O O O O O O 00000 Carnitine Content of I. bovina. . . . . . . . . . . . . . . Effect of D-carnitine and Possible L-carnitine Precursors O on I. bovina Growth . . . . . . . . . . . . . . . . . . . . . Assay Conditions for I. bovina CAT. . . . . . . . . . . . . Factors Affecting CAT Production in I. bovina . . . . . . . Substrate Specificity of I. bovina CAT. . ........ . . Percent of Acylcarnitine as Acetylcarnitine in 1. bovina. . Distribution of 14C in Cells Grown with [14C]Acetylcarnitine. Solubilization of 14C from [14C]Acetate-labeled Cell Walls. . Distribution of 14c in Saponified Cells ..... . . . . . Distribution of 14C in Fractions from Homogenized Cells . . Protein Acetylation in Crude Cell Extracts Incubated with [14CJAcetylcarnitine. . . . . . . . . . . . . ....... Isolation of 14C Labeled Protein. . . . . . . . . . . . . . iv 66 77 77 81 86 88 98 104 104 108 . 113 117 119 Table Page 19. Distribution of [14C]Acetylcarnitine Derived Radioactivity in Amino Acid Fractions Separated by TLC. . . . . . . . . . . 126 20. CAT Distribution in Crude Extracts. . . . . ..... . . . . 131 21. Subcellular Distribution of CAT in I. bovina. . . . . . . . . 140 LIST OF FIGURES £19m £3.92 1. Effect of Carnitine on the Growth Rate of I. bovina . . . . . 52 2. Covariance of Cell Number and Culture Absorbance. . . . . . . 54 3. The Amount of Glucose Fermented to Ethanol During Exponential Growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4. Effect of Aerobic and Anaerobic Culture Conditions on Growth. 61 5. Difference Spectrum of I. boxing Mitochondrial Cytochromes. . 65 6. Effect of Dilution of Cell—Associated Carnitine on Growth Rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 7. Declining Growth Rate in Carnitine Starved Cells. . . . . . . 71 8. Dependence of Growth Rate on Media Carnitine Concentration. . 73 9. Uptake of DL-[methyl-3Hjcarnitine by Growth Arrested Cells of I. bovina. . . . . . . . . . . . . . . . . . . . . . . . . 76 10. Stimulation of CAT by Increasing Ionic Strength . . . . . . . 83 11. Linear Dependence of CAT on Protein Concentration . . . . . . 85 12. Formation of Radiolabeled Acylcarnitines by Cell-free Extracts of I. bovina . . . . . . . . . . . . . . . . . . . 90 13. Determination of the Km for AcetleoA of I. boxing CAT. . . . 93 14. Determination of the Km for L-carnitine . . . . . . . . . . . 95 15. Uptake and Metabolism of Carnitine by I. bovina . . . . . . . 97 16. Incorporation of [14C]Acetylcarnitine in Growing Yeast Cells. 101 17. Distribution of [14C] in Cell Lipids. . . . . . . . . . . . . 106 18. Effect of Carnitine on [14C]Acetate Incorporation into Neutral Lipids. O O O O O O O O O O O O O O O O O O O O O O O O 0 vi . 111 Figure 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Chromatography of RNA Nucleotides . . . . . . . . . . . . . Identification of 14c Labeled Amino Acids by HPLC . . . . . . TLC Analysis of Radioactive Labeled Amino Acids Derived from I. bOVina Ce1‘l PrOteTnS O O O O O O O O O O O O O O O O O 0 Effect of Phosphate Concentration on Sedimentation Behavior Of CAT. 0 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 Isopycnic Sorbitol Density Gradient Analysis of CAT in Extracts from Mechanically Disrupted I. bovina Cells. . . . Effect of High Phosphate Concentration on the Isopycnic Dens‘ity 0f CAT. 0 0 O O O O 0 O O O O O O O O O O O O O O 0 Electron Micrographs of Peak CAT Fraction (D420=1.138). . . Subcellular Localization of I. bovina CAT in Mitochondria Isolated by Isopycnic Sorbitol Gradient Centrifugation of a Spheroplast Lysate. . . . . . . . . . . . . . . . . . . . . Chromatography of Mitochondrial Preparations Incubated with Radioactive Acetate or Acetylcarnitine. . . . . . . . . . . Schematic Representation of the Metabolic Role of Carnitine in I. bOVina. O O O O O O O O O O O O O O O O O O O O O O O Pathway of Biosynthesis of Leucine and Valine . . . . . . . vii 125 130 134 137 139 143 144 155 159 ATCC ATP CAT CPM acleoA Ci CoASH DNA DPM DTN D42 EDTA G6PDH HPLC Ki Km NAD NADH Nz/COZ pI RNA TCA TLC Tris Emii LIST OF ABBREVIATIONS American Type Culture Collection Adenosine triphosphate Carnitine acetyltransferase (EC 2.3.1.7) Counts per minute Acylcoenzyme A Curies Coenzyme A Deoxyribonucleic acid Disintegrations per minute 5,5-'dithiobis-(2-nitrobenzoic acid) Density measured at 20°C and corrected to 4°C, in g/cm (Ethylenedinitrolo)-tetraacetic acid Glucose-6-phosphate dehydrogenase High pressure liquid chromatography Inhibition constant Michaelis constant Nicotinamide adenine dinucleotide, oxidized Nicotinamide adenine dinucleotide, reduced 5% C02 in nitrogen isoelectric pH Ribonucleic acid Tricarboxylic acid (citric acid) cycle Thin layer chromatography Tris (hydroxymethyl) aminomethane Maximum velocity Concentration of viii INTRODUCTION THESIS STATEMENT The growth of the yeast, Torul0psis bovina ATCC 26014, is stimulated by carnitine (1-4). One knownfunction of carnitine is to shuttle long-chain fatty acids into mitochondria for 8 oxidation (5-8). However, a preliminary study showed that I. boxing lacked fatty acid oxidase (9). Thus carnitine had an unknown function in this yeast. The purpose of this thesis was to determine the metabolic role of carnitine in I. bovina. LITERATURE REVIEW BIOCHEMISTRY 0F CARNITINE, ACETYLCARNITINE, AND CARNITINE ACETYLTRANSFERASE (CAT) Carnitine participates in the transport of long-chain acleoA derivatives across the acleoA impermeable inner membrane of mitochondria and thereby facilitates oxidation of fatty acids (6-8,15,16). However, other roles for carnitine must exist since carnitine acyltransferase is associated with peroxisomes and microsomes as well as mitochondria (70), and since all mammalian tissues contain significant quantities of short-chain acylcarnitines (28,130,224). Furthermore, carnitine affects branched-chain amino acid metabolism in mammals (44,45), and pyruvate oxidation in the fatty acid oxidase-deficient flight muscle of the blowfly, Phormia regina (79). Some possible roles for carnitine in intermediary metabolism have recently been summarized (225). For an understanding of carnitine metabolism in I, boxing, the possible functions of acetylcarnitine and CAT are most relevant. Carnitine, acetylcarnitine and CAT are present in numerous animal tissues, some plant tissues and some microorganisms although few analyses have been made in the latter two categories (10—14). Fraenkel observed that since carnitine was not present to the same extent in all tissues and species, it may be involved "in two different processes, one of which requires it in minute quantities and the other in large quantities" (17). I, bovina and Tenebrio molitor are two organisms that require small quantities of carnitine. CARNITINE AS A GROWTH FACTOR Tenebrio Carnitine, Vitamin BT, was first recognized as a growth factor for larvae of the mealworm, Tenebrio molitor (18), although the BT factor was not identified as carnitine until 1951 (19). Other beetle larvae belonging to the family Tenebrionidae require carnitine (20-22). Optimal survival of I. molitor was obtained with L-carnitine concentrations of 0.35 “9/9 dry weight of diet (inner salt, 19) while 1.5 pg L-carnitine/g dry weight of diet was required for fully successful growth (23). Under certain culture conditions the beetles ceased to show a carnitine requirement but the deficient state could be induced by feeding the larvae 4-N-trimethylaminobutyrate. This provided the first clue that the B-hydroxyl group was important to carnitine metabolism. The 4-N-trimethylaminobutyrate induced deficient state showed symptoms similar to carnitine deficiency with disturbances in the casting of the skin during molting and also bulges in the midgut (22). The function of carnitine in Tenebrio is unknown. Fraenkel and Chang found that in starved larvae the fat content decreased from 43% to 12% while the water content increased from 58.5% to 71%. In contrast, in carnitine deficient larvae the fat content only decreased to 24% and the water content fell to 42% (24). McFarlane found no decrease in fat content in carnitine deficient larvae (25). These results suggest that carnitine deficiency blocks lipid catabolism. No histological deficiency symptoms were observed in muscle or nervous tissue (26) although it is uncertain that lipid droplets would have been observed around the muscle mitochondria as has been described in recent reports of muscle carnitine deficiency in humans (27). Carnitine concentration must be greater than 10 uM in order to stimulate fatty acid or fatty acleoA oxidation by mitochondria (26). This represents a tissue concentration of at least 10 nmol/g wet weight or 50 nmol/g dry weight assuming 80% water content. When fed a diet containing carnitine, Tenebrio contained about 142 nmol L-carnitine/g dry bodies (23) or nearly three times the minimum amount necessary to stimulate fatty acid oxidation. However, McFarlane found no difference in the rate of oxygen consumption or in the respiratory quotient of carnitine deficient larvae compared to either normal young larvae or normal older larvae (25). It seems doubtful, therefore, that carnitine-stimulated fatty acid oxidation is the immediate cause of death. The carnitine deficient larvae appear to die because of water loss due to improper hardening of the cuticle following molting (24). It would be interesting to determine if the insect contains CAT or carnitine palmityltransferase and what carnitine derivatives form 1_ vivo. An intriguing possibility is that acetylcarnitine participates in acetylation reactions necessary for proper cuticle hardening. Support for the existence of CAT in Tenebrio is provided by the data of Bhattacharyya gt a1. (29) indicating that acetylcarnitine substitutes for carnitine in the nutrition of this insect. Torulopsis The structural similarity of carnitine and choline prompted some investigators to search for lipid bound carnitine but instead of finding phosphatidylcarnitine, long-chain acylcarnitines were isolated and identified (16,30,31). Phosphatidyl-B-methylcholine was found when carnitine replaced choline in the diets of some insects (32) although carnitine does not generally have a choline-sparing function (16). For example, carnitine did not support the growth of choline-less mutants of Neurospora crassa (33). However, Travassos gt a1. (1) were able to isolate from a choline-requiring yeast a carnitine-requiring strain of Torulopsis bovina. Their isolate, I; 221122 ATCC 26014, was sensitive to 0.1 ug% DL-carnitine or 2.5 pmol/ml L-carnitine with optimum growth requiring >1.5 pg% DL-carnitine (37.5 pmol/ml in L-carnitine). Travassos and his colleagues studied the growth requirements of 1. ‘leiga ATCC 26014 in detail paying particular attention to those compounds that might serve as precursors of carnitine, as carnitine was thought to be synthesized in 1961, and to the possible methyl denoting capacity of carnitine's quaternary ammonium group. The yeast's growth was stimulated by 5 mg% L-glutamic acid but none of the other intermediates in the biosynthesis of carnitine proposed by Guggenheim in 1951 (gl utami c acid—> 4-ami nobutyrate—D4-N-trimethyl ami nobutyrate—> crotonobetaine—§carnitine) stimulated growth (42). Maximum growth stimulation required both carnitine and choline although methionine substituted for choline. The combination of leucineplus methionine stimulated growth in the absence of choline and carnitine. Somewhat later, Miranda gt al. (2) discovered that carnitine accumu- lated in the growth media and concluded that carnitine biosynthesis was not impaired in this yeast but that it must "leak" carnitine and thus grow more poorly than the parent strain where a "retention mechanism" operates. However, their study did not actually measure the carnitine content of the yeast or the media. Nor did their study find any lipid bound carnitine in the lipid extract of cells grown 48 h with DL-[methyl-14C]carnitine. Except for a preliminary study by Bieber gt a1. (9), the reports on I. boyiga_ATCC 26014 have all been by members of the laboratory from which the original report issued. They did not perform any biochemical analyses of the yeast although they observed that acetylcarnitine substituted for carnitine suggesting, as demonstrated in the preliminary report from this laboratory, the existence of CAT l".I-.22liflé ATCC 26014. Phosphorylcarnitine also stimulated yeast growth (3) leading one to imagine a high energy storage function for carnitine although the intervention of a non-specific phosphatase might be explanation enough. Phosphorylcarnitine is hydrolyzed by crude phosphatase preparations from sperm (3). Other Organisms The initial description of carnitine as a "growth factor" for Tenebrio caused many investigators to test the growth promoting characteristics of carnitine on a variety of systems. For example, Liebecq-Hutter, who worked with Tenebrio also claimed an effect of carnitine on the development of embryonic chick bone but the effect was later attributed to crotonobetaine (149). Sakuguchi(150) reported that 10 ug/ml DL-carnitine permitted growth of the soy sauce lactic acid bacterium, Pediococcus soyae, but no additional reports followed. Strack and Rotzsch tested carnitine on a variety of animals (151,152): tadpoles grew faster in water containing carnitine; carnitine induced weight gain in young rats presumably by stimulating appetite; and carnitine antagonized the effects of thyroxine on tadpoles and rats. Reynier (135) found that carnitine lowered the ratio of nitrogen catabolism and increased survival time in starving rabbits. Many other examples could be cited but too little is known about the biochemistry of these effects to make a listing worthwhile. Discovery of CAT During their work with Tenebrio, Friedman and Fraenkel (34) discovered that OL-carnitine inhibited the acetylation by acetleoA of p-aminobenzoate catalyzed by preparations from pigeon liver acetone powders. DL-carnitine was inhibitory at a concentration of 10 uM and appeared to be specific for carnitine with acetylcarnitine formed in the absence of p-aminobenzoate. Furthermore, acetylcarnitine itself acetylated p-aminobenzoate thereby demonstrating the equivalent bond energy of acetleoA and acetylcarnitine which was not expected of a secondary o-acetyl ester group (35). In fact, acetylcarnitine formed acetleoA when the liver extracts were incubated with CoASH and the following reversible enzymatic reaction was proposed: Forward O-acetyl-L-carnitine + CoASH;=->_ acetleoA + L-carnitine (eqn. 1) Reverse At the time (1955) acetylcarnitine had not been found in animals although it had not really been looked for. Acetylcarnitine was known to replace carnitine in the diet of Tenebrio (29) and it seems peculiar that Friedman and Fraenkel did not look for either CAT or acetylcarnitine in this insect. CAT Involvement in Fatty Acid Oxidation The purification of CAT was not reported until almost a decade after the above report. In the meantime, a number of studies implicated carnitine having a role in lipid metabolism. Fritz (52,53,36) found that DL-carnitine stimulated rat liver slices and homogenates to oxidize palmitate to C02 and ketone bodies. Carnitine augmented fatty acid oxidation in liver particulate fractions but not in soluble systems, a finding which first suggested that carnitine had some transport function. Similar results were obtained using skeletal and cardiac muscle with carnitine stimulating fatty acid oxidation of isolated mitochondria (55,56). Indeed, mitochondria very actively oxidized long-chain acylcarnitines (15,16) which were formed by mitochondrial preparations (40,16) and also identified in the lipid extracts of various tissues (30,31). Carnitine did not act by stimulating the long-chain acleoA synthetase reaction (36). In these early preparations, carnitine did not stimulate palmitleoA oxidation but further research showed that carnitine did in fact stimulate mitochondrial long-chain acleoA oxidation (16). From these data came the proposal that fatty acid oxidation was compartmentalized inside the mitochondria behind a CoASH impermeable barrier that was permeable to acylcarnitines (6). The inner mitochondrial membrane is impermeable to CoASH (57) although carnitine is restricted to the same mitochondrial space as CoASH (57-60). Thus, there was a physiological need for a reversible long-chain carnitine acyltransferase catalyzed reaction analagous to CAT. Some evidence for a long-chain carnitine acyltransferase had been obtained that suggested the enzyme was different from CAT (61). Shortly thereafter, carnitine palmityltransferase was purified from calf liver (62). Thus it was established that carnitine stimulated long-chain acleoA oxidation via acylcarnitine formation and transport into the mitochondrial matrix with subsequent transfer of the acyl group back to CoASH at the site of the fatty acid oxidase system. The physiological need for a short-chain carnitine acyltransferase such as CAT was not apparent from the discovery of the role of carnitine in fatty acid oxidation. Carnitine did not stimulate octanoate or butyrate oxidation in rat liver slices or particulate fractions (36) and has not been found to be obligatory for the oxidation of medium- or short-chain fatty acids. However, the oxygen consumption of rat kidney, heart, brain, and testis mitochondria was strongly stimulated by acetyl-DL-carnitine and in particular by acetyl-L-carnitine (37).. Other studies also showed that short- and medium-chain acylcarnitines are oxidized by mitochondria (15,16,38). Bremer observed that when presented with an excess of pyruvate, isolated mitochondria convert carnitine to acetylcarnitine but acetylcarnitine oxidation is inhibited when pyruvate is added (37). These results suggested that the reversible enzymatic acetylation of carnitine was responsible for the stimulation by carnitine of fatty acid oxidation. More importantly, however, Bremer pointed out that acetylcarnitine formed during pyruvate oxidation might be a "loophole" in the compartmentation of the cell allowing "active acetate" derived from carbohydrate breakdown to leave the mitochondria and thus be available for extramitochondrial biosyntheses, e.g., fatty acids and cholesterol. Citrate was not recognized as an acetyl carrier for fatty acid synthesis at the time and Bremer's proposal sparked a flurry of research (see below). After Fritz and Yue discovered that carnitine stimulated palmitleoA oxidation (16), they again investigated the oxidation of acetate by heart muscle mitochondria and found that indeed unlike acetate, acetleoA oxidation was carnitine dependent (63). Similar results were obtained with butyrylcoA and hexanoleoA and the lack of stimulation by carnitine of short-chain fatty acid oxidation was attributed to the intracellular localization of the different chain-length acyl thiokinases. Carnitine presumably stimulated acetleoA oxidation by facilitating its transfer into mitochondria. However, acetleoA is ordinarily generated inside the mitochondria by the operation of the fatty acid and pyruvate oxidase systems. Thus it made sense to reason that under physiological conditions, carnitine facilitated the transfer out of the mitochondria of the acetyl groups in the intramitochondrial acetleoA pool. This view is indirectly supported by the fact that (+)-acetyl-0-carnitine, a potent inhibitor of CAT (see below) has no effect on carnitine stimulated palmitate oxidation by rat heart mitochondria (64) but this result should be interpreted with caution because (+)-acetyl-0-carnitine exchanges very poorly with intramitochondrial L-carnitine (41). Strictly speaking, therefore, carnitine-stimulated palmitate oxidation does not require any CAT activity external to the inner mitochondrial membrane. 10 CAT Purification and Properties CAT was partially purified from pig heart by Fritz gt 31. (35) developing four different assay procedures in the process for both the forward and reverse direction (see eqn. 1). The enzyme remained particulate in homogenate prepared in 0.25 M sucrose but was easily solubilized using 0.1 M K2HP04. The enzyme preparation had slight or no fatty acleoA hydrolase or acetylcarnitine esterase activity and did not react with choline or its derivatives. The apparent equilibrium constant at pH 7 was 0.6 in the direction of acetleoA formation and the apparent Km value for acetyl-DL-carnitine was 6.2 x 10'4M. The high group potential of the O-acetylester bound (AF°' = -7.9 kcal) was unexpected but emphasized the "active" acetate nature of acetylcarnitine. High specific activities of CAT were found associated with the isolated mitochondria from rat, pigeon, and locust muscle with much lower activities in rat liver and kidney mitochondria and no detectable activity in bee flight muscle (46). As in pig heart, the CAT activity in the other tissues remained particulate when the mitochondria were prepared in isotonic sucrose but was easily solubilized by homogenization in 0.1 M phosphate buffer, pH 7.2. Under the conditions of the assay, butyryl-, decanoyl-, and palmitylcarnitine inhibited the CAT reaction from which it was erroneously concluded that the same enzyme transferred acyl groups from short-, medium-, and long-chain acylcarnitines to CoASH. The presence of CAT in locust flight muscle which utilizes fatty acids for energy and its absence from a carbohydrate utilizing tissue, bee flight muscle, caused the authors to stress that CAT was only important in fatty acid oxidation. 11 The above study showed that pigeon breast muscle was a much better source of CAT than pig hearts and Chase gt 31. (47) quickly reported the preparation of crystalline CAT from pigeon breast muscle. The molecular weight of the enzyme determined by Sephadex chromatography was 55,000. Further characterization of the enzyme's kinetic behavior (48), its dependence on pH (49) and its substrate specificity (50) were quickly reported. The enzyme is strictly a short-chain carnitine acyltransferase with the interesting property that the Michaelis constants for acleoA substrates from acetyl- to octanleoA are experimentally identical (38 i 6 uM), whereas Vmax for the catalyzed reaction decreases about 10-fold over this range of substrates. The equilibrium constant for the reaction measured in the direction of acetleoA formation is 0.6 (65), identical to the value obtained for pig heart CAT (35). Long-chain acleoA's reversibly inhibit the pigeon breast enzyme, mostly by decreasing CAT's affinity for L-carnitine (50). The Ki for palmitleoA is 0.43 uM, a value which is probably physiologically significant. Palmitylcarnitine (100 uM) did not inhibit the enzyme but bromo-acetleoA was a potent irreversible inhibitor at nearly a 1:1 ratio of enzyme to inhibitor (66). Fritz and Schultz (51) found that acetyl-D-carnitine is a potent inhibitor of pig heart CAT with a Ki of 2.5 x 10'4M when assayed in the direction of acetylcarnitine formation. The enzyme is inhibited competitively by D-carnitine with an apparent K1 of 2.1 x 10‘3. The Km for DL-carnitine is 6.2 x 10'4M. They reported that comparison of reciprocal plots for DL-carnitine with those for L-carnitine showed an increased Km for L-carnitine and an unaltered Vmax for the racemic mixture. 0n the other hand, Chase and Tubbs 12 (48) found that a fixed ratio of D- to L-carnitine produces a parellel decrease in Vmax and Km in the pigeon breast muscle enzyme and that the Km's for the L-forms of carnitine and acetylcarnitine are approximately equal to the Ki's for the D-forms. In addition acetyl-L-carnitine was a competitive inhibitor of L-carnitine in pigeon breast CAT whereas it was a non-competitive inhibitor for pig heart CAT. These differences might have been due to Fritz and Schultz measuring the rate of thiol release from acetleoA while Chase and Tubbs measured the reverse reaction by coupling acetleoA formation to citrate formation. CAT was recently purified from beef heart mitochondria as part of a larger study to isolate carnitine octanyltransferase activity as a separate enzyme (67). The CAT activity was purified over 400-fold and was greater than 95% pure. The enzyme had similar molecular weights determined by Sephadex G-200 chromatography (60,500) and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (62,600). Isoelectric focusing produced a single peak at pH 8.2. Studies of the forward and reverse reaction kinetics revealed a very significant difference in the relative activities measured with increasing acyl chain lengths. In the direction of acylcarnitine formation, the activity with octanyl- and decanoleoA is 27 and 5% respectively of the rate with acetleoA whereas in the direction of acleoA formation the corresponding relative activities are 54 and 26%, respectively. Mittal and Kurup noted that treating animals with hypolipidemic drugs causes major increases in CAT and proceeded to purify CAT from hepatic mitochondria of rats fed clofibrate (68). Although gel filtration indicated the molecular weight of the enzymes was 56,000, two non-identical subunits were obtained using 505 gel electrophoresis. 13 Solberg was unable to purify calf liver CAT because the carnitine acetyl-, octanyl- and palmitoyl-transferases all eluted as a single peak with molecular weight about 68,000 (69). Thus, it is not known if the two subunit native of the clofibrate induced hepatic CAT is the result of proteolysis, a reflection of a difference between muscle CAT and hepatic CAT, or the result of clofibrate treatment. In all other respects, the clofibrate induced hepatic CAT was very similar to the partially purified CAT from pig heart (35,51). Mitochondria contain 60-70% of the cellular CAT (see below) but significant levels of extramitochondrial CAT exist (70-76) and the microsomal and peroxisomal activities have been partially purified by Markwell and Bieber (74). The microsomal enzyme activity was very labile in sucrose or dilute phosphate buffer solutions but stable in the presence of 0.4 M KCl. Gel filtration of the enzymes on Sephadex G-100 resulted in the recovery of only 10% of the applied CAT activity which eluted at an apparent molecular weight of 59,000. Both the peroxisomal and microsomal enzymes had similar pI values of 8.3 with the microsomal preparation exhibiting a minor peak at pI 5.3 under some conditions. Both enzyme preparations had the same apparent Km for acetleoA (69 uM) and L-carnitine (146 pH) with nearly identical substrate specificities. However, the reported relative activities with hexanoleoA or octanoleoA are much lower than the corresponding activities of beef heart and pigeon breast muscle CAT preparations (67). The peroxisomal and microsomal enzymes were also reported by Markwell and Bieber (74) to function with malonleoA and acetoacetleoA at 50 and 75% respectively of the rate with acetleoA. Scholte's study of liver malonleoA decarboxylase concluded that malonleoA is not a substrate for CAT but, although not specified, 14 this result was probably obtained using the commerical preparation of CAT from pigeon breast muscle (77). Bressler and Katz (78), in their study of fatty acid synthesis in guinea pig liver, reported that CAT is 23% as active with acetoacetleoA as it is with acetleoA but they too used a CAT enzyme derived from muscle tissue. Distribution of CAT The preceeding sections demonstrate the presence of CAT in pig heart (35), pigeon breast muscle (47), rat liver (74), and liver of clofibrate-treated rats (68), pigeon and sheep liver (34), calf liver (69) and beef heart (67). CAT was present in locust flight muscle but absent from bee flight muscle (46), a result confirmed by Childress gt .al. (79). The tissue distribution of CAT was indirectly determined by Beenakkers and Klingenberg (46) in their survey of mitochondrial preparations. Actual study of the CAT distribution in various tissues of rat was done by Marquis and Fritz (80). Highest activity was present in heart and testis, somewhat lower activity in brown adipose tissue and relatively low levels in skeletal muscle, kidney and brain. CAT was extracted most successfully from the tissues using a buffer (pH 8.0) containing 0.1 M KZHP04, which seems to be a universal finding. Skeletal muscle CAT activity was lower but the specific activity in mitochondria isolated from skeletal muscle (410 nmol/min/mg protein) was essentially equal to the specific activity in heart muscle mitochondria (440 nmol/min/mg protein). These values are higher but qualitatively similar to those of Beenakkers and Klingenberg. 15 It is noteworthy in the data of Marquis and Fritz that of all the tissues only testis contains a much higher ratio of CAT to carnitine in an equivalent weight of tissue. A closer study of male rat reproductive tissues revealed that epididymal sperm contain the highest CAT activity of any tissue so far examined (276 umol/min/g dry weight). Ram and human sperm (88) and presumably bovine sperm (100) are similarly rich in CAT. In rat relatively high levels of CAT were also associated with testis, caput epididymis, caudal epididymis, and vas deferens with no activity in cell-free epididymal fluid (81). A more extensive tissue distribution study was performed by McCaman .2E.§l- (82) using a unique assay system yielding results in good agreement with Marquis and Fritz even though the two groups assayed the reaction in reverse directions. Sciatic nerve, lung, spleen, small intestine, thymus, and liver were the least active tissues while heart and testis were the most active. Kidney, skeletal muscle, brain, and adrenal gave intermediate values. In contrast, choline acetyltransferase was absent from kidney, liver, thymus, intestine, and lung with activities in heart, testis, brain, and skeletal muscle 1 x 10'2 to 5 x 10"5 times the CAT activities in these tissues. There was little variation in the CAT levels present in 9 different regions of rabbit brain with no difference between white and gray matter. Four day old rabbit brain cortex only contained 10% of the adult level of CAT. As part of a study to determine if mitochondrial CAT was accessible to externally added substrates, Barker gt a1. (83) compared the relative rates of the CAT catalyzed reaction in both the forward (acetylcarnitine formation) and reverse direction using three different assays and thereby demonstrated the variability of the results obtained by the different 16 methods. CAT was present in the mitochondria of sheep liver and the mitochondria of liver and mammary gland of guinea pig, goat, and rat. CAT values in rat liver mitochondria were 20- to 40-fold lower than in the other animals' livers. Barker gt 31. claim their rat liver value is much lower than that found by Marquis and Fritz (80, see above) but they must have confused the units because the measured values were 5.1 and 4.2 nmol/min/mg protein respectively in the two reports. The levels of CAT as well as the isobutyryl-, isovaleryl- and octanylcarnitine acyltransferase activities in tissues of fed and fasted rats were also recently examined by Ch°l.§£.£l° (84). The carnitine acyltransferases in liver increased after 8 days of fasting while the levels in heart, skeletal muscle, kidney, and testes remained unchanged. Variations in CAT activity in tissues of rat exposed to fasting and cold were studied by Kerner gt al. (85). Significant increases of CAT activity were observed in brown fat and liver of cold adapted rats compared to controls. In another study by the Pecs group, CAT was found in gastrocnemius muscle of the frog (86). CAT is present in both normal and dystrophic murine skeletal muscle (94) and in mouse liver and cardiac tissue (95). A good correlation exists between CAT activity and acetylcarnitine levels in developing embryonic tissues as first demonstrated by Casillas and Newburgh (96) using embryonic chick brain, heart, and liver. CAT was also present in the yolk sac. CAT is absent in immature rat ovary but the enzyme appears during development induced by pregnant mare gonadotropin and remains active during steroidogenesis (97). Fetal and adult monkey skeletal muscle, heart, liver, and brain fat all contain CAT (98). CAT activity is relatively high in liver, soleus, tibialis, fetal 17 liver, and fetal heart of female guinea pigs and rabbits (99) and, so too in brown adipose tissue (89). Human arterial and venous tissue from various locations all contain low levels of CAT (101) as do human platelets (87). ‘Relative to adults, lower activities of CAT are present in human fetal heart, liver, brain, and skeletal muscle (102). CAT is present in human placenta (102) and also in the placenta of mouse, marmoset and tamarin (103). Human kidney and fibroblasts also contain CAT and in one case a general tissue deficiency of this enzyme was implicated as the cause of fetal ataxic encephalopathy (104). There is an overwhelming interest in the CAT levels of mammalian tissues. CAT has been reported in yeast (9,90,91) and, with the recent interest in peroxisomal metabolism, CAT activity has been reported in goldfish intestine (92) and carp liver (93). However, CAT is reported absent from a number of plant tissues (70) including spinach leaf peroxisomes (76). African trypanosomes do not oxidize fatty acids. Yet the blood stream form of Trypanosoma lewisi has an extremely high level of CAT activity (105) and other trypanosomes contain CAT (106). Intracellular localization of CAT Differential centrifugation of a large number of different tissues has repeatedly confirmed that the majority of cellular CAT activity sediments with the mitochondrial fractions (37,80,82,83,107). Given the prevailing view that CAT facilitates mitochondrial export and import of acetleoA, it is not surprising to find CAT localized predominantly in mitochondria. However, Markwell and coworkers noted that CAT was significantly skewed to higher densities than was the mitochondrial 18 marker enzyme in a rat liver homogenate fractionated by isopycnic centrifugation in a very steep sucrose gradient (109). Careful investigation of similarly fractionated homogenates of rat and guinea pig kidney and liver revealed that CAT was indeed a mitoChondrial enzyme in kidney but in liver substantial (nearly 50%) activity was also present in the peroxisomes and microsomes (70). Since then other investigators have looked for extramitochondrial CAT. The enzyme is only present in the mitochondria of guinea pig intestine (108) while in rat heart a Small but definite percentage of the total CAT resides in the microsomes (75). Peroxisomal CAT has been demonstrated in mouse liver (110), goldfish intestinal mucosa (92), carp liver (93), brown adipose tissue (111), and alkane-grown yeast (91). Rat liver peroxisomal CAT is a soluble matrix enzyme (74) while the microsomal activity is membrane associated. Part of the microsomal activity faces the cytosol and part is exposed on the lumenal surface (112). The picture concerning the intramitochondrial localization of CAT is confusing. Pearson and Tubbs argue, and many agree, that the role of acetylcarnitine is to provide an "acetyl sink" for mitochondrial matrix acetleoA (10), a role that requires CAT only on the matrix side of the mitochondrial inner membrane. Although histochemical data obtained from rat heart (113) and skeletal muscle (114) indicate CAT occupies the space between the outer and inner mitochondrial membrane, most other reports conclude there is very little CAT in the outer mitochondrial compartment, most of the activity being present on the matrix side of the inner mitochondrial membrane. 19 In one study, Tubbs and Chase (115) obtained preparations of rat heart mitochondria that oxidized acetylcarnitine but would not oxidize acetleoA without the addition of carnitine. Based on the pattern of inhibition of oxidation of these substrates by bromoacetleoA and bromoacetylcarnitine, suicide inhibitors of CAT (116), these workers concluded that there were two pools of CAT in mitochondria, one accessible to acetleoA (outer compartment) and one accessible to acetylcarnitine (matrix compartment).. However, in a later study (117) liver mitochondria oxidized acetleoA + carnitine at an insignificant rate and they concluded the carnitine dependent oxidation observed for rat heart was probably due to stimulation of endogenous fatty acid oxidation. Two separate studies indicate the rate of oxygen consumption does not adequately reflect the activity of external CAT. In the first study, Fritz and Yue (63) showed that even though the rate of oxygen consumption increased only 1.6 times, carnitine stimulated the degradation of [1-14CJacetleoA by rat heart mitochondria 50-fold. The second study deals with the oxidation of isobutyrleoA which is also a substrate for CAT. In their study, Choi gt al. (39) report that beef and rat liver mitochondria oxidize isobutyryl CoA + carnitine at a negligible rate due to inhibition of isobutyrylcarnitine translocation rather than lack of formation of isobutyrylcarnitine. By one method these workers showed that nearly 50% of mitochondrial CAT was external to the inner membrane while a second method indicated 10-20% was external. The formation of acetylcarnitine from pyruvate by mitochondria of rat heart (41); rat heart, kidney, and liver (37); and bovine sperm (100) demonstrates that CAT is accessible to acetleoA generated in the matrix 20 compartment. At least 90% of mitochondrial CAT is reported to be latent and therefore presumably in the matrix compartment in sheep liver, kidney cortex, heart, and skeletal muscle (107). Similarly, 10% of the CAT activity of disrupted mitochondria was measurable in intact mitochondria of goat and guinea pig liver and mammary glands, sheep liver, and rat mammary gland (83). A somewhat higher percentage (25%) of mitochondrial CAT was assigned to the outer compartment based on subfractionation studies of rat liver and pig kidney mitochondria with digitonin (118). These workers reported that the rate of oxidation of acetleoA + carnitine by pig kidney mitochondria decreased 3-fold when the outer compartment CAT was removed by digitonin treatment but that oxidation capacity could be restored if purified CAT was added back to the depleted mitochondria. Solberg also used digitonin fractionation and found little or no outer compartment CAT in rat and mouse liver mitochondria (119). Likewise in blowfly flight muscle mitochondria, separate investigators (120,79) reported the total absence of outer CAT because, whereas acetyl- carnitine was rapidly oxidized, acetleoA + carnitine was not. External CAT may depend upon developmental age. Mitochondria from fetal bovine heart do not oxidize acetleoA + carnitine whereas the mixture is oxi- dized by calf heart mitochondria. Both tissues oxidized acetylcarnitine (121). CAT isozymes In general, the above studies suggest two non-interchangeable pools of CAT in mitochondria. Also, the peroxisomal CAT activities might represent different enzymes and tissue specific isozymes of CAT might explain the proposed differences in the function of CAT in different 21 tissues. However, all the evidence obtained to date indicate a single type of CAT under all these conditions. Rat liver peroxisomal CAT is a soluble matrix enzyme and microsomal CAT is a membrane bound enzyme, but both enzymes have similar molecular weights, similar isoelectric points, similar chromatographic properties, and similar kinetic constants (74). The similarities between pigeon breast muscle CAT (47,48,50) and pig heart (35) CAT have already been discussed and the somewhat different molecular weight and subunit composition of clofibrate induced rat liver CAT (68) has been noted. Although species differences in the isoelectric points of partially purified CAT from rabbit, human, and pigeon tissues were reported by White and Wu (122), essentially identical isoelectric focusing patterns were observed for CAT from different tissues of the same species. Edwards gt l. (117) found that CAT from ox heart, ox liver, sheep liver, and pigeon breast muscle were separarted similarly by isoelectric focusing, ion-exchange chromatography and centrifugation. They observed two interconvertible forms of the enzyme, one attributed to its being membrane associated. Clarke and Bieber (67) and Markwell gt a1. (74) have confirmed these findings in beef heart and rat liver respectively. Thus, only a single type of CAT appears to exist within a given tissue. It is noteworthy that, whereas White and Wu (122) found commercially prepared pigeon breast muscle CAT to contain three different enzyme peaks after electrofusing, several other investigators report only a single peak near pH 8 for this same enzyme preparation (67,74,117). Thus it is uncertain that species differences in CAT actually exist. 22 Acetylcarnitine metabolism Marquis and Fritz (11) were the first to observe that tissues containing a high level of CAT usually contain a high concentration of carnitine and acylcarnitine. Assuming that CAT operates near equilibrium, changes in the ratio [acetylcarnitineJ/[carnitine] are expected to reflect identical changes in the [acetleoAJ/[CoASH] ratio. ‘ However, it is not proven that in different metabolic states CAT operates at equilibrium in each compartment of the cell in all cell types. These data are really only available for perfused rat heart, locust flight muscle, and for several tissues of sheep. Perfusing rat hearts with appropriate substrates causes very large changes in acetleoA levels but the corresponding changes in acetylcarnitine are compensatory with the ratio approaching the theoretical value of 0.6 under most conditions (10). Very similar results were obtained by Whitmer gt al. (123) using control, ischemic and hypoxic perfusion conditions with either glucose or glucose + palmitate as substrates. Direct measurements of metabolite compartmentation shows approximately 95% of the cellular CoA but only 10% of cellular carnitine is in the mitochondrial compartment in rat heart (124). This data is supported by the fact that the plot of the mass action ratio of CAT by Pearson and Tubbs (10) passed through the origin. Thus not only does CAT appear to operate near equilibrium in perfused rat heart, but the whole of the acetleoA pool in this tissue appears to be in equilibrium with the acetylcarnitine pool. These results caused Pearson and Tubbs to propose that acetylcarnitine buffers the acetleoA/CoA ratio allowing a larger input of substrate into the cell without depleting the free CoASH 23 which would destroy the ability of the mitochondria to function especially under conditions of high energy requirement. Worm gt_gl. (125) applied this same reasoning to a study of the metabolite changes occurring during flight in the flight muscle of Locusta migratoria. Plots of the mass action ratio for CAT versus flight time indicated the reactants were not in equilibrium in resting insects or during the first minute of flight although equilibrium was attained immediately thereafter. The data in this study also suggested that there were two distinct pools of acetleoA one of which was not in equilibrium with acetylcarnitine. Snoswell and Kaundakjian (107) performed a similar study of normal and diabetic sheep tissue. Not finding any other obvious function for CAT in sheep, these authors concluded that the enzyme buffered the acetleoA/CoA ratio. The apparent equilibrium constants for the CAT reaction calculated for both normal and diabetic sheep heart and skeletal muscle were somewhat higher (range 1.3-4.2) then the expected value (0.6). The calculated intracellular equilibrium constant for CAT in sheep liver and kidney cortex was nearly 30-fold higher (direction of acetleoA formation) than under in 11539 additions. Liver and kidney perform different functions than the heart and carnitine could have other roles in the liver than buffering the acetleoA/CoA ratio. Liver cells maintain a fairly equal distribution of their cellular CoASH between the extra- and intra-mitochondrial compartments (126). The carnitine content of liver is one-half to one-quarter the carnitine content of heart muscle and would not be expected to represent the same buffering capacity as exists in heart. Thus it is not surprising that the situation in sheep liver appears more complex. 24 Pearson and Tubbs measured the levels of free carnitine and acetylcarnitine in a large member of tissues including liver and kidney taken from rats in various nutritional and metabolic states. Whereas the total acid soluble carnitine content of each tissue remained relatively constant, major changes in the acetylcarnitine/carnitine ratio (from (.04 to 6) were observed for the various tissues in different metabolic states (10). Still assuming that CAT operates near equilibrium, Bohmer and coworkers (127,128) measured significant changes in the acetylcarnitine/carnitine ratio in the liver, kidney, heart, and adipose tissues of rat in various nutritional and metabolic states. Ciman gt 21° (129) performed a similar analysis of resting and exercised rat muscle. Under normal conditions acetylcarnitine is the predominant short-chain acylcarnitine in rat tissues (130), however, it is more precise to be concerned with the total acylcarnitine composition of a tissue and whether or not it is in equilibrium with the cellular acleoA pool. For CAT it is approximately the C2 to C5 short-chain acylcarnitine/carnitine ratio that is relevant and this ratio does change with nutritional status (10,12,131) although over a smaller range than the acetylcarnitine/carnitine ratio. For example, the ratio of acetylcarnitine to carnitine in rat liver drops from 0.24 in the fed animal to less than 0.05 in the starved, carbohydrate re-fed rat while the acid soluble acylcarnitine/carnitine ratio remains constant at 0.7 (10). Determining the acylcarnitine composition would more precisely reflect the metabolic state of these cells. In a study of anoxia in rat heart and pig heart, fairly minor changes in the short-chain 25 acylcarnitine/carnitine ratio were recorded while major changes in the tissue levels of some specific acylcarnitines occurred (133). One other aspect of acylcarnitine metabolism explored by Brass and Hoppel (132) using rat liver is the extent to which the cellular acylcarnitine pools turnover and mix with the acleoA pool. In isolated rat liver mitochondria oxidizing palmitylcarnitine, exogenous carnitine has no effect on the mitochondrial content of acetleoA, acid-soluble 00A or acid-insoluble CoA, or no effect on oxygen consumption or citrate formation even though acetyl units are shunted to acetylcarnitine. Approximately 5% of the acetyl groups generated from palmitylcarnitine by B-oxidation appeared in acetylcarnitine without affecting the acleoA/CoA ratio. From these data plus data obtained ifl.!i!9 (131) the authors concluded that carbon flux through the short-chain acylcarnitine pool is sluggish compared to the flux through the CoA pool. However, Brass and Hoppel made a very interesting calculation; the rate of acetylcarnitine formation by mitochondria in state 3 oxidizing palmitylcarnitine is about 1 nmol/min/mg mitochondrial protein very nearly equal to the specific activity of CAT in rat liver mitochondria. Thus, this study indicates that CAT can shuttle acetylcarnitine out of the mitochondrion at a rate nearly equal to its specific activity without affecting the intramitochondrial acetleoA/CoA ratio. In other words, it appears as if CAT couples the cytosolic acetylcarnitine/carnitine ratio directly to the intramitochondrial ratio of acetleoA/CoA. Metabolism of acetylcarnitine at a rate equal to its rate of delivery to the cytosol would make this system behave like an acetyl pump exporting “active acetate" from the mitochondrion. 26 Involvement of acetylcarnitine in fatty acid synthesis and biological acetylations Carnitine is thought to play a central role in the control of fatty acid utilization and fatty acid synthesis (134,143). Reynier (135) reported that carnitine is antiketogenic in fasting rabbits as has also been seen to occur in other animals (137,139,142). 0n the other hand, some investigators report that carnitine stimulates acetoacetate production (138,140,36). These contradictory results may be caused by carnitine having opposite effects in fed and fasting animals (136). Moreover, the carnitine concentration determines if ketogenesis is stimulated or depressed. (137,138). Fritz found that carnitine stimulated fatty acid oxidation in rat liver homogenates and reported that acetoacetate production was stimulated simultaneously (36). Carnitine-stimulated acetoacetate production was studied by Bressler and Katz using fasted, pregnant guinea pig liver homogenates (78). They attributed the increased acetoacetate production to a faster rate of delivery of long-chain fatty acids to the site of a-oxidation, a result readily understood in terms of the known role of carnitine in fatty acid oxidation. Bressler and Katz also suggested that acetoacetleoA transport out of the mitochondria was carnitine dependent but this scheme has not been substantiated and it is accepted that acetoacetate is produced in the mitochondrion via the HMG-CoA cycle (141). During their study of acetoacetate production, Bressler and Katz noted that exogenous carnitine stimulated the conversion of L2-14C]pyruvate to fatty acids by liver homogenates of fed guinea pigs. Bremer had already pr0posed that activated acetyl groups exported 27 out of mitdchondria as acetylcarnitine could be available for fatty acid or cholesterol biosynthesis (37). In a subsequent study, this time using fed male guinea pig liver homogenates, Bressler and Katz (145) showed that carnitine stimulates the conversion of pyruvate to acetylcarnitine and to long-chain fatty acids and that in vivo carnitine stimulates the conversion of glucose, pyruvate, and acetate to fatty acids in liver and adipose tissue. Carnitine had no effect on the conversion of citrate to fatty acids. It should be pointed out that although DL-carnitine caused a 2- to 3-fold increase in the amount of pyruvate carbon incorporated into fatty acids, the absolute rate of incorporation was only of the order of 0.002 nmol/min/mg protein. Under .l_.1119 conditions the rate of incorporation was even lower, 0.0005 nmol/min/mg protein. A year later Bressler and Brendel (144) reported a similar investigation of fatty acid synthesis in pigeon liver but this time the rate of incorporation of added substrates into fatty acids were between 0.025 to 0.05 nmol/min/mg protein. The purified fatty acid synthesizing system of pigeon liver incorporates acetleoA into fatty acids at the rate 6.5 nmol/min/mg protein (146). In this second study, Bressler and Brendel concluded that in the absence of added carnitine, 75% of the pyruvate converted to fatty acids probably went via the indirect citrate pathway but that this decreased to 60% when L-carnitine was added. These values were calculated using assumptions which if not true would have overestimated the percentage of pyruvate converted to fatty acid via the citrate pathway. Thus in the presence of exogenous carnitine, at least 40% of fatty acid carbons are derived from pyruvate by a non-citrate pathway, presumably as acetylcarnitine. Furthermore, the acetylation of 28 sulphanilamide at a rate of about 0.5 nmol/min/mg protein by pigeon liver derived nearly 50% of its acetyl groups from pyruvate by a non-citrate pathway. The role of carnitine in biological acetylations is discussed further below. Lowenstein (147,148) absolutely refutes any role for acetylcarnitine in fatty acid synthesis. However, he used progressively purer preparations of the fatty acid synthesizing system in his studies which by his own data lack anydetectable CAT activity. It is not surprising, therefore, that acetylcarnitine emerges as a very poor substrate for fatty acid synthesis in his studies. Marquis gt 31. (143) demonstrate that carnitine and palmitylcarnitine have complex effects on the rate of fatty acid synthesis in partially purified systems unrelated to the function of CAT. Lowenstein claims (147) that CAT is not active enough to account for the cell's rate of fatty acid synthesis. The maximum rate of acetleoA incorporation into fatty acids by high speed supernatant fractions of livers is about 1 or 2 nmol/min/mg protein. But even in rat liver where the level of CAT is low compared to other animals, Brass and Hoppel showed (see previous section) that acetylcarnitine is produced and pumped out of the mitochondrion at a rate nearly equal to the specific activity of CAT in the mitochondria, 1/nmol/min/mg protein. These arguments do not prove that acetylcarnitine is formed and transferred out of the mitochondrion and then used for fatty acid synthesis. They do indicate that some of the older literature should be re-evaluated on this point. A related issue that has not received much attention is the role of cytosolic acetylcarnitine in biological acetylations. Biological acetylations are a heterogenous group of 29 reactions some of which appear to have important functions in the control of cellular growth, development and metabolism. The most obvious example is the acetylation of histone and non-histone chromosomal proteins which reactions require extramitochondrial acetleoA that may even have to be transported into the nucleus as acetylcarnitine. As a class the sialyl residues on secretory proteins are O-acetylated and there are many more specific examples of O-acetylated or N-acetylated proteins. It is very tempting to speculate that microsomal CAT may play a direct role in these acetylation reactions. The mitochondrial CAT may simply deliver active acetate to the cytosol but whereas cytosolic citrate may be committed to fatty acid synthesis, cytosolic acetylcarnitine may be available for other purposes. It is amazing that so much could be known about an enzyme and yet its importance in metabolism be so poorly understood. Yeast metabolism Typically, glucose fermenting yeast are studied because of their economic value. However, glucose has a very particular effect on the metabolic state of yeast. The classical glucose effect was described many years ago by Epps and Gale (153) and J. Monod (154). As the biochemistry of the glucose effect came to be better understood, Magasanik coined the term "catabolite repression" (155). In its simplest terms, glucose or one of its "catabolites" causes the repression of other catabolic enzymes not related to glucose fermentation. In modern terms "catabolite inactivation" is defined as "the loss in 1112 of catalytic activity of certain enzymes subsequent to the addition of glucose or related sugars to cells adapted to a non-sugar carbon source or to 3O starved cells" (156). Some of the enzymes inactivated include fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, cytoplasmic malate dehydrogenase, a-glucoside-(maltose) permease, the galactose uptake system, uridine nucleosidase, and aminopeptidase I. The glyoxalate pathway is also suppressed and although cytochromes may be synthesized they may not be assembled into a functional electron tranSport system. In this thesis it is catabolite repressed yeast that are studied. It is quite certain that most yeast growing on glucose would, if fatty acids were added, still use only glucose for their energy metabolism. Under these conditions, most of the added fatty acid would be incorporated into the yeast lipids (157). ‘I.‘bgvina ATCC 26014 is no exception, [1-14CJpalmitate, when added to the growth media with glucose, is recovered almost completely in the cell lipids and protein/cell wall residue (9). The ability of Saccharomyces species to use long-chain fatty acids as sole carbon sources has not been investigated. There is a group of alkane utilizing yeast of which Candida tropicalis is a well-studied example. This yeast contains peroxisomes (158) and extremely high levels of CAT (91), probably even higher than reported because the enzyme was assayed with subsaturating concentrations of acetleoA (assuming its Km for acetleoA is similar to that reported for CAT purified from other sources, see above). CAT is present in both the peroxisomes and mitochondria of Q. trgpicalis and Kawamoto gt a1. (91) propose that CAT participates in shuttling "activated acetate" from the peroxisome where it is formed exclusively to the mitochondria where it is a substrate for the TCA cycle. 31 At the other extreme are the oleaginous yeast, a group of yeast that accumulate lipids and are so far the only yeast found to contain ATchitrate lyase (159). Non-oleaginous yeast grown on glucose must synthesize fatty acids to grow. Kohlhaw and Tan-Wilson (90) found high levels of CAT in a commercial preparation of Baker's yeast and suggested that acetylcarnitine shuttles acetyl groups out of the mitochondria for use in fatty acid synthesis when yeast are grown on non—fermentable substrates. Presumably in yeast grown on glucose, pyruvate is decarboxylated and dismutated to acetate and ethanol, the acetate being activated to acetleoA in the cytosol and used for fatty acid synthesis. Direct evidence for either of these hypotheses is lacking. Severe biosynthetic requirements are placed on yeast grown on glucose. As a result a number of synthetic pathways for various compounds depend on a condensation reaction between acetleoA and an a-keto acid. Lysine is biosynthesized via the a-aminoadipate pathway and the first intermediate homocitrate, is formed by the condensation of acetleoA with a-ketoglutarate catalyzed by homocitrate synthase (160). AcetylcoA is also required to condense with oxaloacetate forming citrate in order to produce a-ketoglutarate. The condensation of a-ketoisovaleric acid with acetleoA catalyzed by arisopropylmalate synthase is the first reaction in leucine biosynthesis (161). O-Acetylhomoserine is an intermediate in methionine biosynthesis in Neurospora (162) and N-acetylglutamate is a key intermediate of arginine biosynthesis in a number of organisms including Saccharomyces (163). In 1957 Van Uden and Do Carma-Sousa (164) described a yeast isolated from the caecum of a cow and named it Candida bovina CBS 2760 (it should be noted that Candida bovina has been transferred to the genus 32 Torulopsis). The yeast does not form spores, ferments and assimilates only glucose, does not split arbutin, and does not assimilate ethanol or nitrate. C, bovina was very similar to Torulopsis pintolopesii and Kreger-vanRij took 9. bovina to be the imperfect stage of Saccharomyces tellustris (165). Thus it is not unusual to compare the metabolism of I. b91153 to that of Saccharomyces. These three yeast are part of a group of thermophilic yeast that are regarded as obligate saprophytes of warm blooded animals growing best at around 40°C although they do grow well at 30°C but poorly at 20°C or lower (165). Cury gt 31. (166) studied the nutritional requirements of this group of yeast and discovered that some of them including 1. bovina needed choline to grow. The isolation of a carnitine-requiring strain of T. bovina has already been described (see above). Watson and coworkers have studied the lipid and cytochrome content and composition of the wild type I. boyifla_ATCC 22987 with the following results. The yeast is a facultative anaerobe (167) but it is competent of respiration and its cytochrome content is significantly greater than that of other thermophilic yeast (168). The major phospholipids in I. bgyiga are phosphatidylcholine, cardiolipin, and phOSphatidylethanolamine in nearly equal amounts. Palmitoleic acid is the predominant fatty acid component followed by oleic acid (169). The above data are presented for comparisons sake; there are no data to indicate that the carnitine-requiring strain has the same properties as the wild type yeast. In fact, Watson's data suggests that I. bovina ATCC 22987 assimilates ethanol whereas the type description stated that ethanol was not assimilated. Thus Watson's data were collected with 33 yeast grown on ethanol and would not be expected to represent the biochemistry of glucose grown cells. Restatement of the problem With this introduction then, the problem is to determine the role of carnitine in a yeast grown on glucose, thus not oxidizing fatty acids nor presumably requiring any shuttle mechanism for acetleoA in order to synthesize fatty acids. Because the electron transport chain is suppressed by glucose, carnitine is not expected to act as a high energy reservoir of acetyl units for consumption in the TCA cycle; the TCA cycle is used to supply growing yeast cells with biosynthetic intermediates, not to oxidize acetyl groups. Determining the metabolic role of carnitine I”.Ir.22!iflé ATCC 26014 may provide the first unequivocal demonstration of a function for CAT and its possible importance in the metabolism of other organisms. EXPERIMENTAL METHODS AND RESULTS MATERIALS AND METHODS Materials ‘ Sodium [1-14CJacetate (58.3 Ci/mol) and standard [14C]toluene were purchased from New England Nuclear (Boston, MA). The [14C]acetate was used for the synthesis of [1-14C3acetylcarnitine as recently described (170). Phthaldialdehyde and ethanethiol were obtained from Pierce Chem. Co. (Rockford, IL). Avicel coated uniplates were purchased from Analtech, Inc. (Newark, DE) and most of the solvents were purchased from Mallinckrodt (St. Louis, MO) except the acetonitrile used for HPLC analysis which was glass distilled, Omnisolv from MCB Manuf. Chemists, Inc. (Cincinnati, OH). Glass beads (0.45 mm) were purchased from Thomas Scientific Co. (Philadelphia, PA. Nephelometer flasks were purchased from Bellco Glass, Inc. (Vineland, NJ). L-carnitine°HCl was generously provided by Otsuka Pharmaceutical Co., Japan. All acleoA derivatives were purchased from PL Biochemicals Inc. (Milwaukee, WI) but [1-14C]acetleoA as well as DL-[methyl-3H]carnitine was purchased from Amersham (Arlington Heights, IL). Type H-2 B-glucuronidase from Helix pomatia was purchased from Sigma Chemical Co. (St. Louis, MO). Prepurified nitrogen and 5% C02 in nitrogen were purchased from Airco, Inc. (Montvale, NJ). All other chemicals were of reagent grade from commercial sources. 34 35 Organism Torulopsis bovina ATCC 26014 was used throughout this study. It is the carnitine-requiring mutant of the parent I. bovina ATCC 22987 isolated as described by Travassos gt El. (1). Growth media Yeast were grown on a modified Wickerham's yeast carbon base medium (171). Four stock solutions (A,B,C, and D) were prepared. Their composition is indicated in Table 1. Solution C was used in the preparation of Solution 0. A stock solution of ten-times concentrated media was prepared consisting of 5 parts solution 0 supplemented with 2% (w/v) L-asparagine and 3 parts distilled water. This was sterilized by autoclaving 20 minutes at 15 psi and then combined with 1 part A and 1 part B. Growth media was prepared by dissolving the carbon source in 5 parts 0.1 M potassium phthalate buffer, pH 4.5, adding 4 parts water, autoclaving to sterilize, and then diluting the sterile concentrated media ten—fold into this solution. The carbon source was glucose at 1% (w/v) final concentration. When present, L-carnitine was added to the media with the carbon source to a final concentration of 1 pg/ml or to the final concentrations indicated in the specific figures and tables. Basal media lacks carnitine. Maintenance and growth conditions .1. bovina was grown at either 37°C or 30°C with L-carnitine included in all maintenance cultures. For most experiments, cells were grown in 7 ml of synthetic media in 18 x 150 mm test tubes or in 100 ml of media in 500 ml nepholometer flasks with an 18 mm diameter side arm. Cultures 36 TABLE 1. COMPOSITION OF STOCK SOLUTIONS FOR T. BOVINA MEDIA PREPARATION Aa Bb Ingredient Ag/ZOOml Ingredient g/500ml Choline-Cl 0.050 L-Phenylalanine 1.60 Biotin 0.005 Ca-Pantothenate 0.100 L-Tryptophan 0.50 Thiamin 0.100 Pyridoxine 0.100 L-Histidine 0.20 Nicotinic Acid 0.100 Inositol 0.500 L-Methionine 0.20 Cytosine 0.500 Adenine 0.500 cc Dd Ingredient mg/L Ingredient g/L KH2P04 20.0 H3803 500 M9304 7H20 10.0 CuSO4 5H20 40 NaCl 2.0 FeS04 7H20 40 KI .002 NazMOO4 ZOO CaClz 2H20 1.0 ZnSO4 400 CoClz 6H80 40 Trace Elements MnSO4 H7 400 Solution C 20 ml a100x Concentrated Amino Acid Solution: dissolve in the smallest volume of 1 N HCl, dilute to 180 ml, adjust the pH to 4.5, make the volume to 200 ml and autoclave to sterilize. 100x Concentrated Vitamin Solution: dissolve the nucleosides in 1 N HCl and add to the vitamins in 400 ml water. Adjust the pH to 4.5 make the volume to 500 ml and autoclave to sterilize. C1000x Concentrated Trace Elements Solution: add trace elements in order to 800 ml water, make the volume to 1 Liter and store in a brown glass bottle. d20x Concentrated Salts Solution: in 750 ml water. drops 1 N HCl. dissolve the salts in order Predissolve the CaCl2°2H20 in water + 4-5 and add 4 ml CHCl3 as preservative. Adjust the pH to 4.5, make the volume to 1 Liter .u. .‘ 37 were shaken continously on a New Brunswick gyratory shaker and growth was monitored by measuring the absorbance of the cell suspension at 600 nm in a Coleman Jr. II spectrophotometer. Growth rates were calculated as the slope of the least squares line fitted to a plot of the logrithm of the absorbance (LogloA) versus incubation time in hours. In Figure 1 the equation for this line was used to calculate relative incubation time by taking as time zero that point at which the cells reached a density equivalent to 0.01 absorbance. Exponential phase cells were used for the inoculation of all test media. Cells were harvested by centrifugation and washed three times with phthalate buffered saline (potassium phthalate, 10 mM; KH2P04, 7.4 mM; NaCl, 137 mM; KCl, 2.7 mM; pH 4.5). The cells were suspended in phthalate buffered saline to an absorbance of 0.1, the cell density being about 3 x 106 cells/ml. This suspension was diluted 100 fold and a 0.1 ml sample was used to inoculate a 7 ml culture and a 1.0 ml sample was used to inoculate a 100 ml culture. Anaerobic cultures were obtained by purging flasks containing media with gas filtered first through sterile cotton then through sterile water to saturate it with water vapor and finally through a glass tube plugged with sterile cotton. The flasks were sparged with gas for 1-2 hours, inoculated with cells, and then gassed another 30 minutes before sealing them with a bunsen valve. A fish tank air pump hooked to a similar gas train was used to continuously aerate aerobic cultures. Uptake of radioactive carnitine Cells were grown in 100 ml of the synthetic media without added carnitine. They were harvested by centrifugation and suspended in 5 ml of ice cold uptake buffer (KH2P04, 10 mM; MgSO4°7H20, 1 mM; 38 NaCl, 50 mM; KCl, 2.7 mM; pH 4.5). A 0.5 ml sample was removed for dry weight determination. Carnitine uptake was determined by adding 0.2 ml of DL-[methyl-3H]carnitine solution (1.18 pCi at .25 Ci/mmol diluted with 55.6 nmol unlabeled L—carnitine-HCl) to 5 ml of uptake buffer warmed to 37°C and mixing this solution with 4.5 ml of cold cell suspens- ion at time zero. The cells were incubated in 25 ml Erlenmeyer flasks at 37°C in a Dubnoff water bath. At the times indicated (the first time point was 10 sec) 1 ml samples were removed, collected on glass fiber filters using a millipore apparatus, dried at 60°C for 8 hours, weighed and then placed into scintillation vials. The filters were wetted with 1 ml water, 10 ml of Triton X-100 based scintillation cocktail was added (173) and the cell associated tritium counted with 15% efficiency. Carnitine analysis Carnitine analysis was a modification of the methods of Cederblad and Lindstedt (174) and Parvin and Pande (175). The reaction was buffer- ed at pH 7.6 and contained in 0.2 mlz288 pmol [1-14C]acetylcoenzyme A (0.0167 uCi), 50 ug N-ethylmaleimide (prepared fresh on day of use), 20 pmol potassium phosphate, and the sample (in 100 pl) to be analyzed. This was used to assay 1-20 pmol L-carnitine. Unlabeled acetleoA (333 pmol) was added to assay 10-200 pmol of L-carnitine. The reaction was initiated by the addition of 25 pt of CAT (0.425 units/assay) and was terminated by putting a 200 pt sample into a column (5 x 25 mm) of Dowex 1-x8 (100-200 mesh) in chloride form, washing it through with 1 ml of water and collecting the total eluate in a scintillation vial for detection of 14C. The enzymatic blanks were very low and equal to the non-enzymatic blanks when phosphate buffer was used. 39 Duplicate analyses were performed on each sample with and without a known amount of L-carnitine added and the sample values were corrected to 100% recovery of the added L-carnitine. Carnitine was generally extracted from cells by boiling for 3 min. The data in Table 2 shows that boiling carnitine and/or acetylcarnitine solutions made up in basal media did not interfere with their analysis and that essentially 100% of the added carnitine was recovered after saponification of the sample. Total carnitine was determined in all samples after saponification of the extract in 0.2 N KOH for at least 30 min at 50°C after which a solution of potassium phosphate, pH 7.6, was added to 0.1 M final concentration to aid neutralization to pH 7.6 with HCl prior to analysis. The endogenous carnitine content of I. bgvigg was determined as follows. Cells were grown for 36 h in basal media (no carnitine present), harvested, washed, and then inoculated into three flasks each containing 1 L of basal media. The cells were harvested in the late exponential phase of growth (0.5-0.6 absorbance), collected by centrifugation and extracted by boiling 5 min with 2 volumes of distilled water. Cell debris was removed by centrifugation and the pellet washed twice with 2 volumes of water. ~The hot water extracts were lyophilized and then made to a known volume with water. This solution was assayed directly for carnitine and total carnitine was assayed after saponification of a measured sample of the extract. Carnitine uptake and its esterification by whole cells was measured as follows. Cells were grown at 30°C in media supplemented with 5 pM L-carnitine. Samples were taken at various times after inoculation and the cells collected by filtration on Whatman GF/C glass fiber filters 40 TABLE 2. CARNITINE RECOVERIES IN BOILED MEDIA SOLUTIONS 1. 2. 3. 5. 6. Sample Stock L-carnitine solution (5 pM) 5 pM L-carnitine basal media Basal media 5 uM acetylcarnitine 5 pM carnitine + 5 pM acetylcarnitine in basal media Same as 5 plus boil 3 min Same as 6 plus saponification Carnitine Found nmol/100 ml culture 470 468 ' <1 5 <15 502 519 1006 41 using a Millipore apparatus. The filtered media was assayed directly for free carnitine while the cells and filters were immersed in 2 ml water in a capped culture tube and placed into a boiling water bath for 3 min. The filter and cell debris were removed by centrifugation and the hot water extract analyzed for free carnitine. Total carnitine was determined in saponified samples of the media and cell extracts and esterified carnitine was calculated from the difference between the total and free carnitine values. The amount of acetylcarnitine synthesized by the yeast was determined in cells grown with 5 pM L-carnitine in 1 L of media in 2.8 L Fernbach flasks st0ppered with cotton.- Cells were harvested by centrifugation in the late exponential phase of growth and extracted by shaking 1 g of wet packed cells 4 min with 10 g of 0.45 mm glass beads in 4 ml of 6% (w/w) HClO4. The extracts were filtered on fritted glass Buchner funnels, the filtrates neutralized to pH 6.5 with KOH and centrifuged to remove salt. Acetylcarnitine was assayed in the acid extracts enzymatically by coupling to the formation of citrate as described previously (176). Fermentation analysis Cells were grown on 1% glucose at 30°C with or without L-carnitine. Samples of the cultures were taken during the measurable stage of exponential growth and the fermentation fluid separated from the cells by centrifugation for 10 min at 10000 x g. The supernatant fluid was analyzed in duplicate for glucose, ethanol, acetate, and lactate. The assay procedures for glucose, ethanol, and lactate were taken from Bergmeyer (177-179). Acetate was assayed by the method of Guynn and 42 Veech (180). Standard curves were generated for each compound in its respective assay and all samples were listed in duplicate for recovery of added standard. In a different experiment, 100 ml cultures were grown without phthalate, one with and one without carnitine added, and the spent media (88 ml) was lyophilized and redissolved in 10 ml of water. Samples (1 ml) were then acidified, saturated with NaCl and extracted with ether. The ether extracts were analyzed by gas chromatography for volatile fatty acids (181). RADIOACTIVE TRACER STUDIES Growing cells were incubated for various times with either [1-14Jacetate or [1-14C3acetylcarnitine (approximately 5 pM with the exact concentrations given in the individual experiments). The cells were then fractionated into various components and analyzed for 14C content as described in the following sections. Lipid extraction Cells were grown in 100-200 ml of media to an absorbance of 0.2 at which time [14C]acetate or [14CJacetylcarnitine was added and the cells allowed to grow for 2 h more. The cells were then collected by centrifugation, washed with cold distilled water, and pelleted in 50 ml, thick-walled glass centrifuge tubes. The cells were treated with 0.5 ml of saturated KOH solution plus 15 ml of 95% ethanol and a couple boileezer glass chips added. The tube was heated, uncovered, in a glycerol bath at 80°C. When the volume was reduced to 0.5-1 ml, the tube was cooled and the neutral lipids extracted with 2 or 3, 10 ml volumes of 43 petroleum ether or until no further 14C was extracted. The residual solution was treated with one drop of cresol red indicator solution (0.1 g in 26.2 ml of 0.01 N NaOH plus 223.8 ml water) and acidified with H2804 (concentrated acid diluted 1 to 2 with water). After the second color change to red, the acidified lipids were extracted as described above with petroleum ether. Warm water (25-50 ml) was then added to the residual cell material most of which dissolved. This solution was centrifuged and decanted to obtain the soluble non-lipid fraction and the insoluble residue. The 14C in the neutral lipids, acidic lipids, soluble non-lipids and insoluble residue was determined by scintillation counting as described below. Phenol extraction Cells were harvested by centrifugation at 2000 x g for 10 min and ruptured by shaking 1 g of cells (wet weight) with 20 9 glass beads and 10 volumes water until >90% of the cells were broken as judged by microscopic observation. The glass beads were removed by filtration on a coarse fritted glass buchner funnel and the homogenate repeatedly centrifuged at 860 x g for 2 min until no further pellet was formed. All the pellets were combined and washed with water by similar repeated centrifugation until negligible radioactivity could be washed from the pellet. This low speed pellet constituted the cell wall fraction. The pellet washes were combined with the original 860 x g supernatant fluid and acidifed with concentrated HCl04 to a final concentration of 6% (w/v). Denatured protein was collected by centrifugation at 10,000 x g for 10-15 min. The supernatant fluid was decanted, neutralized to pH 7 with KOH, removed of salt by centrifugation and assayed for acid soluble 44 radioactivity. For protein isolation, the denatured protein pellet was dissolved directly in phenol. The protein pellet was first treated with chloroform-methanol (1:1) to extract lipids. The lipid extraction was repeated 3 or 4 times with 2 ml solvent, the pellet being dispersed each time by sonication and re-collected by centrifugation. In some instances, the lipid extracted protein pellet was hydrolyzed 18 h at 37°C in 0.3 N KOH (182) to isolate ribonucleotides which were analyzed as described elsewhere (183). Otherwise, the lipid extracted protein pellet was dissolved in ammoniacal phenol and acetone precipitated essentially as desCribed previously (184). Protein hydrolysis About 4 mg of methanol washed, dry protein isolated as described above, was hydrolyzed in constant boiling HCl containing 2% thioglycollate (185) and a crystal of phenol as described by Moore and Stein (186). Hydrolysis was performed for 72-96 h, the hydrolysate centrifuged and transferred to a small boiling flask with 1 ml of 1 N HCl and evaporated under reduced pressure to dryness. The condensing flask of the rotary evaporator contained 25 ml of I N KOH solution cooled in ice to trap any [14C]acetate. The amino acids were dissolved in 0.5 ml of water and the pH adjusted to 7-8 with KOH. Samples were removed for determination of 14C and amino nitrogen. Amino nitrogen was assayed with ninhydrin using leucine as standard (187). Amino acid analysis The amino acids in the protein hydrolysate were isolated by reverse phase HPLC chromatography of the phthaldialdehyde derivatives essentially 45 as described by Schubert and Coker (188) using a Beckman HPLC system connected to an Aminco Fluoro-colorimeter interfaced to a Hewlett-Packard integrator. The eluate from each run was collected in 0.5 min fractions directly in mini vials and combined with 4 ml of Triton x-1oo based scintillation cocktail and enough 67% ethanol to obtain a homogenous solution for detection of 14C. Heyns and Walter (189) described a paper chromatographic procedure in separating isomers of leucine and isoleucine which we combined with solvent system II of vonArx and Neher (190) to effect the specific separation of lysine, leucine, isoleucine, histidine, and proline. The amino acids were separated on cellulose TLC plates by development in the first direction for 3 h with isopropanol formic acid (88%), water (160:9:39) and in the second direction, twice, for 3 h with pyridine, amyl alcohol, water (35:35:30). Amino acids were visualized with ninhydrin spray reagent (191). Radioactivity associated with the amino acids was detected by running two identical plates, spraying one with ninhydrin and using it as a template for the second plate. The second plate was still lightly sprayed with ninhydrin to be sure that leucine and isoleucine were properly separated. The spots were scraped from the second plate, placed into scintillation vials with 1 ml water and 10 ml Triton based cocktail and counted for 14C. Radioactivity measurements Radioactivity in all aqueous and particulate fractions was measured by scintillation counting in a Triton X-100 based cocktail (173) and all lipid fractions were counted in an identical cocktail in which toluene replaced the Triton. Lipid fractions in chloroform were evaporated under 46 a stream of N2 and redissolved in absolute ethanol prior to assaying 14C. DPM were determined by the channels ratio method of quench correction or by inclusion of an internal standard ([14C]toluene). All samples were counted on a Packard Tri-Carb spectrometer. CAT STUDIES Preparation of mechanically disrupted cell-free extracts Cells were collected by centrifugation and washed once with ice cold 50 mM phosphate buffer, pH 6.5 in either a tared or graduated centrifuge tube. The cells were packed by centrifugation at 2000 x g for 10 min and the wet weight or packed cell volume recorded. For disruption, the cell pellet was suspended in 20 volumes of cold buffer and a 4 ml sample combined with 8 9 glass beads (0.45 mm) in an 18 x 150 mm capped culture tube. Higher cell densities, i.e., only 5-10 volumes cold buffer per g wet weight of cells, were used when the extract was to be applied to a sorbitol gradient. The mixture was agitated at full speed with a Vortex-Genie for 2 min unless stated otherwise. The homogenate was siphoned off with a Pasteur pipet and the glass beads washed with 4 ml buffer. The homogenate and washes were combined and "crude extracts" prepared by centrifuging the solution for 10 minutes at 500 x g to sediment whole cells and cell wall debris. Crude extracts were used immediately or frozen at -80°C. Enzyme levels were measured in homogenates prepared in 50 mM phosphate buffer, pH 6.5 and differential centrifugation was performed on crude extracts prepared in sorbitol buffer (sorbitol, 0.45 M; TriS°HCl, 10 mM; KH2P04, 5 mM; KZHPO4, 5 mM; NaCl, 50 mM; pH to 6.5). 47 Samples (about 5-7 mg protein) of cell-free homogenates prepared in 0.25 M sorbitol, 50 mM phosphate buffer, pH 6.5 were layered on top of 32 ml linear gradients of 10-40% (w/w) sorbitol in 50 mM phosphate buffer, pH 6.5, containing 2 ml cushions of 55% (w/w) sorbitol in the same buffer. Centrifugation was performed in a Beckman SW 27 rotor in a Beckman L2 Ultracentrifuge run at 22,000 rpm for a period of 16-18 hr at 4°C. Subcellular particles were collected by puncturing the tube from the bottom and collecting 1.5 ml fractions. The fractions were either kept at -80°C or they were made to 0.1% (w/v) in sodium azide and kept at 4°C. Preparation of spheroplasts and isolation of mitochondria Spheroplasts were prepared and lysed as described by Linnane and Lukins (192) except 1 ml of B-glucuronidase was added to 5 ml of cell suspension and 0.5 ml p-mercaptoethanol was added to 100 ml of cell suspension. Also, spheroplasts were lysed by dilution without further treatment in the French Press. Spheroplast formation was followed by measuring the decrease in turbity and was about 90% effective in 40 min as judged by microscopic observation. Mitochondria were isolated by differential centrifugation or purified by isopycnic density centrifugation in linear gradients of 15-66% (w/w) sorbitol in 1 mM TriS°carbonate buffer, pH 7.0 and 1 mM EDTA. Centrifugation was performed in a Beckman SW 27 rotor at 22,000 rpm for 3 h at 4°C. Subcellular organelles were collected by puncturing tubes from the bottom and collecting 1.3 ml fractions. 48 Enzyme assays CAT was assayed in the presence of 0.4 mM acetleoA in either Buffer I (0.1 M Tris, pH 8.0, 1.1 mM EDTA, 1.25 mM L-carnitine, 0.15 mM DTNB, 0.1% (v/v) Triton X-100) or Buffer II (50 mM glycylglycine, pH 8.2, 1.25 mM L-carnitine, 25 mM MgClz, 0.15 mM DTNB) as previously described (70). Rates were recorded continuously at 25°C using a Gilford 250 or Zeiss PM6 spectrophotometer. Citrate synthase was assayed exactly as CAT using Buffer 11 except 10 mM cis—oxalacetic acid was substituted for L-carnitine. Fumarase was assayed as described by Hill and Bradshaw (193). Catalase was assayed as described in Bergmeyer (194). Isocitrate dehydrogenase was assayed as described by Cook and Sanwall (195) using either NAD+ or NADPT. Glucose-6-phosphate dehydrogenase was assayed in a 0.2 ml reaction mixture containing 4 mM glucose-6-phosphate, 0.26 mM NADPT, 50 mM Tris buffer, pH 7.3, and 0.1 ml of enzyme solution. NADH (and NADPH) dehydrogenase was assayed according to Mackler (196). Malate dehydrogenase was assayed by following the reduction of oxalacetate according to Kitto (197). a-Oxoglutarate dehydrogenase was assayed by following the reduction of NAD+ according to Reed and Mukherjee (198). The assay for a-glucosidase (maltase) was by the method of Halvorson (199). NADPH-cytochrome c reductase was assayed as described by Masters §$_al. (200) assuming an E550 of 19.7 x 106 mole‘lcmz. AcetleoA synthetase was assayed spectrophotometrically by coupling citrate synthase and malate dehydrogenase to the reduction of NAD+ as modified by Pearson (201). The assay contained 100 mM Tris-HCl and 15 mM L—malic acid adjusted to pH 7.8, 2.4 mM dipotassium ATP, 3.0 mM MgCl2, 0.25 mM NADT, 0.1 mM NADH, 1.60 mM CoASH (free acid), 1.8 mM potassium acetate, 1 unit malate dehydrogenase, 0.4 units citrate 49 synthase, and enough enzyme solution and water to bring the final volume to 0.25 ml. The blank lacked acetate. ATP-citrate lyase was assayed by both the hydroxamate method and the spectrophotometric method described by Takedo ££.él- (202) except glutathione was substituted for mercaptoethanol. Citrate lyase was assayed as described by Dagley (203). Protein determinations Protein was determined using Coomassie Blue G250 (204). Protein, 0-50 mg, in a volume of 0.1 ml was mixed with 5 ml of the dye reagent and its absorbance measured at 605 nm. The dye reagent was prepared by dissolving 300 mg Coomassie Blue G250 (Brilliant Blue G, Lot No. 18C-0132, Sigma Chemical Co., St. Louis, MO) in 50 ml absolute ethanol, adding 70 ml of 60% (w/v) HCl04, and making the volume to 2 liters. The reagent was filtered and the absorbance at 605 nm adjusted to 0.2 by dilution with 2.1% HCl04. The dye dissolves readily in absolute ethanol but only with difficulty in 95% ethanol. The 2.1% HCl04 produced the best linear response with BSA as standard, but other batches of dye may require a different concentration of HClO4. RESULTS GROWTH STUDIES Carnitine stimulation of growth Very low concentrations of carnitine promote the growth of the yeast, Torulopsis bovina ATCC 26014 (1) but the published data do not show whether carnitine affects cell size, final cell density, lag time or growth rate. The data in Figure I show that cells grown in media supplemented with 1 ug/ml L-carnitine grow at nearly twice the rate of control cells grown without carnitine. Similar results were obtained with cells grown at 30°C or 37°C; see Table 3. Cultures grown on 1% glucose with or without carnitine entered the stationary phase at approximately the same cell density (0.6 absorbance) indicating that carnitine did not noticeably affect cell yield. One attempt was made to count cells. Cell numbers varied linearly with the absorbance of the culture, see Figure 2, but the ratio of cell number to absorbance (the slopes of the lines in Figure 2) was different when carnitine was present than in the control. Microscopic examination of the cultures revealed that the carnitine supplemented culture contained mostly quadruplet strings of cells whereas the control culture contained mostly doublet and single cells. Thus the accelerated growth rate induced by carnitine caused the morphological appearance of the yeast to change. The data in Table 4 show that the dry weights of cells 50 51 Figure 1. Effect of carnitine on the growth rate of T. bovina. Cells from an exponentially growing culture containing 1 ug7ml L-carnitine were diluted to 400 cells/ml and incubated at 30°C. Ar-ZS; no added carnitine; crqa, 1 pg/ml L-carnitine added. 52 1 I I l I I I J 2 4 6 8 IO I2 l4 RELATIVE INCUBATION TlME(hrs) Figure l 53 Figure 2. Covariance of cell number and culture absorbance. Cells were grown with 6 mM glucose in 100 ml of media at 30°C. Samples were removed at the indicated absorbance values and cells counted using an haemocytometer. The data represent the results obtained from duplicate cultures. H, cells grown in basal media.A—A, cells grown in basal media plus 5 uM carnitine. 54 - _ L i. _ 0 8 6 4. 2 .. _-_e ..o_ x Emssz .38 022 ABSORBANCE 0.6 0.4 Figure 2 55 TABLE 3. THE EFFECT OF L-CARNITINE ON YEAST GROWTH RATE Growth Growth Rate (H‘l) Temperature L-carnitine Control 37°C .286 i .045 .176 i .039 (n=23) (n=18) 30°C .180 i .010 .111 i .013 (n=13) (n=7) For each experiment the same inoculum cells were added to a group of tubes containing either control media (no carnitine added) or media containing 1 pg/ml L-carnitine and the cultures incubated at the indicated temperature. The total number of cultures analyzed in all the experiments is indicated in parenthesis. The L-carnitine rate differs from the control at a significance level of p<0.01 by the Wilcoxon signed rank test (172). TABLE 4. EFFECT OF CARNITINE ON CELL YIELD Experiment mg dry weight per unit absorbancea + Carnitine - Carnitine 1 1.28 1.17 2 1.33 1.20 3 1.35 1.33 Average 1.32 i .04 1.23 i .08 Yeast were grown with or without 5 uM carnitine as shown and harvested in the late exponential phase of growth. A sample of each cell suspension was then filtered and the cells dried 24 h at 60°C before weighing. aValues were normalized by calculating the ratio of the cell dry weight (in mg/ml of culture) to the culture absorbance at the time of harvest. 56 from cultures grown with or without carnitine are the same (7% difference between the average values) and that the absorbance is proportional to the total cell mass. If carnitine affected glycolytic metabolism, this might be reflected in the fermentation balance. The data in Figure 3 demonstrate that I. bgyigg produces ethanol in approximately equal quantity to the amount of glucose consumed. Carnitine did not have a major effect on the fermentation balance. However, in other experiments, it was shown that the pH of the media decreased from 4.5 to 3 in the absence of carnitine but remained at 4.5 when carnitine was added. Addition of 0.05 M phthalate maintained the pH at 4.5 under all growth conditions without altering the growth response. Thus the slower growth rate in the absence of carnitine was not due to a decrease in pH. Analysis of the fermentation liquor indicated that lactic acid was not produced in measurable quantity nor were any standard volatile fatty acids produced. If acetic acid was produced, it was present at less than 40 nmol/L. When phthalate was left out of the media, the amount of titratable acid. produced in cultures without added carnitine was 2 meq/L. Effect of air and anaerobiosis on carnitine stimulated growth The effect of carnitine on growth could be associated with this yeast's aerobic or anaerobic metabolism. To explore this possibility, growth tests were conducted in test tubes containing synthetic media to a height of 15 cm, one series with and one without added carnitine. The yeast grew equally well in both except in the top 1-2 cm of the tubes without carnitine where growth was retarded during the first 24 hours of incubation. This suggested that growth was inhibited by air and 57 Figure 3. The amount of glucose fermented to ethanol during exponential growth. Cells were grown with 6 mM glucose in 100 ml of media at 30°C. Samples of the cultures were taken at the indicated cell densities and the media analyzed for glucose and ethanol as described in the Materials and Methods. The data represent the results obtained from duplicate cultures. A, cells grown in basal media plus 5 pM carnitine. B, cells grown in basal media. 0 O , glucose. A A, ethanol. .h 58 BEE .omonoomm JOZom o» cmptm>coo w:_uwccmofl:mw Pe_u_:v we mmeucoogwa asp macsm mczm_w och .Amxav _wm eu_me uw>osoc ms» we m:_p:=oo :owuwp__u:_om an umpop_uco:c use mmuera asp woo quecum mposuoca :m mcwucoqmmggou asp ecu ucommmc Amcam mgoucwmaco ;u_3 Umuompwv .xrmzomcmu—ze_m :3; ago; mvceucmum .u:m>—om we Awuomuomv o_=oEEc .Pocccpms .Egowoco—gu anew: acamcmouoEoczo Loze— cwsu an mme_u umpmuwvcw ecu pm umom_om_ mew: mmcwu_ccoo—xo< .vmpu_eo mm: mp2s pgmoxm Amy me=o_>qu umnwgommu we mcwu_:toom:mH14c uce pwe_oa z: m.~m Lo .:mpoo Lo .i—aLxusnom_ .ipxumoo 25 H.o sow: umpmnzocw mew: muooeuxw mwcw-__mu .mmflwmm .H.wo muomguxw mmgwi__mo An mmcwp_cgmu_>oe um—wano_me do cowueELod .NH mesmwd 9O 00 2:). dz: 29.—.06. mm O¢ ON d ISIIIIIOO. IIIII OOIIODIII UZ_.—._ZIZm>._bmom_ mz.._..zm._.wo< 0. ON 3N111N8vo [Hg-To 91 The apparent Km for acetleoA of CAT in cell-free extracts was determined from Lineweaver-Burk plots. The concentration of L-carnitine was 1.25 x 10'3 M while the concentration of acetleoA varied from 9.5 x 10-7 M to 9.5 x 10-4 M; a Km value of 6.3 x 10-5 was obtained for yeast grown with L-carnitine and a Km value of 6.7 x 10'5 M was obtained for yeast grown without L-carnitine, see Figure 13. The Km for L-carnitine was determined at an acetleoA concentra— tion of 4 x 10'4 M; a Km value for L-carnitine of 3.0 x 10'4 M was obtained for yeast grown with L-carnitine and 6.7 x 10"4 M for yeast grown without L-carnitine, see Figure 14. Very similar Km's were obtained in other determinations using partially purified enzyme. CARNITINE AND ACETYLCARNITINE METABOLISM Identification of acetylcarnitine as the major acylcarnitine Figure 15 shows that growing cells take up media carnitine. The amount taken up is linear with cell density during the exponential phase of growth (culture absorbance (0.6). Nearly 80% of the media carnitine became cell associated by the time the culture reached 0.4 absorbance. From the value in Table 4, the cell dry weight of a 100 ml culture is 53 mg at 0.4 absorbance. At this absorbance, the cells contained 7.0 pmol carnitine/g dry weight or 1.4 pmol/g wet weight assuming 80% water content. This concentration is 3500 times the carnitine concentration in cells grown without carnitine (see Table 7). During the exponential phase of growth about half the cell-associated carnitine was esterified. Since all the carnitine was extracted by boiling the cells 3 minutes, it was not tightly or covalently bound to any macromolecules. 92 Figure 13. Determination of the Km for acetleoA of T. bovina CAT. CAT was assayed in crude extracts in Buffer I as described in the Methods and the Km V max calculated from the Lineweaver-Burk plots shown. A, cells grown with 5 pM carnitine; 0.79 mg protein/ml. Km =67 pM. B, cells grown without carnitine; 0.85 mg protein/ml. Km= 63 uM. Each assay received 5 pl of crude extract. 93 l0.88~ 8.I6‘ 5.44- 2.72; IO.881 1/v (pmovmh/mn' 5.44« 2.721 1 l .62 0'4 .0'6 .68 VS, Aceiyl CoA WM)“ Figure l3 94 Figure 14. Determination of the Km for L-carnitine. CAT was assayed in crude extracts of I. bovina in Buffer II as described in the Methods. The acetleoA concentration was 0.4 mM while the carnitine concentration was varied as shown. A, cells grown with carnitine; each assay received 5 pl crude extract, 1.29 mg protein/ml. Km = 0.30 mM. B, cells grown without carnitine; each assay received 10 pl crude extract, 0.63 mg protein/ml. Km = 0.67 mM. 95 I/ V (pmol/min/mII' 1 1 1 1 IO 20 30 40 VS, CARNITINE (mi/1)" Figure l4 96 Figure 15. Uptake and metabolism of carnitine in I. bovina. Cells were grown in 100 ml of media with 5 mM L-carnitine and the cell associated carnitine measured at various times after inoculation as described in the Materials and Methods. The results are expressed on the basis of the amount of carnitine in the whole culture. '01 lb CARNITINE, pmol io 97 CELL CARNITINE a . ESTERIFIED CARNITINE L 1 1 1 1 1 I .2 3 4 5 6 Figure l5 CELL DENSITY (ABSORBANCE) 98 TABLE 12. PERCENT OF ACYLCARNITINE AS ACETYLCARNITINE IN T. bovina. CARNITINE, pmol % ESTERIFIED AS EXPERIMENT TOTAL ESTERIFIED ACETYL- ACETYLCARNITINE 1 3.7 1.2 1.2 100 2 4.2 1.5 1.4 93 Cells were grown in 1 L cultures with 5 pM L-carnitine, harvested in the exponential phase of growth, extracted with perchloric acid and assayed for total, free and acetylcarnitine as given in the Materials and Methods section. The yeast dry weight was 521 and 530 mg in experiments 1 and 2 respectively. 99 Due to the substrate specificity of CAT acetyl-, propionyl-, and/or isobutyrylcarnitine were the only likely acylcarnitines formed jg yiyg. The data in Table 12 show that, when assayed enzymatically and specifically for acetylcarnitine, essentially all of the esterified carnitine in extracts of I. bovina is acetylcarnitine. Acetylcarnitine and the synthesis of N-acetylglutamate Besidesforming citrate in the TCA cycle, acetleoA participates indirectly in the formation of arginine in yeast and fungi (163). Coincidently, arginine stimulates I. pgyigg growth (214). Therefore, the ability of acetylcarnitine to act as the acetyl-donor for N-acetylglutamate synthase was investigated. This enzyme was assayed by incubating cell-free extracts with acetylcarnitine and [U-14C]glutamate (see ref. 215) but N-acetyl[14C]glutamate was not formed even when acetylcarnitine was replaced by acetleoA. Another possibility is that CAT functions in the cell to transfer the acetyl group from N-acetylornithine to glutamate but again, when N-acetylornithine was used in the assay for N-acetylglutamate synthesis, no N-acetyl[14C]glutamate formed. Utilization of [14C]acetylcarnitine by growing yeast Since acetylcarnitine was the only acylcarnitine detected in I. .pgyigg, its metabolic fate was determined. [1-14C]acetylcarnitine was synthesized and added to cultures of the yeast growing in the absence of carnitine. For comparative purposes, [1-14C]acetate was added to identical cultures. It has already been shown that I. pgyigg does not synthesize significant quantities of carnitine and thus [14C]acetate 100 Figure 16. Incorporation of [14C]acetylcarnitine into growing yeast cells. Cells were grown in basal media lacking L- carnitine but containing in A, 500 nmol of10. 5 Ci/mol [14&]acetylcarnitine or in 8,500 nmol of 1.46 Ci/mol [14C]acetate. C was measured in samples of the media and then each flask was inoculated with 40,000 cells. Cell density was monitored by turbidity measurements at 600 nm and the C distribution determined at the indicated points. The culture 14C was measured directly on a 1 ml sample of the culture. Another 1 ml sample of the culture was placed into a boiling water bath for 3 min, cooled, and centrifuged to remove debris and the 1 C in the supernatant fluid measured. Cell associated1 C was measured after filtering a 2 ml sample of the culture through a Whatman GF/C glass fiber filter using a Millipore apparatus. The cells and filter were assayed together for C. The filtrate C was a so measured and its value subtracted from the amount of C released by boiling the culture to obtain a measure of the cell associated extracted by hot water. All values are expressed as a percentage of the 4C present in the cultures at the time of inoculation. At least 24 h elapsed before the cultures reached 0.1 absorbance after which only 4 and 6 h of incubation time were required for the cultures to reach 0.5 absorbance in A and B respectively. lOl 100 A.E4C]acetylcarnitine 80 _ Culture 60- Cell Associated 4o 20 Hot Water 4_E xtract A O 4 1 1 1 1 .00 B. fi‘dacetate + 80- Cell Associated Percent of PC] Present at Inoculat1on 20 Hot Water ‘ Extract #8.. O t 1 1 1 1 I t 1 2 3 4 .5 Inoculation Cell Density (Absorbance) Figure l6 102 serves as a control. Figure 16A presents the data for the uptake of [14CJacetylcarnitine as a function of cell density over the exponential phase of growth while Figure 168 presents the results obtained with [14C]acetate. The figure indicates that a substantial portion of the 14C from both acetylcarnitine and acetate were taken y the cells during the period of analysis. The percentage of cell associated 14C in this figure was calculated relative to the amount of 14C added to the culture. Due to the small decrease in total 14C in the cultures during the course of the incubations, somewhat higher percentages for cell associated 14C would be obtained if they were calculated relative to the 14C in the culture at the time of measurement. At any given cell density, a slightly greater quantity of [14C]acetate was taken up than was [14C]acetylcarnitine. From other data it was calculated that acetylcarnitine stimulated the yeast growth rate to the same extent as carnitine. The cultures represented in Figures 16A and B took 24 and 28 h respectively to reach cell densities having an absorbance of 0.1; subsequent growth to 0.5 absorbance occurred within 4 to 6 h respectively. Thus very little 14C was lost from the cultures during the period when most of the cell mass was produced and during which other studies showed the amount of glucose consumed increased from 6% to nearly 60%. These results indicate that a significant portion of the acetylcarnitine was not oxidized to gaseous C02 during the incubation period. Moreover, very little of the cell-associated 14C was extracted by hot water indicating that [14C]acetate and [14C]acetylcarnitine were metabolized by the cell and that the 14C was incorporated into cellular components. 103 Distribution of 14C among cellular components Homogenates prepared from the cultures shown in Figure 16 were separated into cell walls, lipids, and acid-insoluble protein plus nucleic acids. The term cell walls refers to all the cell debris sedimenting at 700 x g in 2 min. A large amount of 14C derived from [14CJacetate was recovered in this cell wall pellet, see Table 13, whereas this fraction was poorly labeled by [14C]acetylcarnitine. Table 14 shows that a combination of 6 N KOH and 1% sodium dodecylsulfate are required to completely extract the 14C in the cell wall pellet from cells labeled with [14C]acetate. In other experiments, the proportion of 14C in the cell wall fraction varied with the preparation, probably due to the heterogeneous nature of this fraction. As expected, very little 14C was found in the acid soluble fraction of either sample after denaturing the homogenates with 6% HCl04, see Table 13. The lipids extracted from the acid insoluble pellets were labeled with 14C especially from cells incubated with [14CJacetylcarnitine, see Table 13. Samples of these lipid extracts were analyzed by TLC on silica gel and as shown in Figure 17, both [14C]acetate and [14]acetylcarnitine contributed 14C to the 3 major classes of lipids, i.e., polar, non-polar, and acidic lipids. Since O-acetyl esters are hydrolyzed by 0.1 N KOH, the lipid-extracted protein was treated with this reagent to determine the extent to which cell protein was O-acetylated. Almost none of the 14C remaining in the lipid-extracted protein pellet was solubilized by 0.1 N KOH in the culture labeled with [14C]acetate while 25% of the label derived from [14C]acetylcarnitine that was in this pellet was solubilized by 0.1 N 104 TABLE 13. DISTRIBUTION OF 14C IN CELLS GROWN WITH [14CJACETYLCARNITINE % of Total L14C1acetate [14C]acetylcarnitine Cell associated 98 83 700 x g cell wall pellet 42.7 2.6 Water soluble 2.5 4.7 Soluble in chloroform/methanol 15.8 34.4 Soluble in 0.1 N KOH 0.4 25.6 Insoluble in 0.1 N KOH 11.1 4.2 Celli were grown in 100 ml Of media containing 5 pM [1-14C]acetate or [1-1 C]acetyl-L-carnitine. Specific actiylties were 2 pCi/pmole for [1-14CJacetate and 0.48 pCi/pmole for [1- C]acetyl-L-carnitine. Cells were fractionated and analyzed for 14C as described in Methods. TABLE 14. SOLUBILIZATION OF 14C FROM [14CJACETATE-LABELED CELL WALLS SolubilizingpAgent % Soluble 0.1 N KOH 24.4 6 N KOH 36.5 1% SDSa 39.1 The cell wall pellet from cells grown with [14C]acetate (see Table 13) was treated in sequence with the solutions shown and the percentage Of 14C removed each time with the supernatant fluid after centrifugation Of the sample reported. The pellet was incubated 30 min at 65°C with 0.1 N KOH and 20 h at 65°C with 6 N KOH. aSodium dodecylsulfate in 0.25 M phosphate buffer, pH 8.0. 105 Figure 17. Distribution of 14C in cell lipids. Cell lipids were isolated as indicated in Table 13 and separated by TLC on silica gel developed with petroleum etherzetherzacetic acid, 70:30:1. Cholesterol and oleate were run simultaneously as shown. Condiolipin, which remained at the origin, and olive oil triglyceride, which migrated with Rf>0.8, were run separately (not shown). Lipids were located by exposing the plates to iodine after which segments of silica gel were removed to scintillation vials and analyzed for 14C as shown above each chromatogram. The numbers above the segments represent the percentage of total 4C recovered. A, cells grown with L 4C]acetylcarnitine; plate developed 30 min. B, cells grown with [14C]acetate; plate developed 2.5 h. 106 A. 48.7 1.0—22.6 \\ - ::E \\ IESJQr §§§$b 93 05— S 21 \ \ 9.4151 4‘ § \ 4 S\ 1111'.- o 000 O 00 Oleate- 0 Cholesterol— 0 LL, 1 LI 1 I 1 I 1 II I, O 0.2 0.4 0.6 0.8 Rf 8. ° 2.0r- \ ‘5 12.9. 1.5— a 2 Lol- J_6_5_, £1. ‘- . \ s 05— a N s is . \ ‘ C 11 \\ \T \ O Lipids- 0 000 O 00 Cholesterol— 0 ()kyome ::___L_ 1 .41 1 U‘IIIII’ I l J J O 2 .4 .6 .8 Figure l7 107 KOH. Although the 14C solubilized by this treatment was presumed to be derived from O-acetylated cell components, it did not CO-Chromatograph on paper with authentic [14C]acetate. The distribution Of [14CJacetylcarnitine in this experiment was markedly different from that produced by [14C]acetate. About 85% of the cell-associated [14C]acetylcarnitine was acid precipitable with one-third of the label being found in the lipids fraction. Since lipids are easily extracted, the amount of acetylcarnitine incorporated into lipids was determined. A series Of cell cultures were grown to an absorbance of 0.2, treated 2 h with [14CJacetate, [14C]acetate + carnitine, or [14C]acetylcarnitine and the cells harvested. The cells were saponified in ethanolic KOH at 80°C until the ethanol evaporated. This basic solution was extracted with petroleum ether to remove sterols and then it was acidified to pH <2 and the saponifiable fatty acids extracted with petroleum ether. The material remaining after ether extraction was treated with warm water and insoluble debris was removed by centrifugation. The data in Table 15 present the results Of two experiments that were averaged together. A negligible amount of 14C remained insoluble after the ether and aqueous extractions. Most Of the 14C derived from acetylcarnitine appeared in the ether extracted aqueous fraction although a significant amount of 14C was recovered in the saponifiable fatty acids fraction. In contrast, [14C]acetate mostly labeled sterols, i.e., the non-saponifiable lipids extracted from basic solution. The saponifiable fatty acids fraction is not the predominantly labeled fraction although [14C]acetylcarnitine contributes more acetyl units to the fatty acid pool than does acetate. The combination [14C]acetate + carnitine 108 .eeueweemme ppee eEeeee s_o>_eooemoc we.mm eea we.~m eowee we eeeoom oeo e_ see eeH x eee.H eea oeoe_coexo omc_w as» cw sec moH x mwm.o ee>weeec meeeupee eeueegu eewuweeeewxuee_oooemoc ae.me eea we.wm ;o_;z we eeooom one e. zae eeA x em.N eea beoe_coexo oee e_ zae eeH x oe.~ ee>weeeL megeypee eeueeeu eueueee ecu mpeemeeeec eewe> :eem .meeguez ece m_ewgepez cw eeeweewwe me u Lew eOpreee ece eewwweeeem ego; m~_ee any news; Lepwe ; N Low 2: w.m we :ewuecpeeeeee e Wm eeeee eewuwegeepxpeeemueflg Le eueueeemuefiu Lesuwe saw: eeueeeecw cusp eee N.o we meeeeeemee :e eu ecwpwceee peespwz ezegm mew: mwpeu .ewceuee Pwee eewwweeeem .eweewem o.mw N.Nm ~.wm Lowe: .eeueecuxe Lezum . eewue_em ewee EeLw e.eH e.e~ e.eH eoooacoxo mewewe eo_w_eoeam :ewuepem ewmee Eecw 8.4 e.AH m.em eoooecexo 88.8.4 eo_ww=oeam oe_o_ecaopaeoo19 A a L 3:: '3 m 214113 < - .. o -I 5 .J a: 4 “J , Lu _J x m x __, z 5 ‘>‘ 3 l J i l 1 1 L l l 500_ 5 10 IS 20 . 25 . 30 .35 4O 45 Retention Time, min 400- (39 o .‘L’ trE 11.1 9 g BOOP d :1 .1 0 :3 . _ m :to 200 .1 100 - («J l J l I l L l 1 1 L l 10 20 so 40 so 60 70 so 90 B a 9 x (D 4 _| g L l 1 l 1 l l l l 5 IO 15 20. 25. 30 .35 4O 45 1501. Retention T1me, min I40. u; (D >- 3 3 8 100- 1:11 vs) 801- : (.9 g _ _.1 El: :3 o 60- “1 WW 1 1 1 0 IO 20 30 4O 50 60 70 80 90 100 Fraction Number Figure 20 123 detection of 14C. For example, the area under the aspartic peak in Figure 20B was 1.5 x 107 units while the same peak still had an area of 1.3 x 107 units when only one-fifth as much sample was derivatized. Although the Chromatographic procedure used in Figure 20 effectively separated glutamate and arginine, leucine was not resolved from isoleucine or tryptophan, histidine was poorly derivatized and therefore inadequately resolved, and proline is inert to phthaldialdehyde derivatization. Therefore, a TLC system was used to specifically separate these amino acids. The protein hydrolysate from the [14C]acetylcarnitine treated cells was passed through a column of Dowex 1-x8 (acetate) resin to remove glutamate. The eluate was evaporated under reduced pressure, dissolved in a small volume of water and Chromatographed as indicated in Figure 21. Praline was identified by the Characteristic yellow color it produces with ninhydrin. Leucine was resolved from both isoleucine and tryptOphan, and lysine, histidine and proline were each individually isolated. The ninhydrin reactive spots were numbered as shown and used as a template for an identical sample Chromatographed similarly. The numbered spots were scraped from the second plate, placed into scintillation vials and counted for 14C. The radioactive amino acids were detected as shown in Table 19. Praline contained 14C as expected if it was derived from glutamate. Histidine was not labeled. Separation of isoleucine from leucine demonstrated that only leucine was radiolabeled. Lysine also contained 14C. The smear of radioactivity in spots 4-6 is attributed to the imperfect separation of arginine in this region of the plate by the TLC solvent systems selected. TLC analysis of the [14C1acetate grown 124 igure 21. TLC analysis of radioactive labeled amino acids derived from . bovina cell proteins. A sample (2 pl) of the [14C]acetylcarnitine abeled protein hydrolysate (described in the legend to Figure 20A) freed f glutamate by treatment with Dowex 1-X8 (acetate) resin was applied to he lower right corner of an AVICEL coated TLC plate. It was developed n the first direction 3 h with isopropanol, formic acid (88%), water 160:9:39) (IPF). It was developed twice in the second direction for 3 h sing pyridine, amylalcohol, water (35:35:30) (PA). Guide strips with tandard amino acids were run as indicated. The amino acids were isualized using ninhydrin spray reagent. The following spot numbers orrespond to: l, histidine; 2, lysine; 9, proline; 14, isoleucine; 15, euc1ne. 125 mOOu m- m- We... Figure 21 126 TABLE 19. DISTRIBUTION OF [14CJACETYLCARNITINE DERIVED RADIOACTIVITY IN AMINO ACID FRACTIONS SEPARATED BY TLC TLC Fraction ‘DPM TLC Fraction (DEM 1 (HIS) 0 9 (PRO) 264 2 (LYS) 481 16 o 3 217 11 o 4 184 12 0 5 651 13 8 6 104 14 (ILE) 42 7 16 15 (LEU) 438- 8 25 A sample (2 pl, 5240 DPM) of the glutamate free protein hydrolysate derived from [14C1acetyl- carnitine treated cells was analyzed by TLC as described in the legend to Figure 21. Some of the amino acids were visualized by spraying lightly with ninhydrin but all 15 numbered spots were removed from the plate using Figure 21 as a template and tgansferred to scintillation vials for detection of 1 C as shown. The numbered TLC fractions exactly correspond to those shown in Figure 21 with the amino acids of interest identified in parentheses. 127 cells' protein hydrolysate confirmed that leucine and lysine were the only amino acids containing measurable levels of radioactivity. Glutamate is derived from the TCA cycle via citrate and 2-Oxoglutarate. If the TCA cycle is fully Operational in this yeast, then the a-carbon of glutamate should be labeled. Glutamate was isolated by anion exchange Chromatography from the hydrolyzed protein sample labeled with [14CJacetylcarnitine and the glutamate decarboxylated with glutamate decarboxylase. The liberated C02 was collected in 1 M hyamine which when analyzed did not contain detectable levels of 14C02. These results demonstrate that acetylcarnitine contributes carbons for the synthesis of amino acids. Glutamate, proline, and arginine form a family of amino acids in E. 9911 labeled by radioactive acetate (222) because of their synthesis from a common precursor, 2-Oxoglutarate derived from the TCA cycle and thus, ultimately from acetleOA and oxaloacetate. The TCA cycle reactions are confined in the matrix of mitochondria in eucaryotes and so in I. bgyigg acetylcarnitine must enter the mitochondrial pool of acetleoA whereas acetate does not. Certainly no evidence has been obtained with I. pgyigg to discourage the view that acetylcarnitine facilitates acetyl group transfer across acleOA impermeable membranes and it is proposed here that acetylcarnitine shuttles acetyl groups into the mitochondria. If this is true, then CAT must be associated with mitochondria. 128 INTRACELLULAR LOCALIZATION OF CAT Evidence for membrane bound (particulate) CAT It had been Observed earlier that during gel permeation Chromatography of crude extracts (Sephadex 6150), more than 50% Of the CAT activity eluted in the void volume. In addition, the duration of cell homogenization did not affect the distribution of CAT during differential centrifugation. Subsequent experiments demonstrated that the sedimentation behavior of CAT depends on the phosphate concentration in the homgenization buffer, see Figure 22. The proportion of CAT in a 27,000 x g pellet fraction was about 50% when the phosphate concentration in the homogenization buffer was less than 50 mM, but less than 5% at 200 mM phosphate. Evidence for compartmentalization of CAT is presented in Table 20. Cells were homogenized for 5 sec in a low phosphate concentration sorbitol buffer isotonic for yeast mitochondria which were collected by centrifugation at 20,000 x g for 10 min and assayed for CAT. Greater than 50% of the extracted CAT was particulate even though most Of the mitochondrial matrix enzyme, citrate synthase, remained soluble. Virtually none Of the cytosolic enzyme glucose-6-phosphate dehydrogenase was associated with the 20,000 x g pellets. Traces of malate dehydrogenase were found in the particulate fraction but 98% Of the activity was soluble. Catalase, fumarase, NADH dehydrogenase, a-ketoglutarate dehydrogenase and isocitrate dehydrogenase (NADT-linked) were not detected. 129 Figure 22. Effect of phosphate concentration on sedimentation behavior of CAT. Crude extracts of I. bovina were prepared by the usual procedure (see Methods) in phosphate buffers at the indicated phosphate concentrations. CAT was assayed in the crude extract and then the extract was centrifuged 10 min at 27,000 x g and the supernatant fluid removed. The pellet was resuspended in buffer and assayed for CAT. CAT was assayed in Buffer II (see Methods). CAT, pmol/min 4s N 130 L_. Crude fxtroc/ 27000 x g Pellet i I 1 1 50 I00 I50 200 [Phosphate], mM Figure 22 131 TABLE 20. CAT DISTRIBUTION IN CRUDE EXTRACTS Citrate Glucose-6-Phosphate CAT Synthase Dehydrogenase pmol/min (%) 500 x g supernate 0.96 (100) .100 (100) .350 (100) 20000 x g supernate 0.44 (46) .079 (79) .320 (91) 20000 x g pellet 0.53 (55) .012 (12) .004 (1) Cells were homogenized for 5 sec with glass beads as described in the Materials and Methods. CAT was assayed in Buffer II supplemented with 0.1% (v/v) Triton X-100. Subcellular fractions were isolated by differential centrifugation. All centrifugations were performed for 10 min in a Beckman J2—21 centrifuge with the pellets being resuspended in the homogenization buffer. The values in parentheses represent the percent of the enzyme activity in the fraction. 132 Isopycnic density gradient analysis of CAT When a cell homogenate (>95% of cells broken) prepared in 50 mM phosphate buffer was subjected to isopycnic centrifugation on a linear, 10-40% sorbitol gradient, more than 87% Of the CAT activity in the gradient was particulate, see Figure 23. This figure shows that CAT has a bimodal distribution with peak activities at equilibrium densities Of 1.13 and 1.09 g/cm3 on either side of the ribosomal protein peaks. AcetleOA hydrolase and glucose-6-phosphate dehydrogenase are located exclusively in the soluble portion of the gradient. A small but consistently measurable fraction of the total CAT activity overlapped into the soluble region. Specific activities of 4.4 and 9.8 pmol/min/mg protein for the lower and higher density CAT peaks respectively were Obtained. These values represent, respectively, a 14-fold and a 31-fold increase in the enzyme activity compared to that in the crude extract. The distribution of some other enzymes in cell-free homogenates are also shown in Figure 23. AcetleoA synthetase migrates very nearly like a soluble enzyme as did NADPH-cytochrome C reductase although NADPH cytochrome C reductase was also present in low but measurable quantities near the bottom of the gradient. The higher density NADPH-cytochrome C reductase activity was not detected without adding sodium azide to the assay. The yeast mitochondrial membrane marker enzyme, NADH dehydrogenase, and the plasma membrane marker enzyme, a—glucosidase (maltase), were not detected. NADPT-linked isocitrate dehydrogenase was detected and it remained in the soluble region. A portion of the Citrate synthase also remained in the soluble region of the gradient indicating that mechanical disruption broke inner mitochondrial membranes. However, there is a second peak of Citrate synthase, note 133 Figure 23. Isopycnic sorbitol density gradient analysis of CAT in extracts from mechanically disrupted I. bovina cells. Cell homogenization, gradient purification and enzymatic analysis were performed as described in the Materials and Methods section. The absorbance ratio is the ratio of the 260 and 280 nm absorbances. G6PDH, glucose-6-phosphate dehydrogenase. All enzyme activities are expressed in pmol/min. 134 28m $0556 mEeOB 5.28: 9: 522%?» 859034619 22:20.9 0. 5 1 2 5 _ 4 4 _ _ I m m 1 b “m. N n .m 1.. C o m MM w w m .4, l. o. 5 2 w w. r. _ ._o _ .1_ - . O O 2 O. m. 05296 . m 0. m O m O L L w + «855% emeweeeem ewe-e6»: % m. 48.28%... o 2.9526 48.38,}; Ioammxl +£94.21 Figure 23 135 fraction 16, reminiscent Of a novel Citrate synthase particle reported previously (see ref. 217). It was found by gradient analysis of other cell homogenates that media carnitine did not alter the CAT distribution seen in control cells. High concentrations of phOSphate solubilize both the microsomal acetleoA synthetase in S. cerevisiae (218) and CAT in mammals (46,80,117). When I. bgyigg was homogenized in 0.2 M phosphate buffer and then analyzed as described above by isopycnic density gradient centrifugation, see Figure 24, CAT remained mostly particulate. A typical amount of CAT activity was extracted from the cells but most of the activity migrated into the gradient as a peak at 1.07 g/Cm3 well separated from the soluble enzyme markers. In another gradient (not shown) the higher density peak fractions of protein, CAT and NADPH-cytochrome C reductase were negatively stained and examined by eleCtron microscopy. The NADPH cytochrome c reductase fraction (D420=1.163) contained a large number Of small vesicles with diameters less than 0.2 pm. The CAT fraction (O420=1.138) contained both small and large vesicles with diameters approaching 1 pm, see Figure 25. The protein peak (D420=1.120) contained only large, 1 pm diameter vesicles. Association of CAT with mitochondria Although these results indicated CAT was primarily a particulate enzyme in I. pgyigg, the cellular origin of this material was not identified with any Of the marker enzymes assayed. The fairly large vesicles observed with the electron microscope suggested the particulate material was membrane ghosts of mitochondria. The homogenization 136 Figure 24. Effect of high phosphate concentration on the isopycnic density of CAT. Crude extracts were prepared in 0.2 M phosphate buffer and centrifuged through a sorbitol gradient. CAT was assayed in Buffer II and the other analyses were performed as described in the Methods. 136R 2E\c_E\_oE$ aoqz 6858.250 22:68. I m 5 0 5 ..1 7 6 O... P .0 253...: :90? To 8:5 2.55.5653 emceeoecgceo 2282a $18 6. 0c P. 8m 6. 4. 2 . 4 A I 20 200" L .1 O O 6 D 2.55.5655 2.4.0 I w 0 5 15 IO Fraction Number 5 Figure 24 137 68898.2: .538? SN weeez mew—.2: e 5.5 8556 use 3363328: 53:; eeewepm Dwipemme .mé :e .mewee 32323 2:. om 552% 35:26 me: pceweeem 36.5.23 e 5 Am 9:6: 33 mEoE mg; we 435:3 oweoaemw :e 92.52 eeBeeLw :3 3235.28 2: .AmEeB mg; n 845 eewuoecw 1:3 xeee we mseeemeeewe 5:85 .3 95m: 138 Figure 25 ’4 1am 139 procedure using glass beads is described as a method for preparing mitochondria from microorganisms but it obviously did not work for I. ‘pgyigg. Thus another method was selected to prepare intact mitochondria from these yeast, i.e., osmotic lysis of spheroplasts. A summary of the sedimentation behavior of CAT from spheroplast lysates is given in Table 21. In experiment 1, a significant portion of the Citrate synthase activity and CAT was pelleted by a relatively low force of 8000 x g. In experiment 2, about 20% of the initial Citrate synthase and CAT activities were not pelleted by centrifugation at 31,000 x g. The pellets contained only traces of the cytosolic marker enzyme in both experiments. Since a large percentage of the mitochondria were left intact after osmotic lysis of the spheroplasts, they were purified by iSOpycnic density gradient analysis as shown in Figure 26. CAT has an identical distribution profile to citrate synthase throughout the gradient with the major peaks for both enzymes located at a density of 1.21 g/cm3, a density typical of yeast mitochondria. Few mitochondria were broken in this preparation as indicated by the extremely low amount of Citrate synthase located at the top of the gradient with the soluble glucose-6-phosphate dehydrogenase. The recovery of Citrate synthase was 82% and the recovery of CAT was 93%. The lack of glucose-6-phosphate dehydrogenase activity in fraction 2 but the presence of CAT, Citrate synthase and NADPH-cytochrome C reductase in this fraction suggests that it may contain spheroplasts that have lost their soluble, cytoplasmic components but not their intracellular organelles. The enzyme activities located near the top of the gradient are probably due to a heterogenous population of membrane particles. AcetleoA synthetase is shown to be a 140 .Lewwe ewe» esp :_ eeeeeememee mcwee mum—wee we» saw: emewwcueee Hmuwe eeExeem e cw ewe oH Lew eefleeweew wee: meewuemewweueee __< .eewpemewwgueee wewueecewwwe we eeuewemw wee: meewueecw Le_:__eeeem .Amomv .Hm.mm.xgzee we eesuee egg on mcweeeeee euewwem wxeeeee Seweem A>\zv RH saw: eepeeeu mepeEem :e vexemme we: :weuece .ooHIX eeuwcw A>\>v RH.o saw: eeueeEe—eeem HH Lewwem cw eexemme me: wmcanm ccaucwum wee maven ocwsm mmwcucozoouve .osz mammFQouao .op>u ME:_:o_me owemc_aov:m ..z.m ”crow ovumonopmxo .<=L»q .x>m mwpc>2L2q_ocwozamoca .ama m< mEANcmOQF>amoc .mown asp cw pcmuoeme m an op vm>m_Pmn mumsamogaocxa mewEmwsu we m>wpm>_cmu :oneaoum asp cu weave; =mu>smupmpwo< m>wau<= .m:_Fm> use acrosmp do m_mm;u:>mown mo anguma .mm we:m_d 159 @5384 9:8: O O) _ mc__o> limeemaseosxsxllnAmheufiémwéxlleoagemé-69A @ 992$ @ «8284 290E§9aom76 as 923.3050an ab 28588.29. :8 .2 0583 Figure 29 160 except for a small percentage in the cytosol, CAT has the same density distribution in a sorbitol gradient as citrate synthase. The data do not localize CAT to the inner and/or outer faces of the mitochondrial inner membrane. Instead, I. bgliga_CAT remains tightly bound to the mitochondrial membrane with less than 5% soluble activity in crude extracts. Mitochondrial outer membranes have an isopycnic density of 1.09 g/cm3 while ruptured mitochondria band at a density of 1.14 g/cm3 (223). Therefore, it seems that the two particulate CAT peaks in mechanically disrupted cells (see Figure 24) are derived from CAT bound to outer mitochondrial membranes at 1.09 g/cm3 and CAT bound to the membranes of ruptured mitochondria in the 1.13 g/cm3 region. In Figure 28, CAT is shown as an integral membrane protein with separate transporter proteins for carnitine and acetylcarnitine although a single translocase could perform both functions. These details require further experimentation. The discovery that carnitine is involved in the synthesis of amino acids is both new and novel and was never imagined prior to its discovery. Carnitine also affects the proportion of exogenous acetate that becomes incorporated into sterols or fatty acids. Carnitine stimulates the synthesis of glutamate and arginine rather than vice versa, thus explaining how glutamate and arginine stimulate growth without increasing the carnitine content of the yeast. It seems likely that 1. bovina is unable to synthesize carnitine but the yeast compensates by efficiently extracting carnitine from its environment. The yeast's carnitine transport system in conjunction with its exceptionally high levels of CAT help assure its survival. 161 T. bovina demonstrates that CAT and acetylcarnitine are not exclusively involved in fatty acid catabolism. In addition, the yeast system is now defined enough that its manipulation may provide new insight into how carnitine affects metabolism especially the compartmentation of acetyl group metabolism. 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USA, 47:2795-2798. 225. Bieber, L.L., R. Emaus, K. Valkner, and S. Farrell. 1982. Fed. Proc., in press. 226. Hollenberg, C.P., W.F. Riks, and P. Borst. 1970. Biochim. Biophys. Acta 201:13-19. 227. Kresze, G.B. and H. Ronft. 1981. Eur. J. Biochem. 119:573-579. 228. Cazzulo, J.J. and A.0.M. Stoppani. 1969. Biochem. J. 112:747-754. 229. Tracy, J.W. and G.B. Kohlhaw. 1975. Proc. Natl. Acad. Sci. USA, 72:1802-1806. 230. Holzer, H. and G. Kohlhaw. 1961. Biochem. Biophys. Res. Comm. 5:452-456. 231. Bergmeyer, H.U. and E. Bernt. 1963. In H.U. Bergmeyer (ed.) Methods of Enzymatic Analysis, pp. 324-327. Academic Press, New York. 232. Korff, R.W. 1969. In J.M. Lowenstein (ed.) Methods in Enzymology, Vol. 13, pp. 425-430. Academic Press, New York. APPENDIX I 174 ANALYTICAL BIOCHEMISTRY 119, 261—265 (I982) Preparation of Radioactive Acetyl-L-Carnitine by an Enzymatic Exchange Reaction R. EMAUS AND L. L. BIEBER Michigan State University. Department of Biochemistry, East Lansing, Michigan 48824 Received May 18, 1981 A rapid method for the preparation of [1-"C]acetyl-L-carnitine is described. The method involves exchange of [l-"C]acetic acid into a pool of unlabeled acetyl—L-carnitine using the enzymes acetyl-CoA synthetase and carnitine acetyltransferase. After isotopic equilibrium is attained, radioactive acetylcarnitine is separated from the other reaction components by chro- matography on Dowex 1 (Cl') anion exchange resin. One of the procedures used to verify the product [l-"C]acetyl-L-carnitine can be used to synthesize (3S)-[5-"C]citric acid. Acetylcarnitine is found in many mam- malian tissues (1), but its metabolic fate has not been clearly elucidated. Isolated mito- chondria can oxidize acetylcarnitine (2). It can be formed outside the mitochondrion by peroxisomal beta-oxidation (3) or in the matrix of mitochondria via pyruvate oxida- tion (2,4), B-oxidation or oxidation of some amino acids. Acetylcarnitine can be ex- ported from mitochondria and possibly used in the synthesis of fatty acids or biological acetylations (5). Carnitine acetyltransferase activity is associated with mitochondria, per- oxisomes, and microsomes (6-8). Determin- ing the fate of radioactive acetylcarnitine in tissues and cell preparations should help de- termine its function(s). However, a source of radioactive acetylcarnitine is required for such studies. Previous methods for the preparation of labeled and unlabeled acetylcarnitine relied upon their net synthesis from carnitine and an activated acetyl group. The product ob- tained by enzymatic methods (9) is contam- inated with free carnitine unless the reaction is forced to completion with a large excess of reactants. This also is expensive when ra- dioisotopes are used. The same technique is needed to ensure high yields of acetylcar- nitine synthesized by chemical procedures 261 (10,11) but even then the chemical method is difficult to use when only a few milligrams of products of high specific radioactivity are desired. These problems are avoided by an alternative approach to the synthesis of ra- diaoctive acetylcarnitine that is analogous to the method of enzymatic synthesis of [7-32P]ATP from ”P, and ATP via glycer— aldehyde-3-phosphate dehydrogenase and 1,3-diphosphoglycerate kinase (12). In this approach, radiolabeled acetate is exchanged into the unlabeled acetyl group of acetyl- carnitine by coupling two enzymatic reac- tions as indicated below (* denotes a radio- active residue). :acetate + CoASH + ATP H [l] acacetyl-CoA + AMP + PP, acetyl-L-carnitine + tacetyl-CoA H [2] eacetyl-L-carnitine + acetyl-CoA Both reactions have equilibrium constants near one. At isotopic equilibrium, nearly all of the inexpensive radioactive isotope, ace- tate, exchanges into the product, acetyl-L- carnitine, when a large pool of acetyl-L-car- nitine is used with a small pool of high spe- cific activity radioactive acetate. In addition, contamination of acetylcarnitine with free 0003-2697/82/020261-05$02.00/0 Copyright C" I982 by Acadchc Press. Inc. All rights of reproduction In any form reserved. 175 262 carnitine is kept to a minimum by starting with analytically pure acetyl-L-carnitine as a reactant and using catalytic amounts of CoASH to effect the exchange reaction. A method is described herein using [1- "'C]acetate, although [3H]acetate. [2-‘4C] acetate. or [1.2-”C]acetate would serve equally well. MATERIALS AND METHODS Materials. Sodium [l-"C]acetate (58.3 Ci/mol) was obtained from New England Nuclear Corporation (Boston, Mass). CoASH was obtained from PL Biochemicals (Milwaukee, Wis.) The following chemicals and enzymes were obtained from Sigma Chemical Company (St. Louis. Mo.): glu- tathione (reduced), NADT. ATP. Trizma— HCl, L-malate, citrate synthase (85 U/mg), malic dehydrogenase (7150 U/mg). and car- nitine acetyltransferase (85 U/mg). Acetyl- L-carnitine hydrochloride was kindly pro- vided by Otsuka Pharmaceutical Company, (Naruto, Tokushima. Japan) and was judged to be >98% pure by assay of both free and total carnitine content. Acetyl-CoA synthe- tase was purchased from Boehringer-Mann- heim (West Germany. 0.95 U/mg). The Dowex resins were obtained from Bio- Rad Laboratories (Richmond, Calif). Other reagents were from Mallinckrodt (St. Louis, Mo.). Preparation of [1-"C]acetyl-L-carnitine. The final reaction volume of 1.0 m1 con- tained: acetyl-CoA synthetase, 1 mg: sodium phosphate buffer. pH 7.6. 150 pmol; diso- dium ATP, 17.5 pmol: reduced glutathione. 2 pmol; CoASH. free acid, 0.3 pmol; MgClg, 6.6 pmol; acetyl-L-carnitine, 3.8—42 pmol (added from a neutralized solution): [14C]- sodium acetate, 0.34—0.84 pmol. The ex— change reaction was initiated by adding about 2.5 units of carnitine acetyltransferase and the reaction incubated 25 h at room tem- perature (22°C). The reaction was termi— nated by placing the reaction mixture into a boiling water bath for 3 min. Denatured EMAUS AND BIEBER protein was removed by centrifugation. [”C]Acetyl-L- carnitine was purified by passing the solution through a l X 4-cm col- umn of Dowex 1-X8 (Cl‘ form. 100—200 mesh) resin, washing the column with 5 ml water, and collecting the product [”Cl- acetylcarnitine in the eluate. This solution was used to demonstrate that ['4C]acetate had exchanged into acetylcarnitine as de- scribed below. This solution can be stored frozen (‘80°C) after adjusting the pH to 6. The progress of the reaction was followed by removing 2-al samples of the reaction mixture and applying them to 5 X 25-mm columns of the Dowex. The columns were washed twice with 0.5 ml of water, the total eluate collected in scintillation vials, and the radioactivity counted. Verification that ["C]acetale is in ace- tylcarnitine. Cation-exchange column chro- matography was performed as described (10). A 0.2-ml sample of the ["C]ace- tylcarnitine solution (2.886 X 10'5 dpm) was diluted to 2 ml with absolute ethanol, acid- ified with concentrated HCl to 0.05 N HCl, and applied to a 5 X 25-mm column of Dowex 50W-X8 (l-I+ form. 100—200 mesh) resin. The sample was washed onto the col- umn with 2 ml of 0.01 N HCl in 20% ethanol and eluted with 8 m1 of l N NH4OH in 20% ethanol. The eluate was collected in 1 ml fractions in scintillation vials, combined with scintillation fluid, and counted. The radiochemical purity of the ["C]- acetylcarnitine was checked by thin-layer chromatography on silica gel G plates (sol- vent: CHCl3, CH3OH, 17% NH3; 222:1) de- veloped to 16 or 17 cm. Radioactivity on the plate was detected with a Berthold thin-layer chromatography scanner. Further verification that the ['4C]acetate had exchanged into the acetyl residue of acetylcarnitine was obtained using an en- zyme—coupled spectrophotometric acetylcar- nitine assay (l3). The formation of Citrate from acetylcarnitine in this assay is coupled to the reduction of NADT, the amount of NADH produced being proportional to the 176 RADIOACTIVE ACETYL-L-CARNITINE amount of acetylcarnitine. To show that the "C was actually associated with acetylcar- nitine, citrate was isolated and its l"C-con- tent measured. The assay tube contained 0.5 ml of 200 mM TRIS—HCl. pH 7.8, 60 mM L-malate, 4 mM NazEDTA; :0.2 ml of 2.5 mM NAD“; 0.2 ml of 1.5 mM CoASH in a 10 mM solution of reduced glutathione; 1.0 ml water; 10 [1.1 citrate synthase; 5 a1 car- nitine acetyltransferase; l a] malate dehy- drogenase; and 75 a] of the ["C]ace- tylcarnitine solution (1.0637 X 106 dpm). A control tube contained 0.125 pmol ["Cl- acetate in 25 #1- The reactions were termi- nated by placing the tubes in a boiling water ' bath for 5 min, after which they were cooled and centrifuged to remove denatured pro- tein. The deproteinized solutions were com- bined with 1 ml of 8 mM citrate, pH 6.9. and a sample from each reaction mixture was Chromatographed on a 1 X 5-cm column of Dowex 1-X8 (Cl'). Acetate and citrate were retained by the resin and were eluted with an 80 ml, 0.01-0.075 N HCl linear gradient. Citrate was detected with acetic anhydride and pyridine (14) and the "C quantitated by scintillation counting of a small sample of each 1.9-ml fraction collected. Radioactivity was measured in 10 ml of a Triton X-100 based cocktail (15) and the dpm calculated by the channels ratio method of correction for quenching. RESULTS AND DISCUSSION A time-course study for the exchange of ["C]acetate into acetylcarnitine showed that within 25 h greater than 99% of the ["C]acetate exchanged into the large pool of acetyl-L-carnitine (Table 1). In the ex- periment shown, the ["C]acetylcarnitine had a specific activity of 0.476 mCi/mmol. Both the yield and specific activity of ["C]acetylcarnitine depend on the relative amounts of ["C]acetate and acetylcarnitine in the reaction mixture. In other experi- ments, ["C]acetylcarnitine has been pre- pared with a specific activity as high as 6.24 mCi/mmol. 263 TABLE 1 EXCHANGE OF [l-"C]ACETATE INTO ACETYL-L-CARNITINE [ l-"C]Acetyl- Incubation carnitine Percent time (h) formed (pCi) exchange 1.5 6.2 31 3.5 10.8 55 25 20.3 102 Note. The reaction contained 343 nmol [l-"C]acetate (58.3 uCi/amol) and 42 pmol acetyl-L-carnitine. Sam- ples were taken at the times indicated and assayed for ["]acetylcarnitine as described under Material and Methods. . In the radiochemical assay for carnitine (l6), acetylcarnitine is separated from ace- tyl-CoA by passing the reaction mixture over Dowex 1 anion exchange resin. Acetyl-CoA is retained by the resin and acetylcarnitine passes through. Phosphate was chosen to buffer the ["C]acetylcarnitine synthetic re- action mixture and glutathione was used because Dowex l binds both these com- pounds and also binds the ATP, AMP, PP,, and CoASH in the reaction mixture. This allows the [”C]acetylcarnitine to be sepa- rated by passing the deproteinized reaction solution through a column of Dowex 1 (Cl'). Controls have shown that Dowex 1 (Cl‘) retained greater than 99.8% of the "C in a solution of 0.0343 pmol ['4C]acetate in 0.2 ml water when the column was washed with either 1 ml of water or 1 ml of 0.1 M phos- phate buffer, pH 8.0. Greater than 99.5% of the 1"C was also retained by the resin when the reaction mixture lacking carnitine acetyltransferase was applied to and washed through the column. Thus the amount of "C in the Dowex 1 eluate represents the amount of ['4C]acetylcarnitine formed. The [”C]acetylcarnitine prepared as described above contains NaCl and MgClz, which are not removed by the resin. [“C]Ace- tylcarnitine could be separated from these salts by passing the solution through a col- umn of Dowex 50 (NH?) prior to column 177 264 2 Z a Q .. 2 4 .6 Rf FIG. I. Thin-layer chromatography of [”C]ace- tylcarnitine prepared by the exchange reaction de- scribed under Materials and Methods. A Z-pl aliquot of each of the following solutions were applied to a silica gel G plate: I, ["C]acetylcarnitine, 6.5 aCi/ ml; 11, ("C]acetate, 10 aCi/ml; III, acetyl-L-carnitine, 0.39 mmol/ml; and IV, D.L-carnitine-HC1, 1 mmol/ml. The plate was developed to 16 cm in CHC13, CHJOH, 17% NH,; 222:1, dried in air, and exposed to I; to visualize III and IV. The "C-labeled compounds (I and II) were measured with a thin-layer chromatography scanner. chromatography on Dowex 1 (HCO;) and evaporating the eluate containing acetyl-L- carnitine under reduced pressure at 50°C to eliminate (NH4)HC03.This procedure avoids extremes of pH that might otherwise hydro- lyze the ester linkage of acetylcarnitine. At pH values less than 4, acetylcarnitine has a net positive charge causing it to bind to a Dowex 50 cation-exchange resin. When a sample of the ["C]acetylcarnitine solu- tion was acidified, 98% of the l"C bound to Dowex 50 and subsequently eluted with ammonium hydroxide (data not shown). This behavior is typical of short-chain acyl- carnitines (10) and demonstrates that ["C]acetate had exchanged into the acetyl residue of acetylcarnitine. Thin-layer chro- matography of a sample of the ["C]- acetylcarnitine solution showed that the "C comigrated with acetylcarnitine as a single peak well separated from reactant ["C]- acetate (Fig. l). The amount of acetylcarnitine in the ["C]acetylcarnitine solution was also mea- sured by an enzyme-coupled assay that forms citrate from acetylcarnitine (13). One "C-labeled compound was formed from [”C]acetylcarnitine in this assay which eluted with carrier citrate (Fig. 2B). EMAUS AND BIEBER ["C]Acetate was unchanged in the control assay and its elution pattern was different from citrate (Fig. 2A). The trail of 1“C in Fig. ZB after the apparently complete elu- tion of citrate was due to the high specific activity of citrate in this sample. The data in Fig. 2A indicate that the trail of citrate could be chemically detected when more car- rier citrate is applied to the column. In the experiment, the amount of citrate produced was 0.364 pmol (determined by column chromatograph); the amount of acetylcar- nitine was 0.366 pmol (measured by the amount of NAD“ produced in the assay). Both these independently measured values agree to within experimental error with the calculated amount of 0.361 pmol of ["C]acety1carnitine present in the assay. These results also demonstrate that the assay could be used to prepare (35)-[5- l"C]citrate in nearly quantitative yield from ["C ]acetylcarnitine. nmol N __ __ §__ 6 ‘8 m e -- A 25 '9 8 B I). .2. . . 1: i Q) U I 1 2‘ 3°? '( u _ . ., I 2’ 20+ . I, 20 l : ,‘-' '0' "1L '0 "5‘ "is' '2'5’35 as Fraction Number FIG. 2. Dowex l elution profile of ["C]citrate formed from ["C]acetylcarnitine. ["C]Acetylcarnitine was converted to ["C]citrate as described under Materials and Methods. Samples of the boiled reaction mixtures were applied to Dowex 1 (Cl') columns and washed onto the column with 20 ml water (not shown). No more than 0.5% of the l‘C-label appeared in the wash. The samples were eluted with a linear HCl gradient. Sixty— drop (1.9 ml) fractions were collected and conductivity was measured on a type CDM3 radiometer. Copen- hagen instrument. (A) Z-ml sample of ["C]acetate con- trol reaction mixture; (B) l-ml sample (354.556 dpm) of the ["C]acetylcarnitine assay solution. 0, conduc- tivity; A. "C-label; O, citrate. 178 RADIOACTIVE ACETYL-L-CARNITINE The procedure for the preparation of ra- dioactive acetylcarnitine is rapid, quantita- tive, and simple. The data show that ["C]acetate is converted to [MC]- acetylcarnitine and that the ["C]acetyl- carnitine can be purified to greater than 98% radiochemical purity in a single step. Con- tamination of the radiolabeled acetylcarni- tine by free carnitine was minimized by us- ing a small, catalytic amount of CoASH in the reaction mixture. Another advantage of this enzymatic pro- cedure is that if D,L-acetylcarnitine is sub- stituted for the pure L-isomer, only the bi- ologically active L-isomer will be labeled. This is desirable because D,L-acetylcarnitine is cheap and readily available and it has been our experience that at equimolar concentra- tions the D-isomer does not effectively com- pete with the L-isomer. The method could be adapted as a general method for the synthesis of short-chain acyl-L-carnitines since other aliphatic short- chain radioactive acyl groups could be sub- stituted for acetate by using the appropriate thiokinase and acylcarnitine derivative. Pi- geon breast carnitine acetyltransferase is active with acyl residues up to 6 carbons in length (17). ACKNOWLEDGMENT Supported in part by Grant AM18427 from the Na- tional Institutes of Health. 265 REFERENCES . Marquis, N. R., and Fritz, I. B. (1965) J. Biol. Chem. 240, 2193—2196. . Bremer, J. (1962) J. Biol. Chem. 273, 2228-2231. . Kawamoto, S., Ueda, M., Nozaki. C., Yamamura, M., Tanaka, A., and Fukui. S. (1978) FEBS Lett. 96, 37-40. . Childress, C. C., Saktor, B., and Traynor. D. R. (1966) J. Biol. Chem. 242, 754—760. . Bressler, R., and Brendel, K. (1966) J. Biol. Chem. 241, 4092-4097. . Markwell. M. A. K., McGroarty, E. J., Bieber. L. L., and Talbert, N. E. (1973) J. Biol. Chem. 248, 3426-3432. . Bieber, L. L., Sabourin, P., Fogle, P. J., Valkner. K., and Lutnick. R. (1980) in Carnitine Biosyn- thesis, Metabolism and Functions. (Frenkel, R., and McGarry, J. D., eds), pp. 159-169, Aca- demic Press. New York. Valkner, K. J., and Bieber. L. L. (1981) Fed. Proc. 40. 1643. . Choi. 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