A Mitochondrial Carnitine Acylcarnitine Translocase System

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp , March 1975 A Mitochondrial Carnitine Acylcarnitine Translocase System [carnitine acylcarnitine transport/exchange diffusion/acyl(+)carnitine inhibition/fatty acyl transport] SHRI V. PANDE Department of Intermediary Metabolism, The Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, P.Q., Canada H2W 1R7 Communicated by Albert L. Lehninger, December 3, 1974 ABSTRACT Acetylation.of added (-)carnitine by heart mitochondria coupled to the oxidation of pyruvate in the presence of malonate was inhibited, apparently competitively, by long chain acyl(+)carnitines although the activity of carnitine acetyltransferase (EC ) itself was not affected. Mitochondria have been found to possess a translocase system that allows the transport of carnitine and acylcarnitines by exchange diffusion, and interaction with this transport appears to be the cause of long chain acyl(+)carnitine inhibition. These conclusions are based on the following observations: (a) exposure of intact, but not of denatured or disrupted, mitochondria to [14C]- carnitine led to retention of radioactivity with mitochondria that effluxed either in the presence of carnitine or acylcarnitines, or by sonication or freeze-thawing of mitochondria; (b) the [14C]carnitine efflux was saturable at low levels of carnitine or acylcarnitines, showed temperature dependence, and was more rapid with acyl(-)carnitines than with acyl(+)carnitines such stereospecificity was not noticeable with free carnitine; (c) long chain acyl- (+)carnitines inhibited the carnitine-carnitine exchange and higher concentrations of carnitine decreased this inhibition; (d) direct estimations showed the presence of endogenous (-)carnitine in mitochondria that effluxed by freezing and thawing of mitochondria; (e) the amount of total endogenous (-)carnitine present was not affected by prior exposure of mitochondria to (-)carnitine or acetyl(-)carnitine. These results indicate that the carnitine-dependent translocation of acyl groups across mitochondrial inner membrane involves the participation of a carnitine acylcarnitine translocase system. Carnitine, by the participation of mitochondrial carnitine acyltransferase(s), is known to facilitate the entry of fatty acids through the mitochondrial inner membrane. However, it is not yet clear exactly how acylcarnitine formation promotes fatty acid transport despite extensive research, as seen METHODS Rat heart mitochondria were prepared and oxygen consumption was determined as described (13). Loading of Mitochondria with [14C]Carnitine. To freshly isolated mitochondria (30-40 mg of protein) from rat hearts (from three or four rats) was added 200,ul of 200 mm mannitol-50 mm Tris- HCl (ph 7.4) containing 6 yg of rotenone, 10,ug of oligomycin, and 6,ug of antimycin A; and after suspension of the mitochondrial pellet, 10,ul of (i4) [carboxyl- '4C]carnitine (0.5 blmol, 2.75 jci) was added. After 4 min of incubation at 280, 30 ml of ice-chilled 200 mm mannitol-50 mm Tris HCl (ph 7.4) containing 2.1,ug of rotenone plus 1.ug of antimycin A per ml was added and the tubes were centrifuged at 00 for 3 min at 27,000 X g. The resulting pellet was rinsed twice with 15 ml of the above medium before it was suspended in 30 ml of the same medium. After the process of centrifugation and rinsing, etc., described above, was repeated, the pellet was suspended in ml of the medium used for washing of mitochondria. Efflux Incubations. Efflux was initiated (in duplicate, at 00) by the addition of mitochondria loaded with [14C]carnitine to a rapidly stirring (using 10 X 3 mm magnetic bar) solution having 200 mm mannitol, 50 mm Tris*HCl (ph 7.4), 0.5 jg of rotenone, 1.25 jug of oligomycin, and 0.25 jg of antimycin A, and other compounds (except in control) as described. Final volume was 250 jl. After 15 sec, the tubes were centrifuged at 00 for 90 sec in an Eppendorf 3200 centrifuge, and an aliquot of the supernatant was removed for the measurement of radioactivity. Percent efflux was calculated as follows: by the number of schemes put forward to explain the role of carnitine in fatty acid oxidation (1-9). Many of these schemes have postulated the existence of two different carnitine acyltransferases on the two sides of the mitochondrial permeability barrier (i.e., inner membrane) and have further assumed that such a barrier, although impermeable to CoA and acyl-coa esters, would be permeable to carnitine and acylcarnitines. While the former postulate has been amply supported, several investigators now believe that the mitochondrial inner membrane is as impermeable to carnitipe and acylcarnitines as to CoA and its esters (10-12). The results described here show that the mitochondrial permeability barrier is traversed by carnitine and acylcarnitines by a process of exchange diffusion. This transport is facilitated by- the presence of a translocase system in mitochondria. % Efflux - Supernatant dpm in experimental - supernatant dpm in control X 100 Total dpm in mitochondria - supernatant dpm in control Details of other methods are given in the legends. Stereoisomers of carnitine were from Mann Research Laboratories or General Biochemicals. Decanoylcarnitine esters were a gift from Otsuka Pharmaceuticals, Japan. Other acylcarnitines were synthesized (14) and had >95% ester content (15). (in) [Carboxyl-14C]carnitine was obtained from ICN, California. RESULTS AND DISCUSSION Although malonate is known to inhibit the citric acid cycle, the effect of malonate on pyruvate-dependent oxygen consumption showed that in intact mitochondria, malonate inhibited the pyruvate oxidase system as well. Thus, malonate lowered oxygen consumption to 1/12th that of the control (from 160 to 13, Fig. 1) while in theory, if the pyruvate ox- 883

2 884 Biochemistry: Pande Proc. Nat. Acad. Sci. USA 72 (1976) MITO. IImM ie 20 2.Omim PALMITOYL (+1 < FIG. 1. Effects of malonate, (-)carnitine, and palmitoyl(+ )- carnitine on the pyruvate-dependent respiration of heart mitochondria. 1.7 ml of air-saturated medium (230 mm mannitol, 70 mm sucrose, 20 mm Tris - HCl, 20 MM EDTA, 5 mm potassium phosphate, 2 mm ADP, ph 7.2, 280) was supplemented with 1 mm pyruvate. Mitochondria (1.0 mg of protein) and other compounds were added as shown. Numerals below the tracing, the rate of 0 consumption in natoms per min/mg of protein. Mito., mitochondria. idase system was unaffected, malonate should have decreased oxygen consumption to no more than 1/5th. Failure of CoA generation from acetyl-coa was the cause of inhibition because addition of (-)carnitine-which is capable of CoA regeneration in heart mitochondria (16)-stimulated pyruvate oxidation (Fig. 1). That (-)carnitine stimulation accompanied acetyl(-)carnitine formation is evident from Table 1. Unexpected were the observations, however, that the stimulatory effect of (-)carnitine was inhibited by low levels of palmitoyl(+)carnitine (Fig. 1) or decanoyl(+)carnitine (Table 1) in a specific manner. Thus, with a rise in the concentration of long chain acyl(+)carnitine, the (-)carnitine stimulation of pyruvate oxidation (with malonate present, Fig. 1) was steadily decreased until it was completely abolished; no further inhibition was seen thereafter by increase in TABLE 1. Effect of long chain acyl(+)carnitines on the stoichiometry of pyruvate-dependent conversion of (-)carnitine to acetyl(-)carnitine by rat heart mitochondria Additions (-)Carnitine-stimulated ratio A pyruvate: A oxygen: A acetyl(-)carnitine A. (-)Carnitine 1: 0.49: 0.95 B. + Decanoyl(+)- carnitine 1: 0.46: 0.95 C. + Palmitoyl(+)- carnitine 1: 0.49: 0.92 Mitochondria (0.96 mg of protein) were added to 1.8 ml of medium (as in Fig. 1) containing 1 mm malonate. In addition, 0.6 mm pyruvate, 3 mm (-)carnitine, and 6 pam long chain acyl(+)carnitines were also present, as appropriate. Incubations were for 15 min at 280. Oxygen consumption was determined polarographically (13). Pyruvate (17) and acetyl(-)carnitine (18) were estimated enzymically. In a control lacking (- carnitine, pyruvate and oxygen used were 136 and 69 nmol, respectively, and acetyl(-)carnitine formation was not detectable. Presence of 6 MuM long chain acyl(+)carnitines had no effect on these parameters. The (-)carnitine-stimulated pyruvate consumption in A, B, and C was 648, 419, and 299 nmol, respectively. PM H- FIG. 2. Effect of (-)carnitine concentration on the etimulation of pyruvate-dependent respiration of heart mitochondria in the presence of malonate and its modification by palmitoyl(+)- carnitine. Mitochondria (0.87 mg of protein) were added to 1.7 ml of air-saturated medium (as in Fig. 1) containing 1 mm pyruvate and 1 mm malonate. (-)Carnitine and palmitoyl(+ )- carnitine were added 2 and 3.5 min later, respectively. The values shown have been corrected for the rates observed in the absence of (-)carnitine. TABLE 2. Lack of long chain acyl(+)carnitine effect on carnitine acetyltransfera-se activity of rat heart mitochondria Enzyme activity as formation of the concentration of long chain acyl(+)carnitine. The inhibitory effect of long chain acyl(+)carnitines was not modified (not shown) by substitution of uncouplers for ADP, or by the presence or absence of Pi, nor did long chain acyl(+)- carnitines, even at much higher levels, affect the rate of respiration with pyruvate plus malate as substrates. The long chain acyl(+)carnitine inhibition of pyruvatedependent oxygen consumption in the presence of malonate and (-)carnitine accompanied a corresponding decrease in the conversion of (-)carnitine to acetyl(-)carnitine and pyruvate disappearance, so that the stoichiometry of the reaction (Table 1) was not affected by long chain acyl(+)- carnitines. Thus, long chain acyl(+)canitines appeared to be specifically inhibiting the formation of acetyl(-)carnitine from added (-)carnitine; subsequent findings (Fig. 2) that Acetyl- (-)carnitine (-)Carnitine (nmol/min (nmol/min Addition per mg) per mg) None Decanoyl(+ )carnitine (10-300MM) Palmitoyl(+ )carnitine (10-100M&M) Mitochondria were made 20 mm with respect to potassium phosphate (ph 7.4), subjected five times to freezing and thawing, and then centrifuged at 00 for 10 min at 27,000 X g. The carnitine acetyltransferase of the supernatant was assayed in a final volume of 250 Ml at 30. For acetyl(-)carnitine formation, the assay system (19) contained: 100 mm Tris HCO (ph 7.4), 0.2 mm 5,5'- dithiobis-(2-nitrobenzoic acid), 0.6 mm (-)carnitine, 0.08 mm acetyl-coa, and mitochondrial extract (24 Mg of protein). For (-)carnitine formation the assay system (18, 19) contained: 200 mm Tris-HCl (ph 7.8), 1 mm KCN, 0.1 mm EDTA, 10 mm imalate, 0.25 mm NAD+, 0.13 mm NADH, 1 mm acetyl- (-)carnitine, 30 ;&g of CoASH, 20 pg of citrate synthase, 10 pg of malate dehydrogenase, and mitochondrial extract (15 pg of protein).

3 Proc. Nat. Acad. Sci. USA 72 (1975) palmitoyl(+)carnitine increased the s0o. for (-)carnitine for the overall reaction without affecting the Vm. further supported this view. The effect of long chain acyl(+)carnitines on the activity of carnitine acetyltransferase (EC ; acetyl-coa:carnitine O-acetyltransferase) was, therefore, examined both in the forward and the reverse directions at below saturating concenrations of (-)carnitine and acetyl(-)carnitine. However, even relatively high levels of long chain acyl(+)carnitines did not affect the activity of the enzyme from pigeon breast muscle (not shown) or that extracted from heart mitochondria (Table 2) (see ref. 20). The preceding observations, that long chain acyl(+)- carnitines inhibited the conversion of (-)carnitine to acetyl- (-)carnitine but only in intact mitochondria, could then be accommodated by one or both of the following hypotheses: (I) that long chain acyl(+)carnitines were inhibitory for the membrane-bound carnitine acetyltransferase of intact mitochondria but not for the enzyme dissociated from its native environment*; (II) that mitochondria had a pool of (-)- carnitine and acyl(-)carnitines in the matrix and a carnitine acylcarnitine translocase in the inner membrane which allowed, by exchange diffusion, the permeation of the mitochondrial inner membrane by carnitine and acylcarnitines and that long chain acyl(+)carnitines interfered with the transport of (-)carnitine and its esterst. Results of experiments supporting the latter hypothesis are described below, According to hypothesis II it was to be expected (a) that the exposure of mitochondria to ["4C]carnitine would allow labeling of the carnitine pool in matrix, and (b) that this [4C]carnitine would efflux on subsequent incubation of loaded mitochondria with unlabeled carnitine and/or acylcarnitines. These expectations were borne out. Incubation of mitochondria with ["4C]carnitine for 4 min at 280 (see Methods for details-prolonging incubation did not increase carnitine retention; loading was slower at 00, however) led to the retention of a fairly reproducible amount of radioactivity with mitochondria; the amount of ["4C]carnitine so retained (calculated from the specific activity of the ["4C]carnitine added and ignoring small dilution by endogenous carnitine) averaged 1.64 nmol/mg of protein (range for 10 experiments with mitochondria from three or four rat hearts in each experiment). Table 3 shows that the presence of unlabeled carnitine in the incubation system containing [14C]- carnitine-loaded mitochondria led to an efflux of radioactivity that showed both temperature dependence (compare Exps. I and II) and concentration dependence with approach to saturation at higher levels of added carnitine (Exp. I). A similar efflux and concentration dependence was seen with acylcarnitines as well (see Table 5). These observations did not show unequivocally, however, that the observed influx and efflux of carnitine involved a transport. For instance, concentration dependence and sat- * A similar postulate was put forward by Fritz and Marquis (20) for the inhibition of membrane-bound carnitine palmitoyltransferase based on the observations that palmitoyl(+ )carnitine competitively inhibited the (-)carnitine stimulation of fatty acid oxidation but did not affect the activity of the solubilized carnitine palmitoyltransferase. t Long chain acyl(+)carnitines could interfere by inhibiting the entry of (-)carnitine, the exit of acetyl(-)carnitine, and/or by substituting for intramitochondrial (-)carnitine. Carnitine Acylcarnitine Translocase 885 TABLE 3. Carnitine-dependent release of radioactivity from [I4Clcarnitine-loaded mitochondria of rat heart Concentration dpm in Efflux Addition (mm) supernatant % Exp. I at 00 None 1240 (-)Carnitine (+ )Carnitine Exp. II at 220 None (-)Carnitine For efflux, each tube contained loaded mitochondria (0.89 mg of protein) containing dpm. uration could result from the reversible adsorption of carnitine in a lipid-protein-water milieu having limited adsorption sites. This case applied, as pointed out by Zahiten et al. (21), to the kinetics of retention of ["4C]pyruvate by mitochondria inasmuch as heated or acid-precipitated mitochondria showed retention of pyruvate in a manner similar to that exhibited by intact mitochondria. Such was not the case, however, for the retention of ["4C carnitine by mitochondria, for the amount of radioactivity retained by acid-precipitated, heated, or frozen-thawed mitochondria was an insignificant portion of that retained by intact mitochondria (Table 4, Exp. I). Furthermore, Exp. II of Table 4 showed that while incubation of ["4C]carnitine-loaded mitochondria at 0 for 30 min led to only a small (16%) rise in radioactivity in the supernatant, sonication or freeze-thawing led to the release of nearly all of the radioactivity in the supernatant. Additional evidence favoring translocation was provided by the observations that ["4C]carnitine efflux induced by different acylcarnitines showed stereospecificity, since the (-) isomers were more active than the corresponding unnatural (+) isomers (Table 5). However, such specificity was not seen with free carnitines (Table 3). Insofar as at nearly saturating levels long chain acyl(+)- carnitines were less effective than carnitine in promoting ["4C]carnitine efflux from mitochondria, it was to be anticipated that if carnitine and acylcarnitines shared the same translocase system, then the relatively faster carnitine-carnitine exchange would be inhibited by long chain acyl(+)- carnitines. Table 6 shows that this was so and furthermore, that the inhibition of carnitine-carnitine exchange induced by long chain acyl(+)carnitine became less marked at higher concentrations of added carnitine. The possibility of a carnitine (acylcarnitine) pool in mitochondrial matrix was supported by the following observations (Table 7): (a) the endogenous content of total (-)carnitine obtained in different mitochondrial preparations was relatively constant-approaching about 2 nmol/mg of protein

4 886 Biochemistry: Pande TABLE 4. Effects of different treatments on the ability of rat heart mitochondria for loading and retention of [14C]carnitine TABLE 6. Proc. Nat. Acad. Sci. USA 72 (1975) Inhibition of carnitine-carnitine exchange by long chain acyl(+)carnitines dpm in loaded Supernatant dpm as Treatment of mitochondria % of initial mitochondria per mg of protein mitochondrial dpm Exp. I. Treatment before loading None 22,653 Acid treatmerat 87 Heating 272 Freezing and lthawing 521 Exp. Treatment after loading offresh mitochondria Incubation for 15 sec 4 10 min 7 30 min 16 Incubation for 30 min but mitochondria frozen and thawed in the meanwhile 99 Incubation for 10 min but mitochondria sonicated in the meanwhile 92 Acid treatment: Mitochondria were mixed with equal volume of 10% trichloroacetic acid and kept at 0 for 10 min. Then a 10-fold excess of 200 mm mannitol, 50 mm Tris- HCl (ph 7.5) was added and the tubes were centrifuged at 29,000 X g for 3 min at 00. The precipitate was suspended in the same volume of mannitol-tris HCl as before (final ph was 7.4) and "mitochondria" were recovered by centrifugation. Heat treatment: Mitochondria were heated for 2 min in a boiling-water bath, then chilled in an ice-water bath. Sonication: Mitochondria were sonicated three times for 30 sec each time at 00 at 60% of maximum setting with a sonic Dismembrator (Quigley Rochester, Inc., Rochester, N.Y.). In Exp. II, each tube contained 0.82 mg (protein) of loaded mitochondria containing 16,340 dpm. (as described above, similar amounts of [14C]carnitine entered mitochondria on loading); (b) the endogenous total (-)- carnitine was not readily or completely lost by additional washings of mitochondria; (c) freeze-thawing led to the release of an appreciable portion of the mitochondrial (-)- carnitine; and (d) the amount of total (-)carnitine associated TABLE 5. Comparison of the effects of stereoisomers of different acylcarnitines on the efflux of [14C]carnitine from rat heart mitochondria % Exchange with Concentration Acyl- Acyl- Addition mm (-)carnitine (+ )carnitine Acetylcarnitine Decanoylcarnitine Palmitoylcarnitine Additions to incubation medium (-)Carnitine, mm Acyl(+ )carnitine, um Efflux Decanoyl(+ )carnitine, Decanoyl(+)carnitine, Decanoyl(+ )carnitine, Decanoyl(+)carnitine, Decanoyl(+ )carnitine, Palmitoyl(+ )carnitine, Palmitoyl(+)carnitine, Palmitoyl(+ )carnitine, Palmitoyl(+)carnitine, Palmitoyl(+ )carnitine, with mitochondria was not affected by prior exposure of mitochondria to (-)carnitine or acetyl(-)carnitine (compare lines A, E, and F, Table 7) although, as described above, under similar conditions an efflux of loaded [14C]carnitine against added (-)carnitine or aeetyl(-)carnitine readily took place. The results outlined above (and similar results were ob- TABLE 7. Effects of different treatments on the endogenous total (-)carnitine content of rat heart mitochondria Total (-)carnitine associated with mitochondria (nmol/mg of protein) Treatment of mitochondria Exp. I Exp. II A. None B. Three more washings C. Incubated at 00 for 30 min, then as in B D. Five successive freezing and thawings, then as in B E. Incubated 10 min at 00 with 5 mm (-)carnitine, then as in B F. Incubated 10 min at 0 with 5 mm acetyl(-)carnitine, then as in B Mitochondria from 5 (Exp. I) or 3 (Exp. II) hearts were taken up in 230 mm mannitol, 70 mm sucrose, 20 mm Tris * HCl (ph 7.2), 20 M&M EDTA to a protein concentration of 40 mg/ml and treated as described. For washing, a 20-fold excess of 230 mm mannitol, 70 mm sucrose, 20 mm Tris-HCI (ph 7.2), 20 ;&M EDTA was mixed with mitochondrial suspension and the tubes were centrifuged for 90 sec in the Eppendorf 3200 centrifuge at 00. For freezing and thawing, the tubes were alternately immersed in dry ice/acetone and ice-water baths. The (-)carnitine content is expressed on the basis of the protein recovered after the treatments of the mitochondria; the protein loss ranged 2-5% in B, C, E, and F and 14-17% in D. To hydrolyze any acyl(-)carnitines present, the mitochondrial preparations were diluted six times with 0.25 M KOH and kept at 210 for 30 min. After neutralization, (-)carnitine was estimated according to a modification (in preparation) of published procedure (22).

5 Proc. Nat. Acad. Sci. USA 72 (1975) ACYLCoA INNER MEMBRANE MATRIX ACYLCoA ACYL TRANSFERASE CoA ACYL ACYL CoA ACYL TRANSFERASE FIG. 3. A scheme for the (- )carnitine-dependent transport of fatty acyl groups across mitochondrial inner membrane. Although only the inward transport is depicted above, the reactions involved, being readily reversible, would facilitate outward transport as well. tained with liver mitochondria) permit an updating of the original scheme of Fritz and Yue (1) for depicting the role of (-)carnitine in mitochondrial fatty acid transport (Fig. 3). This modified scheme takes into account the following: (a) that the mitochondrial permeability barrier to CoA and acyl- CoA esters is the inner membrane (11, 23, 24); (b) that carnitine acyltransferases are largely mitochondrial enzymes and associated with the inner membrane (5, 8, 11, 24, 25); (c) that mitochondria exhibit two different carnitine long chain acyltransferase activities, one of which is accessible to CoA and its esters, while the other one is accessible to CoA and its esters only from the matrix side of mitochondria but with which added carnitine and acylcarnitines can react somehow (5, 8, 23, 24, 26, 27); and (d) that the mitochondrial inner membrane, while not readily permeable to carnitine and its esters (10-12), allows their transport by a process of exchange diffusion facilitated by the presence of a carnitine acylcarnitine translocase in mitochondria (this paper). Further evidence consistent with the involvement of a carrier in this transport process and the properties of carnitine acylcarnitine translocase shall be described elsewhere. I am grateful to Dr. Jacques Genest for encouragement and thankful to Mrs. Lise D'Aragon for excellent technical help. Supported by grants from the Medical Research Council of Canada (MA-4264) and the Quebec Heart Foundation. Carnitine Acylcarnitine Translocase Fritz, I. B. & Yue, K. T. N. (1963) J. Lipid Res. 4, Tubbs, P. K. & Garland, P. B. (1968) Brit. Med. Bull. 24, Greville, G. D. & Tubbs, P. K. (1968) in Essays in Biochemistry, eds. Campbell, P. N. & Greville, G. D. (Academic Press, New York), Vol. 4, pp Garland, P. B., Haddock, B. A. & Yates, D. W. (1969) in Mitochondria Structure and Function, FEBS Symposium, eds. Ernster, L. & Drahota, Z. (Academic Press, London), Vol. 17, pp Yates, D. W. & Garland, P. B. (1970) Biochem. J. 119, Klingenberg, M. (1970) in Essays in Biochemistry, eds. Campbell, P. N. & Dickens, F. (Academic Press, New York), Vol. 6, pp Bressler, R. (1970) in Comprehensive Biochemistry, eds. Florkin, M. & Stotz, E. H. (Elsevier Publ. Co., New York), Vol. 18, pp Hoppel, C. H. & Tomec, R. J. (1972) J. Biol. Chem. 247, Levitsky, D. 0. & Skulachev, V. P. (1972) Biochim. Biophys. Acta 275, Bremer, J. (1967) in Protides of the Biological Fluids, ed. Peeters, H. (Elsevier Publ. Co., Amsterdam), Vol. 15, pp Haddock, B. H., Yates, D. W. & Garland, P. B. (1970) Biochem. J. 119, Brosnan, J. T. & Fritz, I. B. (1971) Biochem. J. 125, 94P. 13. Pande, S. V. & Blanchaer, M. C. (1971) J. Biol. Chem. 246, Ziegler, H. J., Bruckner, P. & Binon, F. (1967) J. Org. Chem. 32, Skidmore, W. D. & Entenman, C. (1962) J. Lipid Res. 3, Pande, S. V. (1971) J. Biol. Chem. 246, Bucher, T., Czok, R., Lamprecht, W. & Latzko, E. (1965) in Methods of Enzymatic Analysis, ed. Bergmeyer, H. (Academic Press, New York), pp Pearson, D. J., Chase, J. F. A. & Tubbs, P. K. (1969) in Methods in Enzymology, ed. Lowenstein, J. M. (Academic Press, New York), Vol. 14, pp Fritz, I. B., Schultz, S. K. & Srere, P. (1963) J. Biol. Chem. 238, Fritz, I. B. & Marquis, N. R. (1965) Proc. Nat. Acad. Sci. USA 54, Zahlten, R. N., Hochberg, A. A., Stratman, F. W. & Lardy, H. A. (1972) FEBS Lett. 21, Cederblad, G. & Lindstedt, S. (1972) Clin. Chim. Acta 37, Brdiczka, D., Gerlitz, K. & Pette, D. (1969) Eur. J. Biochem. 11, Brosnan, J. T., Kobec, B. & Fritz, I. B. (1973) J. Biol. Chem. 248, Norum, K. R. & Bremer, J. (1967) J. Biol. Chem. 242, West, D. W., Chase, J. F. A. & Tubbs, P. K. (1971) Biochem. Biophys. Res. Commun. 42, Chase, J. F. A. & Tubbs, P. K. (1972) Biochem. J. 129,