Interaction of Malonyl-CoA and 2-Tetradecylglycidyl-CoA with ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1986 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 23, Issue of October 15, pp. 1251612522 1985 Printed in &LA.

Interaction of Malonyl-CoA and 2-Tetradecylglycidyl-CoA with Mitochondrial Carnitine Palmitoyltransferase 1%

(Received for publication, March 18,1985)

Peter E. DeclercqS, Michael D. Venincasas, Scott E. MillslI, Daniel W. Foster, and J. Denis McGarryl/ From the ~ e ~ r t ~ n ~ of I n t e ~ ~ e ~ i c i ~ and 3 i o c ~ ~ i s € ~ , U ~ u e ~ i ~ of Texas ~ e a l ~ ~ Science Center at D a ~ , D a t h , Texas 75235

Malonyl-CoA and 2-tetradecylglycidyl-CoA (TG- CoA) are potent inhibitors of mitochondrial carnitine palmitoyltransferase I (EC 2.3.1.21). To gain insight into their mode of action, the effects of both agents on mitochondria from rat liver and skeletal muscle were examined before and after membrane disruption with octylglucoside or digitonin.

Pretreatment of intact mitochondria with TG-CoA caused almost total suppression of carnitine palmitoyltransferase I, -with concomitant loss in malonyl-CoA binding capacity. However, subsequent membrane solubilization with octylglucoside resulted in high and equal carnitine palmitoyltransferase activity from control and TG-CoA pretreated mitochondria; neither solubilized preparation showed sensitivity to malonyl- CoA or TG-CoA. Upon removal of the detergent by dialysis the bulk of carnitine palmitoyltransferase was reincorporated into membrane vesicles, but the rein- serted enzyme remained insensitive to both inhibitors. Carnitine palmitoyltransferase containing vesicles failed to bind malonyl-CoA.

With increasing concentrations of digitonin, release of carnitine palmitoyltransferase paralleled d i s~p t ion of the inner m i t o c ~ o n ~ a l membrane, as reflected by the appearance of matrix enzymes in the soluble fraction. The profile of enzyme release was identical in control and TG-CoA pretreated mitochondria even though carnitine palmitoyltransferase I had been ini- tially suppressed in the latter. Similar results were obtained when animals were treated with 2-tetradecylglycidate prior to the preparation of liver mitochondria.

We conclude that malonyl-CoA and TG-CoA interact reversibly and irreversibly, respectively, with a common site on the mitochondrial (inner’) membrane and that occupancy of this site causes inhibition of carnitine palmitoyltransferase I, but not of carnitine palmitoyltransferase 11. Assuming that octylglucoside and digitonin do not selectively inactivate carnitine palmitoyltransferase I, the data suggest that both malonyl-CoA and TG-CoA interact with a regulatory locus that is closely juxtaposed to but distinct from the active site

* This work was supported by National Institutes of Health Grant AM 18573. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Senior Research Assistant of the Belgian National Fund for Scientific Research and supported by Grants-in-Aid from NATO and F~ibrjght /~ays.

5 Recipient of a fellowship from the Chilton Foundation, Dal- las,TX.

1 Recipient of National Institutes of Health Grant AM 07139. I/ To whom correspondence should be addressed.

of the membrane-bound enzyme.

The initial steps in mitochondrial long chain fatty acid oxidation are thought to be catalyzed by the sequential action of carnitine palmitoyltransferases I and I1 (EC 2.3.1.21), located on the outer and inner aspects, respectively, of the inner mitochondrial membrane (14). Although extensively studied, this enzyme system has eluded definitive characteri- zation. For example, in their membrane-bound states carnitine palmitoyltransferase I and carnitine palmitoyltransferase 11, both from a given tissue and across tissue lines, exhibit very different kinetic properties towards their substrates, It is not clear whether these differences reflect intrinsically distinct properties of separate enzymes or represent altera- tions imposed on a single enzyme protein by differing membrane environments (2, 4, 5-7). We and others are trying to resolve this problem.

The focus of the present article is on the inhibitory interaction between malonyl-CoA and carnitine palmitoyltransferase I as the enzyme exists in its natural membrane environ- ment. First described for liver (8,9), this inhibition has since been reported for a wide variety of mammalian tissues and, curiously, is p~ icu Ia r ly p rono~ced in ~ t o c ~ o n ~ i a from skeletal muscle (5, 10, 11). The precise mechanism of the malonyl-CoA effect has proved difficult to establish. Key observations have been the following. First, inhibition of carnitine palmitoyltransferase I by a fixed concentration of malonyl-CoA can be overcome by raising the concentration of palmitoyl-CoA (a substrate for the enzyme), but not in a classically competitive manner (9). Second, when released from the membrane with detergents the enzyme loses all sensitivity to malonyl-CoA (7,9, 12, 13). Third, in mitochondria from rat liver, heart, and skeletal muscle a shift in pH from 6.8 to 7.6 greatly reduces the inhibitory potency of malonyl-CoA but has little effect on the apparent K,,, of carnitine palmitoyltransferase I for palmitoyl-CoA, suggest- ing that the affinity of the active site for its acyl-CoA substrate is not pH-sensitive over this range (6). In skeletal muscle mitochondria this loss of inhibitory potential is accompanied by (a) a fall in the affinity of the membrane binding site for malonyl-CoA and (b ) enhancement of the ability of palmitoyl- CoA to displace bound malonyl-CoA (6). Taken together, these findings suggested, but did not prove, that the competitive interaction between substrate and inhibitor occurs not at the active site of carnitine palmitoyltransferase I, but at a closely juxtaposed regulatory domain.

To clarify this issue we have made use of the compound, TG-CoA1, developed by McNeil P h ~ a c e u t i c ~ s and shown

The abbreviations used are: TG-CoA, 2-tetrad~ylglycidyl-Co~ EGTA, ethylene glycol b i s { ~ - ~ i n o e t h y l ~ t h e r ) - ~ , ~ , ~ ~ , ~ ‘ - ~ t r a a c - etic acid; Hepes, 4-(2~hy~xyethyl)-l-piper~ineethanesulfonic acid.

12516

12517

by Tutwiler and colleagues (14,15) to be a powerful, irreversible inhibitor of carnitine palmitoyltransferase I. In this paper we compare the effects of TG-CoA and malonyl-CoA on mitochondrial carnitine palmitoyltransferase activity before and after solubilization of carnitine palmitoyltransferase I (and other membrane components) by detergents. Mito- c h o n ~ a from two tissues, liver and skeletal muscle, were studied because they represent two extremes of sensitivity to malonyl-CoA inhibition. The results indicate that both inhibitors interact at a common locus on the inner mitochondrial membrane, but that this locus is distinct from the active site of carnitine palmitoyltransferase I.

EXPERIMENTAL PROCEDURES

A n ~ ~ - M a l e S p r ~ e - ~ a w l e y rats, maintained on a high sucrose/low fat diet (16) and weighing about 150 g were used in the fed state.

Preparation of Mitochondria-Mitochondria, isolated from liver and hind-leg muscle as described previously (5), were suspended in 0.15 M KC1, 5 mM Tris-HC1, pH 7.4 (medium A). Protein was measured by the method of Lowry et al. (17).

Assay of Carnitine Palmitoyltmnsferase I-Activity was measured at pH 7.2 and 30 "C in the direction: palmitoyl-CoA + [**C]carnitine +. palmitoyl['*G]c~itine + CoASH as described (5). Final concentrations of palmitoyl-CoA, L-carnitine, and fatty acid-free albumin were 50 p ~ , 0.2 or 2 mM, and 1%, respectively. Reaction rates were linear over the time periods studied (generally 4-5 rnin).

Use of TG-CoA-In most experiments with TG-CoA, mitochondria were preincubated at a density of about 3 mg of protein/ml in medium A containing 1% albumin and either 10 (liver) or 16 pM (muscle) of inhibitor. After 20 min at room temperature the mitochondria were sedimented by centrifugation and resuspended in medium A. This generally produced 85-95% inhibition of carnitine palmitoyltransferase I in the resultant preparatio~s. An alternative procedure, which gave identical results, involved replacement of TG-CoA in the preincubation medium with a mixture of free 2-tetradecylglycidic acid (12 p ~ ) , ATP (5.6 mM), MgClz (6.3 mM), and coenzyme A (100 pM). Under these conditions TG-CoA is generated by acyl-CoA synthetase present on the outer mitochondrial membrane (15). In some experiments, particularly those involving freeze-thawing of mitochondria, we wished to avoid the centrifugation step described above for the removal of excess TG-GOA. In such cases the preincubation with TG- CoA was carried out with a %fold higher density of mitochondrial protein (about 9 mg/mI). The mixture was then diluted with 2 volumes of medium A and used directly for measurement of carnitine palmitoyltransferase activity.

In Vivo Use of 8-Tetradecylglycidic Acid-In an effort to reproduce the findings of Tutwiler et al. (18), 2-tetradecylglycidic acid was dissolved to a concentration of 1 mg/ml in 50 mM potassium phos- phate buffer, pH 7.4, containing 10% bovine serum albumin. The mixture was administered to rats intraperitoneally in a volume sufficient to deliver 2 mg of the drug/kg of body weight. Control animals received vehicle alone. Three hours later liver mitochondria were isolated and used for experiments.

Use of OctylgLucoside-Complete solubilization of carnitine palmitoyltransferase was accomplished using the detergent, octylglucoside. To a suspension of intact mitochondria a t a density of about 3 mg of protein/ml in medium A, octylglucoside was added in a concentration of 1%. After stirring in ice for 15 min the mixture was centrifuged at 100,000 X g for 1 h. Little if any pellet was formed and essentially all of the carnitine palmitoyltransferase activity present before centrifugation was recovered in the clear supernatant. In some experiments the supernatant was dialyzed at 4 "C against 80 volumes of medium A for 8 h after which the dialysis was continued in fresh medium A for another 8 h. This resulted in the re-formation of membrane vesicles (as judged by the cloudy appearance) and a distinct pellet was formed upon centrifugation at 100,000 X g for 1 h. When resuspended, this material contained >90% of the carnitine palmitoyltransferase activity present in the uncentrifuged suspension.

Use of Digitonin-To effect the sequential disruption of the outer and inner mitochondrial membranes with concomitant release of carnitine p a ~ m i ~ y l t r ~ s ~ e r a s e I and carnitine p~~itoyltransferase I1 we made use of the membrane disrupting agent, digitonin. In

method 1 intact mitochondria were suspended in medium A to a density of 3.2 mg of protein/ml and dispensed in 3-ml portions into a series of tubes. To each tube was added 300 p1 of 0.25 M sucrose containing sufficient digitonin to provide final concentrations of 0- 0.18% (0-0.62 mg of digitonin/mg of mitochon$ial protein). After 20 min at 0 "C the mixtures were centrifuged at 20,000 x g for 1 h, the supernatants were decanted and the pellets resuspended in medium A. Carnitine p a ~ ~ i t o ~ l t r a n s f e r ~ activity was measured in both fractions.

In method 2 the range of digitonin concentration was expanded while maintaining a relatively dilute suspension of mitochondria. In this case intact mitochondria were suspended to a density of 3.2 mg of protein/ml in medium B (0.25 M sucrose, 1 mM EGTA, 5 mM Tris- HCl, pH 7.4, and 1% albumin). Digitonin was added as in method 1, but over a wider concentration range (0-0.48% or 0-1.65 mg of digitoninlmg of mitochondrial protein) because of the extra protein contributed by albumin. After 20 min at 0 "C the tubes were centrifuged as in method 1. The supernatant fractions were assayed for carnitine palmitoyltransfer~e activity and also for adenylate kinase (a marker of the inter membrane space) and citrate synthase or malate dehydrogenase (enzymes of the mitochondrial matrix) by conventional spectrophotometric techniques.

To approximate more closely the conditions reported by Hoppel and Tomec (19) and subsequently by Bergstrom and Reitz (20) to cause the sequential release of carnitine palmitoyltransferase I and carnitine palmitoyltransferase I1 a much thicker suspension of mitochondria was used (method 3). To 0.3-ml diquots of ~ t ~ h o n ~ i a (62.5 mg of protein/ml of medium A) was added 75 pl of medium A containing increasing amounts of digitonin. This yielded a final mitochondrial protein concentration of 50 mg/ml and a digitonin/ protein ratio (mg/mg) spanning the range 0-0.9. After 15 min of shaking at 0 "C, 3.375 ml of cold medium A was added and the mixtures were treated as in method 2.

Mitochondria prepared from animals previously injected with 2- tetradecylglycidate were treated as described for method 3 except that they were suspended in medium c (70 mM sucrose, 220 mM D- m a n ~ t o ~ , 2 mM Hepes, pH 7.4, and 0.5 mg/ml ~ b ~ i n ) . The digitonin was dissolved in the same medium. This procedure, method 4, was designed to reproduce the conditions employed by Tutwiler et al. (18).

P4C]Malonyl-CoA Binding-The specific binding of [2-"C]malonyl-CoA to muscle mitochondria was carried out as described previously (10).

MateriQb-Octylglucoside and digitonin were from Sigma. The latter was recrystallized from warm ethanol before use. 2-Tetradecyl- glycidic acid and its CoA ester were kindly provided by Dr. Gene F. Tutwiler, McNeil Pharmaceuticals, Spring House, PA 19477,

RESULTS

The inhibitory action of malonyl-CoA on mitochondrial carnitine palrnitoyltransferase I is immediately reversible upon removal of the CoA ester (9), whereas that of TG-CoA is irreversible (15). The effects of both agents are markedly attenuated in the presence of palmitoyl-CoA, a substrate for the enzyme (9, 15). The rate of enzyme inactivation by TG- CoA, a substrate analog, is slowed in the presence of malonyl- CoA (15). Given this background, we wished to determine whether TG-CoA and malonyl-CoA interact with the same or different sites on the mitochondrial membrane and whether the active site of carnitine palmitoyltransferase I is involved, as has been suggested (15,18).

The data of Fig. 1 show that pretreatment of muscle mitochondria with TG-CoA severely reduces the subse~uent binding of labeled malonyl-CoA. This effect of TG-CoA persists after multiple washings of the mitochondria (as does its inhibition of carnitine palmitoyltransferase I (15, 18)). The most likely interpretation is that the agent forms a covalent bond at the malonyl-CoA binding locus, although other sites of TG-CoA interaction are not absolutely excluded.

If TG-CoA reacted irreversibly at the active site of carnitine p ~ m i ~ y ~ t r ~ s f e r a s e I one might expect that after complete detergent solublization of untreated and TG-CoA- pretreat^

’u ,+!GOA , =, 1 0 50 IO0 150 200 250

PRETREATED WITH

**”*

FREE [“e] -MALONYL- CoA (nM 1

FIG. 1. Effect of TG-CoA on binding of malonyl-CoA to skeletal muscle mitochondria. Control or TG-CoA pretreated mitochondria (approximately 3 mg of protein) were incubated at 0 “C for 20 min with [2-14C]malonyl-CoA (5-250 nM) in the presence or absence of 100 PM unlabeled malonyl-CoA. Specific binding is shown.

M - + ~ F M MALONYL-GOA T - PRETREATED WITH TG-COA

INTACT OG-SOLUBILIZED f 6p”- A

U ; f FIG. 2. Effects of TG-CoA, malonyl-CoA and octylglucoside

on carnitine pa lmi toy l t r a~ fe r~e (CPn activity of skeletal muscle mitochondria. Untreated or TG-CoA pretreated mitochondria were assayed for carnitine p a ~ i t o y l t r a n s f e r ~ e activity in the absence or presence of 5 PM malonyl-CoA before and after addition of octylglucoside (OG) in a concentration of 1%. Two hundred micromolar L-carnitine was used in the assay.

mitochondria (providing release of both carnitine palmitoyltransferase I and carnitine palmitoyltransfer~e 11), total assayable activity would be significantly less in the latter case. This was not observed, as illustrated in Fig. 2 for muscle mitochondria. With intact organelles, where the enzyme assay monitors only carnitine palmitoyltransferase I (5), activity was greatly reduced either by inclusion of malonyl-CoA in the assay or by pretreatment with TG-CoA. Combination of the two inhibitors gave no further suppression. We suspect that residual enzyme activity in the presence of TG-CoA andlor malonyl-CoA (generally 5-15%) represents a small contribu- tion of carnitine palmitoyltransferase I1 as a result of me- chanical damage to the inner membrane during the preparation of mitochondria. After octylglucoside treatment carnitine palmitoyltransferase activity increased in control mitochondria, presumably due to the full exposure of carnitine palmi-

toyltransferase 1I.Z Equally high activity was seen in TG-CoA pretreated mitochondria after addition of the detergent; is. the inhibitory effect of TG-CoA was not preserved with soi- ubilization of the enzyme. As expected malonyl-CoA was without effect on solubilized carnitine palmitoyltransferase. The same was true for TG-CoA (data not shown).

The experiment of Fig. 2 was run using 200 PM carnitine in the carnitine palmitoyltransferase assay. This value is well above the apparent K , for carnitine of carnitine palmitoyltransferase I in liver mitochondria at pH 7.2 (-30 PM), but below that of skeletal muscle mitochondria (-530 PM) (5,6), Accordingly, an expanded study was performed with both types of mitochondria using a 10-fold higher concentration of carnitine. As seen from lines 1-4 of Table I, the results were quali~tively similar to those shown in Fig. 2. Also established is that after freeze-thawing of mitochondria the reduced sup- pressibility of assayable carnitine palm~toyltransfer~e by malonyl-CoA, shown here (Table I, lines I and 5) and earlier ($), applies to TG-CoA as well (Table I, lines 1, 3, 5, and 9). Presumably, this results from the generation of a mixture of normally oriented and inside-out vesicles expressing carnitine palmitoyltransferase I and carnitine palmitoyltransferase I1 activities, respectively. Only that fraction of vesicles with normal orientation is inhibited because only carnitine pal- m i t o y ~ t r ~ s f e r a ~ I is sensitive to TG-CoA and malonyl-CoA (9, 14);’ ie. loss of inhibitabili~ is due to inside outness of the membrane, which is impermeable to both palmitoyl-CoA and inhibitors. Treatment of mitochondria with TG-CoA either before or after freeze-thawing produced vesicles in which enzyme activity was essentially resistant to further suppression by malonyl-CoA (Table I, lines 7 and 9). Solubil- ization of both intact and freeze-thawed preparations yielded approximately the same high activity of carnitine palmitoyltransferase regardess of whether they had been exposed to TG-CoA. Again, the implication would be that TG-CoA does not inhibit solubilized carnitine palmitoyltransferase I and that its potent suppression of the membrane-bound enzyme is totally reversed upon release of the latter from the membrane.

As an alternative approach to the problem we used a second membrane disrupting agent, digitonin. The rationale here was based in part on reports from other laboratories that increasing concentrations of this agent causes sequential release of carnitine p~~toyl t ransferase I and carnitine palmitoyltr~s- ferase I1 from the inner m i ~ h o n ~ ~ l membrane (18-20). If this were true, and if TG-CoA reacted irreversibly at the active site of carnitine palmitoyltransferase I, a very different profile of active enzyme solubilization would be expected from control and TG-CoA pretreated mitochondria: ie . the initial enzyme released would be active in the former, but not in the latter. As shown in Fig. 3, this was not the case. Both the

Carnitine p~mitoyltr~nsferase activity as measured in intact and detergent-solubilized mitochondria under our fixed assay conditions should not be compared in absolute terms. This is because the kinetic constants of the enzyme(s) for palmitoyl-CoA and carnitine differ substantially in the two situations and in neither case have we attempted to achieve V,, rates. It is likely that even with a high (2 mM) concentration of carnitine in the assay (Tables I and 11) rates measured in octylglucoside-solubilized preparations will be relatively underestimated compared with those seen in intact mitochondria, since the apparent Km for p~mitoyl-CoA under the two conditions is about 160 and 40 PM, respectively, both for liver and muscle mitochondria (palmitoyl-CoA in the assay is fixed at 50 gM, a nominal value because of the presence of albumin). Similar cautions apply to the interpretation of experiments using freeze-thawed mitochondria (Table I), reconstituted membrane vesicles (Table 111, or agents such as digitonin to disrupt mitochondrial membranes (Fig. 3-5).

Regulation of Carnitine Palmitoyltransferase I TABLE I

Effects of inhibitors on carnitine palmitoyltransferase activity of liver and muscle mitochondria before and after membrane disruption

12519

Mitochondria were pretreated in the manner indicated after which an aliquot from each group was exposed to 1% octylglucoside (OG). Carnitine palmitoyltransferase (CPT) was then assayed in the presence and absence of malonyl-CoA using carnitine at a concentration of 2 mM. Values are means f S.E. for 3-4 experiments.

CPT activity (nmol . min-l. mg protein”)

Pretreatment of mitochondria OG Liver Muscle

Control +50 BM Malonyl-CoA Control +10 @I

Malonyl-CoA

1. None - 6.05 f 0.74 1.28 f 0.27 9.45 f 0.14 1.53 f 0.39 2. None + 12.07 f 0.36 11.66 f 0.36 12.97 f 0.35 13.02 f 0.33

3. TG-COA - 0.08 f 0.07 0.05 f 0.05 1.58 f 1.24 1.08 f 0.85 4. TG-COA + 11.33 f 0.36 11.10 f 0.22 12.53 f 0.32 12.16 f 0.54

5. Freeze-thawed X 3 - 8.93 f 0.66 5.47 f 0.55 12.61 f 0.55 9.79 f 1.03 6. Freeze-thawed X 3 + 10.82 f 0.52 10.18 f 0.58 13.10 f 0.20 13.33 f 0.31

7. TG-CoA, then freeze-thawed X 3 - 5.78 f 0.67 5.18 f 0.73 9.55 & 0.71 9.45 f 0.91 8. TG-CoA, then freeze-thawed X 3 + 11.06 f 0.63 10.46 f 0.88 12.88 f 0.50 12.91 f 0.57

9. Freeze-thawed X 3, then TG-CoA - 5.58 f 0.67 4.85 f 0.59 9.90 f 0.77 9.37 f 0.85 10. Freeze-thawed X 3, then TG-CoA + 10.59 f 0.85 10.53 f 0.88 13.30 f 0.42 13.13 f 0.62

I 1 I I I I 1

160 - - 7 140- E d 120-

100-

80- k L & 60- U t 40-

20 - 0-

.-

- 0 -

0

mg DIGITONIN . mg MIT. PROTEIN“

FIG. 3. Effect of digitonin on carnitine palmitoyltransferase (CPn activity of liver mitochondria in the absence of albumin. Three milliliter aliquots of untreated or TG-CoA pretreated liver mitochondria (containing 9.6 mg of protein) were exposed to the indicated concentrations of digitonin using Method 1 as described under “Experimental Procedures.” Total assayable carnitine palmitoyltransferase activity of the supernatant and pellet fractions is shown. Two hundred micromolar L-carnitine was used in the assay.

pattern and absolute quantities of carnitine palmitoyltransferase solubilized from liver mitochondria with increasing levels of digitonin were identical in the control and experimental groups. Concomitant with the appearance of carnitine palmitoyltransferase in the supernatant fraction, enzyme activity was lost from the residual pellet in the control group.* Before digitonin treatment the pellet from TG-CoA-treated mitochondria showed low carnitine palmitoyltransferase activity (because carnitine palmitoyltransferase I was inhibited). This rose significantly with digitonin addition up to a concentration of 0.2 mg/mg of mitochondrial protein, but with higher concentrations activity fell to very low levels. The biphasic pattern was likely due to initial exposure of carnitine palmitoyltransferase I1 followed by total collapse of the inner membrane with release of both carnitine palmitoyltransferase

I and carnitine palmitoyltransferase 11. As in the experiments with octylglucoside, carnitine palmitoyltransferase activity present in digitonin extracts was totally resistant to inhibition by TG-CoA and malonyl-CoA (data not shown).

The experiment depicted in Fig. 3 was repeated using skeletal muscle mitochondria, with qualitatively similar results (data not shown). Moreover, in no case could we find evidence for an early rise + plateau + later rise sequence of enzyme release with increasing digitonin concentration as reported by Bergstrom and Reitz (20), and interpreted by them to represent the sequential solubilization of carnitine palmitoyltransferase I and carnitine palmitoyltransferase 11. Because of this discrepancy the conditions employed in the experiment of Fig. 3 were modified to allow a more gradual release of carnitine palmitoyltransferase with increasing digitonin concentration. This was achieved by suspending liver mitochondria in medium B, which, unlike medium A, contained 1% albumin. The results are shown in Fig. 4. Four points warrant emphasis. First, with increasing digitonin concentration significant solubilization of carnitine palmitoyltransferase occurred only after the outer membrane had been largely stripped away (as judged by the appearance in the supernatant fraction of the inter-membrane enzyme, adenylate kinase). Second, carnitine palmitoyltransferase release coincided closely with that of citrate synthase, a matrix enzyme, in keeping with the known localization of the former on the inner membrane. Third, results were identical in control and TG-CoA-treated mitochon&ia. Finally, there was no evidence for a break in the curve relating carnitine palmitoyltransferase release to digitonin concentration.

The data of Figs. 3 and 4 were difficult to reconcile with the notion that digitonin first removes the bulk of carnitine palmitoyltransferase I from the inner mitochondrial membrane and then effects the release of carnitine palmitoyltransferase I1 (18-20). It was conceivable that the discrepancy stemmed from differences in the concentration of mitochondria employed during the period of treatment with digitonin (50 mg of protein/ml in the cited studies uersw -3 mg/ml in the experiments of Figs. 3 and 4). Accordingly, the experiment of Fig. 4 was repeated using 50 mg of mitochondrial protein f ml of digitonin extraction medium (method 3 under “Experi- mental Procedures”). The patterns of enzyme release were

12520 Regulation of Carnitine

I ' 6 1 I t 1

loot

80- a $ 60- d 40- 2

20-

w

OL

mg DIGITONIN . mg MIT. PROTEIN"

FIG. 4. Effect of digitonin on enzyme release from liver mitochondria in the presence of albumin. Liver mitochondria (untreated or pretreated with TG-GOA) were exposed to digitonin using method 2 as described under "Experimental Procedures." After centrifugation, the supernatant fractions were assayed for carnitine palmitoyltransferase (Car) (using 200 p~ L-carnitine), adenylate kinase, and citrate synthase activities. Although the data are expressed as a percentage of maximum enzyme released, absolute values were essentially identical in the control and TG-CoA-treated preparations (approximately 0.002, 0.42, and 0.18 pmol.min-'.mg mitochondrial protein" for carnitine palmitoyltransferase, adenylate kinase, and citrate synthase, respectively).

virtually identical with those displayed in Fig. 3. Importantly, the curves relating carnitine pal~toyltransferase solubilization to digitonin concentration were again smooth and sig- moid in shape, and were superim~sable for the control and TG-CoA-treated mitochondria, despite a 90% inhibition of carnitine palmitoyltransferase I in the latter group prior to digitonin addition (data not shown).

Tutwiler et al. (18) prepared liver mitochondria from control and 2-tetradecylglycidate-injected rats and treated them with digitonin under conditions recommended by Hoppel and Tomec (19) for the selective release of carnitine palmitoyltransferase I from the inner membrane ( i e . 0.12 mg of digitonin/mg of mitochondrial protein with a mitochondrial protein concentration of 50 mg/ml). As expected, intact mitochondria from rats given 2-tetradecylglycidate were found to exhibit a greatly reduced activity of carnitine palmitoyltransferase I compared with those from untreated animals. The same difference in enzymatic activity was noted in the digitonin extracts, while assayable carnitine palmitoyltransferase present in the residual mitoplasts (presumed to be carnitine palmitoyltransferase 11) was identical in the two groups (18). The data were taken as further evidence for a covalent interaction of TG-CoA with the active site of carnitine palmitoyltransferase I; they also appeared to strengthen the view that under the conditions specified the enzyme solubilized was largely carnitine palmitoyltransferase I while that remaining membrane bound was mainly carnitine palmitoyltransferase

These findings contrasted sharply with ours. Since the discrepancy might have stemmed from differences in the mode of exposure of mitochondria to TG-CoA (in vivo uersw in uitro) we prepared liver mitochondria from control and 2- tetradecylglycidate-treated rats. When assayed in the intact state (using 200 ~ L M carnitine) values for carnitine palmitoyltransferase I in the two groups were 7.1 and 1.0 nmol. min" . mg of mitochondrial protein-', respectively; i.e. the inhibitor had worked as expected, However, upon solubilization of the mitochondria with 1% octylglucoside the equivalent values for assayable carnitine palmitoyltransferase were 9.5 and 9.4, respectively. Thus, total activity increased and the difference between the groups was eliminated, in keeping with the experiments of Fig. 2 and Table I. Moreover, when the mito-

I1 (18-20).

mg DIGITONIN~mg MIT. PROTEIN"

FIG. 5. Effect of digitonin on enzyme release from liver mitochondria of rats treated with 2-tetradecylglycidic acid in vivo. Liver mitochondria were prepared from a rat treated in uivo with 2-tetradecylglycidic acid and from a control animal as described under ~ E x p e r i m e n ~ Procedures." They were treated with digitonin using method 4 and, after centrifugation, the supernatant fractions were assayed for carnitine palmitoyltransferase (can, (using 200 p~ L-carnitine), adenylate kinase, and malate dehydrogenase activities. Although the data are expressed as a percentage of maximum enzyme released, absolute values were essentially identical in the control and TG-treated preparations (approximately 0.005, 0.64, and 8.6 pmol. min-l. mg mitochondrial protein" for carnitine palmitoyltransferase, adenylate kinase, and malate dehydrogenase, respectively).

TABLE I1 Effect of sequential addition and removal of detergent on carnitine

p ~ ~ m ~ t o y ~ ~ r a n s f e r ~ e activity of muscle mitochondria Control and TG-CoA-pretreated muscle m i t o c h o n ~ a were further

treated as indicated. At each step carnitine p a l ~ i t o y l t r ~ f e r a s e (CPT) was assayed in the absence and presence of 10 pM malonyl- GOA using carnitine at a concentration of 2 mM. Absolute activity in the untreated control (almost exclusively carnitine palmitoyltransferase I) was 10.5 nmol/mjn/mg of mitochondrial protein.

CPT activity (% of untreated control)

Further treatment of mitochondria

Control Pretreated with TG-COA

-Malonyl- +Malonyl- -Malonyl- +Malonyl- CoA CoA CoA CoA

I. None 2. 1% Octylglucoside 3. 100,OOO x g Super-

4. 3 After dialysis 5, l ~ , O O O X g Pellet

from 4

natant from 2

100 4.8 5.2 4.5 157.6 ND" 151.7 ND 154.3 ND 152.1 ND 226.4 226.8 227.4 228.3 205.5 207.0 203.5 202.6

a ND, not determined.

chondria were exposed to increasing concentrations of digitonin using the conditions of Tutwiler et al. (18) (method 4 under "Experimental Procedures") the profiles for enzyme solubilization were again identical in the control and drug- treated groups (Fig. 5).

The data of Table I1 refer to experiments in which octylglucoside-solubilized preparations from control or TG-CoA- pretreated muscle mitochondria were dialyzed extensively to remove the detergent. This resulted in the re-formation of membrane vesicles into which the bulk of solubilized carnitine palmitoy~transferase had been incorporated. Again, despite the potent TG-CoA inhibition of carnitine palmitoyltransferase I in the intact organelles there was no difference between control and experimental groups in measurable carnitine palmitoyltransferase activity either in the detergent-solubilized preparations or in the re-formed vesicles after detergent removal? Furthermore, carnitine palmitoyltransferase associated with the latter was completely resistant to inhibition by malonyl-CoA (and TG-CoA, not shown). When this experiment was repeated using liver mitochondria qualitatively identical results were obtained (data not shown). In the case

12521

of muscle mitochondria the vesicles produced after removal of detergent were also found to have lost their ability to bind labeled malonyl-CoA (data not shown). Binding of TG-CoA could not be assessed since we do not yet have this material with sufficiently high specific activity to allow accurate measurement.

DISCUSSION

It seems reasonable to conclude that malonyl-CoA and TG- CoA bind in reversible and irreversible fashion, respectively, to a common locus on the mitochondrial inner membrane and, in so doing, effect the inhibition of carnitine palmitoyltransferase I. Is this locus the active site of carnitine palmitoyltransferase I? If we accept the commonly held view that treatment of mitochondria with membrane solubilizing agents releases both carnitine palmitoyltransferase I and carnitine palmitoyltransferase I1 as active enzymes the answer to this question would appear to be no, based on the following con- siderations. First, regardless of how it was accomplished? removal. of carnitine palmitoyltransferase I from the inner mitochondrial membrane resulted in complete loss of sensitivity to malonyl-CoA and TG-CoA (7, 9, 12, 13 and present study). Second, after exposure of mitochondria to TG-CoA, which blocked essentially all carnitine palmitoyltransferase I activity, the amount of active enzyme released upon solubilization of the membranes with ~tylglucoside was the same as that from previously untreated mitochondria. This would hardly have been expected had carnitine palmitoyltransferase I been released with a tetradecylglycidyl moiety linked cova- lently at its active site? Third, exposure of control and TG- CoA-treated mitochondria to increasing concentrations of digitonin gave identical profiles for the appearance of active carnitine palmitoyltransferase in the supernatant fractions, Le. there was no evidence for the release of an inactive species of carnitine palmitoyltransferase from the TG-CoA pretreated group.” Fourth, octylglucoside solubilization of mitochondria followed by removal of the detergent yielded reconstituted membrane vesicles with identical properties regardless of whether the original organelles had been exposed to TG-CoA; ie. there was equal activity of carnitine palmitoyltransferase and lack of sensitivity to malonyl-CoA and TG-CoA. Finally, in inverted mitochondria TG-CoA, like malonyl-CoA, failed to inhibit externalized membrane-bound carnitine palmitoyltransferase 11. Accepting for the moment the recent claim that in the same tissue carnitine palmitoyltransferase I and carnitine palmitoyltransferase 11 are very similar, if not identical proteins (20-23, it seems unlikely that TG-CoA would

Free 2-tetradecylglycidic acid does not inhibit mitochondrial carnitine palmitoyltransferase I (15, 27). Presumably its esterification to CoA allows the formation of a covalent bond between the epoxide ring of TG-CoA and a component of the inner m i ~ h o n d r i ~ membrane. However, we suspect that once this bond is formed the CoA group is no longer needed for inhibition of carnitine palmitoyitrans- ferase I. Mitochondrial preparations are generally rich in acyl-CoA deacylase activity which would be expected to hydrolyze the thioester group of the bound TG-CoA, particularly during multiple washing procedures. Moreover, we have exposed TG-CoA-treated mitochondria to hydroxylamine (under conditions that destroyed the thioester group of p~mitoyl-CoA) without loss of carnitine p ~ i ~ y l t r ~ s f e r - ase I inhibition (data not shown).

These findings contrast with those of other workers who con- cluded that under defined conditions of digitonin/mitochondrial ra- tios carnitine palmitoyltransferase I can be solubilized in active form while carnitine palmitoyltransferase I1 remains membrane bound (18-20). We have examined this issue exhaustively, but can offer no explanation for the discrepmcies. We are, however, confident in the ~ p ~ d u c i b i l i t y of the experiments reported here.

have access to the active site of the former but not of the latter.

All of these observations could be readily explained if, as suggested from our earlier kinetic studies (6) and more re- cently by others (7, 23), the binding site for malonyl-CoA (and TG-CoA) on the inner mitochondrial membrane is distinct from the active site of carnitine PaImitoyltransferase I. Such a model would account for the competitive interaction between malonyl-CoA and TG-CoA and for the antagonism exerted against both ligands by palmitoyl-CoA (9,15). Implicit in this construct is that malonyl-CoA and related compounds interact reversibly while TG-CoA (and probably its analogs (24)) form a covalent bond at the “regulatory site.”3 Also required by the model is that palmitoyl-CoA (and other long chain acyl-CoAs) serve both as substrates at the active site of carnitine palmitoyltransferase I and as antagonists to inhibitors at the regulatory locus (6). Binding of the inhibitory ligand could cause a detrimental conformational change in carnitine palmitoyltransferase I or, alternatively, sterically hinder the approach of acyl-CoA substrates to the catalytic center. Presumably, carnitine palmitoyltransferase I1 escapes suppression either because it cannot bind the inhibitors or because it is not associated with a regulatory protein. An understanding of the actual mechanism must await clarifica- tion of whether the active and regulatory loci exist on the same or juxtaposed pol~eptides. On theoretical grounds both scenarios could be reconciled with the data. For example, if only the carnitine palmitoyltransferase I protein is involved, its release from the membrane after detergent treatment might result in a conformational change such that inhibitory ligands could no longer bind, or bind in such a manner that their effectiveness is lost. Failure of the enzyme to return to its normal configuration in the membrane after removal of detergent could then explain the absence of malonyl-CoA (and presumably TG-CoA) binding in reconstituted vesicles and thus the insensitivity of their outer carnitine palmitoyltransferase to both agents. Alternatively, carnitine palmitoyltransferase 1 and the regulatory component might be distinct entities whose close association is disrupted by detergents (with loss of inhibitor effectiveness) and whose reassociation during vesicle reconstitution occurs in a way that precludes the ability of inhibitors to bind and/or inhibit the enzyme. Another possibility is that the regulatory protein is not reincorporated into the vesicles.

A study by Kiorpes et al. (15) may be in accord with a separate regulatory site. These authors showed that labeled TG-CoA bound to a 90?000 molecular weight protein in rat liver mitochondria that was presumed to be a subunit of carnitine palmitoyltransferase I. On the other hand, investi- gators who have extensively purified carnitine palmitoyltransferase from the same source reported its subunit molecular weight on sodium dodecyl sulfate-polyacry~~de gel electro- phoresis to be closer to 6 9 ? ~ 0 (22,25). Is it possible that what Kiorpes et a$. (15) detected was in fact a TG-bound regulatory subunit for carnitine palmitoyltransferase I? The use of highly labeled TG-CoA followed by isolation of its binding protein and comparison of its properties with those of purified carnitine palmitoyltransferase should help in resolving this im- portant question.

Although we have interpreted the present findings to mean that malonyl-CoA and TG-CoA may not be active site-di- rected inhibitors of carnitine palmitoyltransferase I, one ca- veat must be expressed. We have assumed that regardless of whether carnitine palmitoyltransferase I and carnitine palmitoyltransferase I1 are the same or different proteins, both are released from the membrane by octylglucoside and digi-

12522 R ~ g u ~ t ~ n of ~ ~ r n i t i ~

tonin in active form. It is conceivable that the two enzymes are distinct (as suggested by Kopec and Fritz (26)) and that solubilization results in selective inactivation of carnitine palmitoyltransferase I. If so, the species measured. in solubilized preparations would represent only carnitine palmitoyltransferase 11, irrespective of whether carnitine palmitoyltransferase I in the original mitochondria had been inhibited by TG-CoA. While this might be considered a remote possibility it would nevertheless be consistent with the data pre- sented here, and, in our opinion, deserves further exploration. If true, some of our conclusions might require modification. We are presently investigating this issue.

A c k n o ~ ~ ~ m e n ~ - W e are grateful to Karen Thatcher and Mur- phy Daniels for expert technical assistance.

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Interaction of Malonyl-CoA and 2-Tetradecylglycidyl-CoA with ...