Effect of malonyl-CoA on the kinetics and substrate cooperativity of membrane-bound carnitine palmitoyltransferase of rat heart mitochondria

The effect of malonyl-CoA on the kinetic parameters of carnitine palmitoyltransferase (outer) the outer form of carnitine palmitoyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) from rat heart mitochondria was investigated using a kinetic analyzer in the absence of bovine serum albumin with non-swelling conditions and decanoyl-CoA as the cosubstrate. The K0.5 for decanoyl-CoA is 3 microM for heart mitochondria from both fed and fasted rats. Membrane-bound carnitine palmitoyltransferase (outer) shows substrate cooperativity for both carnitine and acyl-CoA, similar to that exhibited by the enzyme purified from bovine heart mitochondria. The Hill coefficient for decanoyl-CoA varied from 1.5 to 2.0, depending on the method of assay and the preparation of mitochondria. Malonyl-CoA increased the K0.5 for decanoyl-CoA with no apparent increase in sigmoidicity or Vmax. With 20 microM malonyl-CoA and a Hill coefficient of n = 2.1, the K0.5 for decanoyl-CoA increased to 185 microM. Carnitine palmitoyltransferase (outer) from fed rats had an apparent Ki for malonyl-CoA of 0.3 microM, while that from 48-h-fasted rats was 2.5 microM. The kinetics with L-carnitine were variable: for different preparations of mitochondria, the K0.5 ranged from 0.2 to 0.7 mM and the Hill coefficient varied from 1.2 to 1.8. When an isotope forward assay was used to determine the effect of malonyl-CoA on carnitine palmitoyltransferase (outer) activity of heart mitochondria from fed and fasted animals, the difference was much less than that obtained using a continuous rate assay. Carnitine palmitoyltransferase (outer) was less sensitive to malonyl-CoA at low compared to high carnitine concentrations, particularly with mitochondria from fasted animals. The data show that carnitine palmitoyltransferase (outer) exhibits substrate cooperativity for both acyl-CoA and L-carnitine in its native state. The data show that membrane-bound carnitine palmitoyltransferase (outer) like carnitine palmitoyltransferase purified from heart mitochondria exhibits substrate cooperativity indicative of allosteric enzymes and indicate that malonyl-CoA acts like a negative allosteric modifier by shifting the acyl-CoA saturation to the right. A slow form of membrane-bound carnitine palmitoyltransferase (outer) was not detected, and thus, like purified carnitine palmitoyltransferase, substrate-induced hysteretic behavior is not the cause of the positive substrate cooperativity.     

Altered sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA in ketotic diabetic rats.

Carnitine palmitoyltransferase of liver mitochondria prepared from ketotic diabetic rats has a diminished sensitivity to inhibition by malonyl-CoA compared with carnitine palmitoyltransferase of mitochondria prepared from normal fed rats.     

Interacting effects of L-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase.

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.     

Increased sensitivity of carnitine palmitoyltransferase I activity to malonyl-CoA inhibition after preincubation of intact rat liver mitochondria with micromolar concentrations of malonyl-CoA in vitro.

Carnitine palmitoyltransferase I in rat liver mitochondria preincubated with malonyl-CoA was more sensitive to inhibition by malonyl-CoA than was the enzyme in mitochondria preincubated in the absence of malonyl-CoA. For carnitine palmitoyltransferase I in mitochondria from starved animals this increase also resulted in the enzyme becoming significantly more sensitive than that in mitochondria assayed immediately after their isolation. Concentrations of malonyl-CoA that induced half the maximal degree of sensitization observed were 1-3 microM.     

Ethanol feeding to rats reversibly decreases hepatic carnitine palmitoyltransferase activity and increases enzyme sensitivity to malonyl-CoA

The effects of prolonged ethanol feeding on both carnitine palmitoyltransferase I activity and enzyme sensitivity to inhibition by malonyl-CoA were studied in rat liver, heart, skeletal muscle and kidney cortex mitochondria. Heart and skeletal muscle enzymes showed the highest specific activity and sensitivity to malonyl-CoA. Carnitine palmitoyltransferase I in extrahepatic tissues showed no changes on ethanol feeding. Only the liver enzyme activity was altered after long term ethanol administration, by suffering a progressive decrease in activity and a parallel increase in sensitivity to malonyl-CoA. These alterations reversed after 10 days of ethanol withdrawal. These results are discussed in relation to the control of carnitine palmitoyltransferase I and the effects of ethanol on fatty acid metabolism.     

Interaction of malonyl-CoA and 2-tetradecylglycidyl-CoA with mitochondrial carnitine palmitoyltransferase I

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 reinserted 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 disruption of the inner mitochondrial 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 initially 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 II. 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 of the membrane-bound enzyme.     

Effects of thyroidectomy and starvation on the activity and properties of hepatic carnitine palmitoyltransferase.

1. Hepatic carnitine palmitoyltransferase activity was measured over a range of concentrations of palmitoyl-CoA and in the presence of several concentrations of the inhibitor malonyl-CoA. These measurements were made in mitochondria obtained from the livers of fed and starved (24 h) normal rats and of fed and starved thyroidectomized rats. 2. In the fed state thyroidectomy substantially decreased overt carnitine palmitoyltransferase activity and also decreased both the Hill coefficient and the s0.5 when palmitoyl-CoA concentration was varied as substrate. Thyroidectomy did not appreciably alter the inhibitory effect of malonyl-CoA on the enzyme. 3. Starvation increased overt carnitine palmitoyltransferase activity in both the fed and the thyroidectomized state. In percentage terms this response to starvation was substantially greater after thyroidectomy. In both the hypothyroid and normal states starvation decreased sensitivity to inhibition by malonyl-CoA.     

Hepatic mitochondrial inner membrane properties and carnitine palmitoyltransferase A and B. Effect of diabetes and starvation.

Intact mitochondria and inverted submitochondrial vesicles were prepared from the liver of fed, starved (48 h) and streptozotocin-diabetic rats in order to characterize carnitine palmitoyltransferase kinetics and malonyl-CoA sensitivity in situ. In intact mitochondria, both starved and diabetic rats exhibited increased Vmax., increased Km for palmitoyl-CoA, and decreased sensitivity to malonyl-CoA inhibition. Inverted submitochondrial vesicles also showed increased Vmax. with starvation and diabetes, with no change in Km for either palmitoyl-CoA or carnitine. Inverted vesicles were uniformly less sensitive to malonyl-CoA regardless of treatment, and diabetes resulted in a further decrease in sensitivity. In part, differences in the response of carnitine palmitoyltransferase to starvation and diabetes may reside in differences in the membrane environment, as observed with Arrhenius plots, and the relation of enzyme activity and membrane fluidity. In all cases, whether rats were fed, starved or diabetic, and whether intact or inverted vesicles were examined, increasing membrane fluidity was associated with increasing activity. Malonyl-CoA was found to produce a decrease in intact mitochondrial membrane fluidity in the fed state, particularly at pH 7.0 or less. No effect was observed in intact mitochondria from starved or diabetic rats, or in inverted vesicles from any of the treatment groups. Through its effect on membrane fluidity, malonyl-CoA could regulate carnitine palmitoyltransferase activity on both surfaces of the inner membrane through an interaction with only the outer surface.     

Response to starvation of hepatic carnitine palmitoyltransferase activity and its regulation by malonyl-CoA. Sex differences and effects of pregnancy.

1. Hepatic carnitine palmitoyltransferase activity was measured over a range of concentrations of palmitoyl-CoA and in the presence of several concentrations of the inhibitor malonyl-CoA. These measurements were made in mitochondria obtained from the livers of fed and starved (24 h) virgin female and fed and starved pregnant rats. 2. In the fed state overt carnitine palmitoyltransferase activity was significantly lower in virgin females than in age-matched male rats. 3. Starvation increased overt carnitine palmitoyltransferase activity in both virgin and pregnant females. This increase was larger than in the male and was greater in pregnant than in virgin females. 4. In the fed state pregnancy had no effect on the Hill coefficient or the [S]0.5 when palmitoyl-CoA was varied as substrate. Pregnancy did not alter the sensitivity of the enzyme to inhibition by malonyl-CoA. 5. Starvation decreased the sensitivity of the enzyme to malonyl-CoA. The change in sensitivity was similar in male, virgin female and pregnant rats. 6. The possible relevance of these findings to known sex differences and changes with pregnancy in hepatic fatty acid oxidation and esterification are discussed.     

The effect of malonyl-CoA on overt and latent carnitine acyltransferase activities in rat liver and adipocyte mitochondria.

1. Carnitine palmitoyltransferase and carnitine octanoyltransferase activities were measured in mitochondria at various acyl-CoA concentrations before and after sonication, thus permitting assessment of both overt and latent activities. 2. Overt carnitine palmitoyltransferase in liver and adipocyte mitochondria and overt carnitine octanoyltransferase in liver mitochondria were inhibited by malonyl-CoA. None of the latent activities were affected by this metabolite. 3. 5,5'-Dithiobis-(2-nitrobenzoic acid) stimulated latent hepatic carnitine palmitoyltransferase at low [palmitoyl-CoA]. 4. Starvation (24 h) decreased overt carnitine palmitoyltransferase activity in adipocyte mitochondria, but did not alter the sensitivity of this activity to malonyl-CoA.     

Feeding of lovastatin to rats increases the activity of the hepatic mitochondrial outer carnitine palmitoyltransferase

Administration of lovastatin to male, Sprague-Dawley rats by addition of the drug to the normal chow diet caused a two-fold increase in the activity of the hepatic mitochondrial outer carnitine palmitoyltransferase, but lovastatin apparently did not affect the sensitivity of the outer carnitine palmitoyltransferase to inhibition by malonyl-CoA. There was also no effect of lovastatin on the activity of the hepatic mitochondrial inner carnitine palmitoyltransferase. Feeding of cholestyramine to rats did not affect either the mitochondrial outer carnitine palmitoyltransferase or the mitochondrial inner carnitine palmitoyltransferase.     

Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat.

The requirement for carnitine and the malonyl-CoA sensitivity of carnitine palmitoyl-transferase I (EC 2.3.1.21) were measured in isolated mitochondria from eight tissues of animal or human origin using fixed concentrations of palmitoyl-CoA (50 microM) and albumin (147 microM). The Km for carnitine spanned a 20-fold range, rising from about 35 microM in adult rat and human foetal liver to 700 microM in dog heart. Intermediate values of increasing magnitude were found for rat heart, guinea pig liver and skeletal muscle of rat, dog and man. Conversely, the concentration of malonyl-CoA required for 50% suppression of enzyme activity fell from the region of 2-3 microM in human and rat liver to only 20 nM in tissues displaying the highest Km for carnitine. Thus, the requirement for carnitine and sensitivity to malonyl-CoA appeared to be inversely related. The Km of carnitine palmitoyltransferase I for palmitoyl-CoA was similar in tissues showing large differences in requirement for carnitine. Other experiments established that, in addition to liver, heart and skeletal muscle of fed rats contain significant quantities of malonyl-CoA and that in all three tissues the level falls with starvation. Although its intracellular location in heart and skeletal muscle is not known, the possibility is raised that malonyl-CoA (or a related compound) could, under certain circumstances, interact with carnitine palmitoyltransferase I in non-hepatic tissues and thereby exert control over long chain fatty acid oxidation.     

Carnitine palmitoyltransferase: separation of enzyme activity and malonyl-CoA binding in rat liver mitochondria

Carnitine palmitoyltransferase activity and malonyl-CoA binding capacity have been studied in Triton X-100 extracts and membrane residues of rat liver mitochondria. Rat liver mitochondria extracted twice with 0.5% Triton X-100 in a salt-free medium showed increased specific binding of [2-14C]malonyl-CoA when compared with intact mitochondria. High malonyl-CoA binding required the presence of salts and was inhibited by albumin. Further solubilization of the membrane residues in the Triton/KCl medium and subsequent hydroxylapatite chromatography gave a complete separation of carnitine palmitoyltransferase and malonyl-CoA binding. The results show that malonyl-CoA binds to mitochondrial component(s) which is different from and more difficult to extract from the mitochondrial membrane than most of the carnitine palmitoyltransferase.     

Carnitine palmitoyltransferase in the heart is controlled by a different mechanism than the hepatic enzyme

Diminished sensitivity of hepatic carnitine palmitoyltransferase to inhibition by malonyl-CoA in the fasting and diabetic states is a well-recognized aspect of the regulatory mechanism forhepatic fatty acid oxidation. Inhibition of myocardial carnitine palmitoyltransferase by malonyl-CoA may play an important role in regulation of fatty acid oxidation in the heart, but there has been a discrepancy in data relating to changes in malonyl-CoA sensitivity of the myocardial carnitine palmitoyltransferase during fasting. Analysis of malonyl-CoA inhibition of myocardial carnitine palmitoyltransferase in fasting and fed states under a variety of conditions has indicated that under no condition could any difference be found in malonyl-CoA sensitivity that was attributable to fasting. Proteolysis of the outer carnitine palmitoyltransferase led to artifactual changes in sensitivity due to the appearance of partial inhibition. We have concluded that the sensitivity of myocardial carnitine palmitoyltransferase to malonyl-CoA does not change during fasting. Changes in fatty acid oxidation in the heart are probably due to changes in malonyl-CoA concentrations or to other inhibitors. (Mol Cell Biochem 116: 39–45, 1992)     

Differential inhibition of ketogenesis by malonyl-CoA in mitochondria from fed and starved rats.

Rates of ketogenesis in mitochondria from fed or starved rats were identical at optimal substrate concentrations, but responded differently to inhibition by malonyl-CoA. Kinetic data suggest that the K1 for malonyl-CoA is greater in the starved animal. These results indicate that, for the regulation of ketogenesis in the starved state, the lower sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA may be more important than the concentration of malonyl-CoA.     

Effects of endurance exercise on carnitine palmitoyltransferase I from rat heart, skeletal muscle and liver mitochondria

Prolonged physical exercise increased the activity of carnitine palmitoyltransferase I in rat heart and skeletal muscle mitochondria, whereas enzyme sensitivity to inhibition by malonyl-CoA remained unchanged. Nevertheless, inhibition of carnitine palmitoyltransferase I activity by small decreases in pH was attenuated in heart and skeletal muscle mitochondria from exercised animals. Liver enzyme did not suffer any alteration by endurance exercise.     

Carnitine acyltransferase activities in rat liver and heart measured with palmitoyl-CoA and octanoyl-CoA. Latency, effects of K+, bivalent metal ions and malonyl-CoA.

1. Liver carnitine acyltransferase activities with palmitoyl-CoA and octanoyl-CoA as substrates and heart carnitine palmitoyltransferase were measured as overt activities in whole mitochondria or in mitochondria disrupted by sonication or detergent treatment. All measurements were made in sucrose/KCl-based media of 300 mosmol/litre. 2. In liver mitochondria, acyltransferase measured with octanoyl-CoA, like carnitine palmitoyltransferase, was found to have latent and overt activities. 3. Liver acyltransferase activities measured with octanoyl-CoA and palmitoyl-CoA differed in their response to changes in [K+], Triton X-100 treatment and, in particular, in their response to Mg2+. Mg2+ stimulated activity with octanoyl-CoA, but inhibited carnitine palmitoyltransferase. 4. The effects of K+ and Mg2+ on liver overt carnitine palmitoyltransferase activity were abolished by Triton X-100 treatment. 5. Heart overt carnitine palmitoyltransferase activity differed from the corresponding activity in liver in that it was more sensitive to changes in [K+] and was stimulated by Mg2+. Heart had less latent carnitine palmitoyltransferase activity than did liver. 6. Overt carnitine palmitoyltransferase in heart mitochondria was extremely sensitive to inhibition by malonyl-CoA. Triton X-100 abolished the effect of low concentrations of malonyl-CoA on this activity. 7. The inhibitory effect of malonyl-CoA on heart carnitine palmitoyltransferase could be overcome by increasing the concentration of palmitoyl-CoA.     

Time-dependence of inhibition of carnitine palmitoyltransferase I by malonyl-CoA in mitochondria isolated from livers of fed or starved rats. Evidence for transition of the enzyme between states of low and high affinity for malonyl-CoA.

The degree of inhibition of CPT I (carnitine palmitoyltransferase, EC 2.3.1.21) in isolated rat liver mitochondria by malonyl-CoA was studied by measuring the activity of the enzyme over a short period (15s) after exposure of the mitochondria to malonyl-CoA for different lengths of time. Inhibition of CPT I by malonyl-CoA was markedly time-dependent, and the increase occurred at the same rate in the presence or absence of palmitoyl-CoA (80 microM), and in the presence of carnitine, such that the time-course of acylcarnitine formation deviated markedly from linearity when CPT I activity was measured in the presence of malonyl-CoA over several minutes. The initial rate of increase in degree of inhibition with time was independent of malonyl-CoA concentration. CPT I in mitochondria from 48 h-starved rats had a lower degree of inhibition by malonyl-CoA at zero time, but was equally capable of being sensitized to malonyl-CoA, as judged by an initial rate of increase of inhibition identical with that of the enzyme in mitochondria from fed rats. Double-reciprocal plots for the degree of inhibition produced by different malonyl-CoA concentrations at zero time for the enzyme in mitochondria from fed or starved animals indicated that the enzyme in the latter mitochondria was predominantly in a state with low affinity for malonyl-CoA (concentration required to give 50% inhibition, I0.5 congruent to 10 microM), whereas that in mitochondria from fed rats displayed two distinct sets of affinities: low (congruent to 10 microM) and high (less than 0.3 microM). Plots for mitochondria after incubation for 0.5 or 1 min with malonyl-CoA indicated that the increased sensitivity observed with time was due to a gradual increase in the high-affinity state in both types of mitochondria. These results suggest that the sensitivity of CPT I in rat liver mitochondria in vitro had two components: (i) an instantaneous sensitivity inherent to the enzyme which depends on the nutritional state of the animal from which the mitochondria are isolated, and (ii) a slow, malonyl-CoA-induced, time-dependent increase in sensitivity. It is suggested that the rate of malonyl-CoA-induced sensitization of the enzyme to malonyl-CoA inhibition is limited by a slow first-order process, which occurs after the primary event of interaction of malonyl-CoA with the mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decrease of fatty acid oxidation, ketogenesis and gluconeogenesis in isolated perfused rat liver by phenylalkyl oxirane carboxylate (B 807-27) due to inhibition of CPT I (EC 2.3.1.21)

Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (B 807-27 or POCA) inhibits ketogenesis from endogenous and exogenous long-chain fatty acids and 14CO2 production from [U-14 C]palmitate, but not from [U-14 C]palmitoylcarnitine or octanoate, and inhibits gluconeogenesis from lactate and pyruvate in perfused livers of starved rats. Inhibition of ketogenesis, 14CO2 production and gluconeogenesis was complete at concentrations of 10 mumol/l POCA, but onset was more rapid for inhibition of ketogenesis and CO2 production than for gluconeogenesis. Infusion of octanoate abolished inhibition of all three processes. Experiments with isolated rat liver mitochondria showed that carnitine palmitoyltransferase I (EC 2.3.1.21) is inhibited by POCA-CoA. The inhibitory process is dependent on time and concentration, and more pronounced in mitochondria from fed than from fasted rats. Concentrations required for 50% inhibition after 20 min preincubation with POCA-CoA are 0.02, 0.06 and 0.1 mumol/l in liver mitochondria from fed, 24-h-fasted and 48-h-fasted rats, respectively. The inhibitor appears to be tightly bound to the enzyme. The extent of inhibition of carnitine palmitoyltransferase I correlates well with the hypoglycaemic and hypoketonaemic effects of the compounds in fasted rats. We conclude that specific inhibition of the enzyme leads, due to inhibition of long-chain fatty acid utilization, to depressed ketogenesis and gluconeogenesis and, in consequence, to hypoglycaemic and hypoketonaemia in vivo under gluconeogenic and ketogenic conditions.     

Carnitine palmitoyltransferase: activation and inactivation in liver mitochondria from fed, fasted, hypo- and hyperthyroid rats

The sensitivity of carnitine palmitoyltransferase to malonyl-CoA is lost when liver mitochondria are preincubated in a KCl-containing medium. This loss of sensitivity is slowed down in mitochondria from hypothyroid rats and accelerated in mitochondria from fasted and hyperthyroid rats. Glucagon seems to enhance the effect of fasting. The loss of sensitivity is significantly slowed down by 50-500 nM malonyl-CoA and accelerated by small amounts of palmitoyl-CoA in the preincubation medium.