Differences in the sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA are due to differences in Ki values

The hepatic carnitine palmitoyltransferase that is present on the outer surface of the mitochondrial inner membrane demonstrates hyperbolic substrate saturation curves with oleoyl-CoA in both fasted and fed rats. However, the addition of malonyl-CoA resulted in sigmoid substrate saturation curves, suggesting that malonyl-CoA induced the cooperative behavior. There was more of the outer carnitine palmitoyltransferase in liver mitochondria derived from fasted rats and that enzyme had a much greater Ki for malonyl-CoA than the enzyme from fed rats, but the Km values were apparently not different. The Dixon plot with mitochondria from fed rats, but not fasted rats, was curved upward, indicating cooperative inhibition by malonyl-CoA. Carnitine palmitoyltransferase of heart mitochondria had a Ki for malonyl-CoA that was much less than that of the liver enzyme and it did not change on fasting. Furthermore, no evidence for cooperative inhibition was found in the heart. The results of these studies indicate that carnitine palmitoyltransferase is not subject to substrate cooperativity and that malonyl-CoA is not a simple competitive inhibitor of this enzyme but inhibits by a mechanism involving cooperative inhibition. The fasting-feeding cycle induces changes in the liver enzyme that alter its affinity for malonyl-CoA without changing its affinity for its acyl-CoA substrate. Carnitine palmitoyltransferase from heart appears to be different from that of liver and is apparently not subject to the same control mechanisms.     

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.     

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)     

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.     

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.     

Effects of DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites.

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.     

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.     

Two mechanisms produce tissue-specific inhibition of fatty acid oxidation by oxfenicine.

Oxfenicine [S-2-(4-hydroxyphenyl)glycine] is transaminated in heart and liver to 4-hydroxyphenylglyoxylate, an inhibitor of fatty acid oxidation shown in this study to act at the level of carnitine palmitoyltransferase I (EC 2.3.1.21). Oxfenicine was an effective inhibitor of fatty acid oxidation in heart, but not in liver. Tissue specificity of oxfenicine inhibition of fatty acid oxidation was due to greater oxfenicine transaminase activity in heart and to greater sensitivity of heart carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate [I50 (concentration giving 50% inhibition) of 11 and 510 microM for the enzymes of heart and liver mitochondria, respectively]. Branched-chain-amino-acid aminotransferase (isoenzyme I, EC 2.6.1.42) was responsible for the transamination of oxfenicine in heart. A positive correlation was found between the capacity of various tissues to transaminate oxfenicine and the known content of branched-chain-amino-acid aminotransferase in these tissues. Out of three observed liver oxfenicine aminotransferase activities, one may correspond to asparagine aminotransferase, but the major activity could not be identified by partial purification and characterization. As reported previously for malonyl-CoA inhibition of carnitine palmitoyltransferase I, 4-hydroxyphenylglyoxylate inhibition of this enzyme was found to be very pH-dependent. In striking contrast with the kinetics of malonyl-CoA inhibition, 4-hydroxyphenylglyoxylate inhibition was not affected by oleoyl-CoA concentration, but was partially reversed by increasing carnitine concentrations.     

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.     

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.     

Milk composition of rats feeding restricted litters.

Continuous assays of carnitine palmitoyltransferase were used to study the hysteretic behaviour of the enzyme. When reactions were started by adding mitochondria to complete reaction mixtures, there was a lag in the assay even in the absence of malonyl-CoA. When mitochondria were preincubated with malonyl-CoA in the absence of palmitoyl-CoA, there was a greater lag period in the assay of carnitine palmitoyltransferase, but this lag was less prominent at 37 degrees C than at 30 degrees C. Preincubation of mitochondria with malonyl-CoA did not change the sensitivity of the enzyme to inhibition by malonyl-CoA.     

Restoration of malonyl-CoA sensitivity of soluble rat liver mitochondria carnitine palmitoyltransferase by reconstitution with a partially purified malonyl-CoA binding protein.

Solubilization of rat liver mitochondria in 5% Triton X-100 followed by chromatography on a hydroxylapatite column resulted in the identification of malonyl-CoA binding protein(s) distinct from a major carnitine palmitoyltransferase activity peak. Further purification of the malonyl-CoA binding protein(s) on an acyl-CoA affinity column followed by sodium dodecyl sulfate gel electrophoresis indicated proteins with Mr mass of 90 and 45-33 kDa. A purified liver malonyl-CoA binding fraction, which was devoid of carnitine palmitoyltransferase, and a soluble malonyl-CoA-insensitive carnitine palmitoyltransferase were reconstituted by dialysis in a liposome system. The enzyme activity in the reconstituted system was decreased by 50% in the presence of 100 microM malonyl-CoA. Rat liver mitochondria carnitine palmitoyltransferase may be composed of an easily dissociable catalytic unit and a malonyl-CoA sensitivity conferring regulatory component.     

Malonyl-CoA inhibits proteolysis of carnitine palmitoyltransferase.

Incubation of isolated mitochondria in the presence of malonyl-CoA prevented proteolysis of the outer carnitine palmitoyltransferase by Nagarse and trypsin. Malonyl-CoA had no direct action on trypsin when present in a chromogenic assay system for proteolysis or when preincubated with the proteases in the absence of mitochondria. As reported previously, Nagarse had a differential effect on carnitine palmitoyltransferase in which malonyl-CoA inhibition was diminished to a greater extent than activity was lost, but all effects were blocked by malonyl-CoA in a concentration-dependent manner. These data suggest a specific effect of binding of malonyl-CoA to carnitine palmitoyltransferase as the protective mechanism.     

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.     

The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive

The data presented herein show that both rough and smooth endoplasmic reticulum contain a medium-chain/long-chain carnitine acyltransferase, designated as COT, that is strongly inhibited by malonyl-CoA. The average percentage inhibition by 17 microM malonyl-CoA for 25 preparations is 87.4 +/- 11.7, with nine preparations showing 100% inhibition; the concentrations of decanoyl-CoA and L-carnitine were 17 microM and 1.7 mM, respectively. The concentration of malonyl-CoA required for 50% inhibition is 5.3 microM. The microsomal medium-chain/long-chain carnitine acyltransferase is also strongly inhibited by etomoxiryl-CoA, with 0.6 microM etomoxiryl-CoA producing 50% inhibition. Although palmitoyl-CoA is a substrate at low concentrations, the enzyme is strongly inhibited by high concentrations of palmitoyl-CoA; 50% inhibition is produced by 11 microM palmitoyl-CoA. The microsomal medium-chain/long-chain carnitine acyltransferase is stable to freezing at -70 degrees C, but it is labile in Triton X-100 and octylglucoside. The inhibition by palmitoyl-CoA and the approximate 200-fold higher I50 for etomoxiryl-CoA clearly distinguish this enzyme from the outer form of mitochondrial carnitine palmitoyltransferase. The microsomal medium-chain/long-chain carnitine acyltransferase is not inhibited by antibody prepared against mitochondrial carnitine palmitoyltransferase, and it is only slightly inhibited by antibody prepared against peroxisomal carnitine octanoyltransferase. When purified peroxisomal enzyme is mixed with equal amounts of microsomal activity and the mixture is incubated with the antibody prepared against the peroxisomal enzyme, the amount of carnitine octanoyltransferase precipitated is equal to all of the peroxisomal carnitine octanoyltransferase plus a small amount of the microsomal activity. This demonstrates that the microsomal enzyme is antigenically different than either of the other liver carnitine acyltransferases that show medium-chain/long-chain transferase activity. These results indicate that medium-chain and long-chain acyl-CoA conversion to acylcarnitines by microsomes in the cytosolic compartment is also modulated by malonyl-CoA.     

Conferral of malonyl coenzyme A sensitivity to purified rat heart mitochondrial carnitine palmitoyltransferase.

An immunoaffinity column against the 86-kDa malonyl-CoA-binding protein of beef heart mitochondria was prepared, and the properties of the eluates were compared to those of eluates of an anti-carnitine palmitoyltransferase immunoaffinity column. Both eluates contain seven to eight major proteins with a malonyl-CoA-binding capacity of approximately 5 nmol/mg of protein; in contrast, the eluates from a preimmune IgG column did not contain any of the major proteins. The eluates from both immunoaffinity columns conferred malonyl-CoA sensitivity to purified rat heart mitochondrial carnitine palmitoyltransferase (CPTi/CPT-II). Addition of phospholipids increased the degree of malonyl-CoA inhibition. Doubling the amount of column eluate approximately doubled the malonyl-CoA sensitivity when added to a fixed amount of CPT; i.e., the inhibition increased from 32 to 67%. These results show that CPTi/CPT-II is capable of exhibiting malonyl-CoA sensitivity in the presence of malonyl-CoA-binding proteins. The results do not support the concept that the 86-kDa malonyl-CoA-binding protein is detergent-inactivated carnitine palmitoyltransferase I;rather, they suggest that it is a regulatory subunit of a carnitine palmitoyltransferase complex.     

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. I. Use of inhibitors

The effects of various inhibitors of carnitine palmitoyltransferase I were examined in mitochondria from rat liver and skeletal muscle. Three types of inhibitors were used: malonyl-CoA (reversible), tetradecylglycidyl-CoA and three of its analogues (irreversible), and 2-bromopalmitoyl-CoA (essentially irreversible when added with carnitine). Competitive binding studies between labeled and unlabeled ligands together with electrophoretic analysis of sodium dodecyl sulfate-solubilized membranes revealed that in mitochondria from both tissues all of the inhibitors interacted with a single protein. While the binding capacity for inhibitors was similar in liver and muscle (6-8 pmol/mg of mitochondrial protein) the proteins involved were of different monomeric size (Mr 94,000 and 86,000, respectively). Treatment of mitochondria with the detergent, octyl glucoside, yielded a soluble form of carnitine palmitoyltransferase and residual membranes that were devoid of enzyme activity. The solubilized enzyme displayed the same activity regardless of whether carnitine palmitoyltransferase I of the original mitochondria had first been exposed to an irreversible inhibitor or destroyed by chymotrypsin. It eluted as a single activity peak through four purification steps. The final product from both liver and muscle migrated as single band on sodium dodecyl sulfate-polyacrylamide electrophoresis with Mr of approximately 80,000. The data are consistent with the following model. The inhibitor binding protein is carnitine palmitoyltransferase I itself (as opposed to a regulatory subunit). The hepatic monomer is larger than the muscle enzyme. Each inhibitor interacts via its thioester group at the palmitoyl-CoA binding site of the enzyme but also at a second locus that is probably different for each agent and dictated by the chemical substituent on carbon 2. Disruption of the mitochondrial inner membrane by octyl glucoside causes inactivation of carnitine palmitoyltransferase I while releasing carnitine palmitoyltransferase II in active form. The latter is readily purified, is a smaller protein than carnitine palmitoyltransferase I, and has the same molecular weight in liver and muscle. It is insensitive to inhibitors where on or off the mitochondrial membrane.     

Distinct kinetics of carnitine palmitoyltransferase i in contact sites and outer membranes of rat liver mitochondria

Carnitine palmitoyltransferase I (CPT I) of rat liver mitochondria is an integral, polytopic protein of the outer membrane that is enriched at contact sites. As CPT I kinetics are highly dependent on its membrane environment, we have measured the kinetic parameters of CPT I present in rat liver submitochondrial membrane fractions enriched in either outer membrane or contact sites. The K(m) for palmitoyl-CoA was 2.4-fold higher for CPT I in outer membranes than that for the enzyme in contact sites. In addition, whereas in contact sites malonyl-CoA behaved as a competitive inhibitor of CPT I with respect to palmitoyl-CoA, in outer membranes malonyl-CoA inhibition was non-competitive. As a result of the combination of these changes, the IC(50) for malonyl-CoA was severalfold higher for CPT I in contact sites than for the enzyme in bulk outer membrane. The K(i) for malonyl-CoA, the K(m) for carnitine, and the catalytic constant of the enzyme were all unaffected. It is concluded that the different membrane environments in outer membranes and contact sites result in an altered conformation of L-CPT I that specifically affects the long-chain acyl-CoA binding site. The accompanying changes in the kinetics of the enzyme provide an additional potent mechanism for the regulation of L-CPT I activity.     

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.     

Cycloheximide blocks changes in rat liver carnitine palmitoyltransferase 1 activity in starvation.

Starvation (24h) increased the maximum activity of carnitine palmitoyltransferase 1 in rat liver and increased the concentration of malonyl-CoA required to cause 50% inhibition of the enzyme (I50). Re-feeding (24h) with a standard cube diet or a high-carbohydrate diet reversed both of these changes, whereas re-feeding with a high-fat diet did not. Administration of cycloheximide (200 micrograms/100 g body wt.) blocked the increases in carnitine palmitoyltransferase 1 activity and I50 on starvation. It is suggested that increase in carnitine palmitoyltransferase 1 activity in starvation may involve synthesis of new enzyme.