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.     

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.     

Sigmoid kinetics of purified beef heart mitochondrial carnitine palmitoyltransferase. Effect of pH and malonyl-CoA

The kinetics of purified beef heart mitochondrial carnitine palmitoyltransferase have been extensively investigated with a semiautomated system and the computer program TANKIN and shown to be sigmoidal with both acyl-CoA and L-carnitine. In contrast, Michaelis-Menten kinetics were found with carnitine octanoyltransferase. The catalytic activity of carnitine palmitoyltransferase is strongly pH dependent. The K0.5 and Vmax are both greater at lower pH. The K0.5 for palmitoyl-CoA is 1.9 and 24.2 microM at pH 8 and 6, respectively. The K0.5 for L-carnitine is 0.2 and 2.9 mM at pH 8 and 6, respectively. Malonyl-CoA (20-600 microM) had no effect on the kinetic parameters for palmitoyl-CoA at both saturating and subsaturating levels of L-carnitine. We conclude that malonyl-CoA is not a competitive inhibitor of carnitine palmitoyltransferase. The purified enzyme contained 18.9 mol of bound phospholipid/mol of enzyme which were identified as cardiolipin, phosphatidylethanolamine, and phosphatidylcholine by thin-layer chromatography. The data are consistent with the conclusion that native carnitine palmitoyltransferase exhibits different catalytic properties on either side of the inner membrane of mitochondria due to its non-Michaelis-Menten kinetic behavior, which can be affected by pH differences and differences in membrane environment.     

Carnitine octanoyltransferase of mouse liver peroxisomes: properties and effect of hypolipidemic drugs

Carnitine octanoyltransferase (COT) in 500g supernatant fluids from mouse liver has a specific activity at least twice that of carnitine acetyltransferase (CAT) or carnitine palmitoyltransferase (CPT). When mice are fed diets containing the lipid-lowering drugs, clofibrate or nafenopin, the specific activity of COT increases 4- and 11-fold, respectively. Liver homogenates from mice fed a control diet, and diets containing clofibrate, nafenopin, or Wy-14,643 were fractionated by sucrose gradient centrifugation, and the subcellular distribution of carnitine acyltransferases was determined. In the controls, peroxisomes contained about 70% of the total COT. The specific activity of COT in the peroxisomal peak was 12-fold greater than either CAT or CPT, and 20-fold greater than the COT activity in the mitochondrial fraction. Treatment with hypolipidemic drugs increased the specific activity of peroxisomal COT 2- to 3-fold and CAT 6- to 12-fold, while mitochondrial COT increased 5- to 11-fold and CAT 19- to 54-fold. COT was purified to homogeneity from livers of mice treated with Wy-14,643. It had an apparent Mr of 60,000 by Sephadex G-100 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a maximum activity for octanoyl-CoA with acetyl-CoA and palmitoyl-CoA having activities of 2 and 10%, respectively, when 100 microM acyl-CoA substrates were used. The Km's for 1-carnitine, octanoyl-CoA, palmitoyl-CoA, and acetyl-CoA were 130, 15, 69, and 155 microM, respectively, in the forward direction; and in the reverse direction were 110, 100, 104, and 783 microM for CoASH, octanoylcarnitine, palmitoylcarnitine, and acetylcarnitine, respectively. With Vmax conditions, acetyl-CoA and palmitoyl-CoA had activities of 8 and 26% of the activity for octanoyl-CoA, and acetylcarnitine and palmitoylcarnitine had activities of 7 and 22%, respectively, of the activity for octanoylcarnitine. It is concluded that COT is a separate enzyme present in large amounts in the matrix of mouse liver peroxisomes, with kinetic properties that greatly favor medium-chain acylcarnitine formation.     

Hepatic mitochondrial inner-membrane properties, beta-oxidation and carnitine palmitoyltransferases A and B. Effects of genetic obesity and starvation.

Hepatic mitochondrial carnitine palmitoyltransferase (CPT) properties, beta-oxidation of palmitoyl-CoA and membrane polarization were measured in lean and obese Zucker rats. The Vmax. of the 'outer' carnitine palmitoyltransferase ('CPT-A') increased with starvation, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA increased with starvation in lean rats, but not in obese rats. The Vmax. of the 'inner' enzyme ('CPT-B'), as measured by using inverted submitochondrial vesicles, increased with starvation in obese rats only, with no change in the Km for either carnitine or palmitoyl-CoA. The Ki for malonyl-CoA was 2-5-fold higher in inverted vesicles than in intact mitochondria, and showed no alteration with starvation. The activities of both enzymes correlated positively with each other and with beta-oxidation, and inversely with membrane polarization. Malonyl-CoA had little effect on gross membrane fluidity in the Zucker rat, as reflected by diphenylhexatriene fluorescence polarization. The results indicate that both enzymes are related and respond similarly to alterations in membrane fluidity. Membrane fluidity may provide a mechanism for co-ordinated control of CPT activity on both sides of the mitochondrial inner membrane.     

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.     

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.     

Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver

The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.     

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.     

Purification and properties of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Peroxisomal carnitine palmitoyltransferase was purified by solubilization using Tween 20 and KCl from the large granule fraction of the liver of clofibrate-treated chick embryo, DEAE-Sephacel and blue Sepharose CL-6B column chromatography. The peroxisomal carnitine palmitoyltransferase was an Mr 64,000 polypeptide; the mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carnitine acetyltransferase was an Mr 64,000 polypeptide. Antibody against purified peroxisomal carnitine palmitoyltransferase reacted only with peroxisomal carnitine palmitoyltransferase, but not with mitochondrial carnitine palmitoyltransferase or carnitine acetyltransferase. In addition, anti-peroxisomal carnitine palmitoyltransferase reacted only with the protein in peroxisomes purified from chick embryo liver by sucrose density gradient centrifugation. Thus, it was confirmed that purified peroxisomal carnitine palmitoyltransferase was a peroxisomal protein. Compared with mitochondrial carnitine palmitoyltransferase, peroxisomal carnitine palmitoyltransferase was extremely resistant to inactivation by trypsin. The pH optimum of peroxisomal carnitine palmitoyltransferase was 8.5, differing from that of mitochondrial carnitine palmitoyltransferase. The Km value of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA (32 microM) was similar to that of the mitochondrial one, whereas those values for L-carnitine (140 microM), palmitoyl-L-carnitine (43 microM) and CoA (9 microM) were lower than those of mitochondrial carnitine palmitoyltransferase. Peroxisomal carnitine palmitoyltransferase exhibited similar substrate specificities in both the forward and reverse reactions, with the highest activity toward lauroyl derivatives. Furthermore, this enzyme showed relatively high affinities for long-chain acyl derivatives (C10-C16) and similar Km values (30-50 microM) for acyl-CoAs, acylcarnitine and CoA, and a constant Km value (approximately 150 microM) for carnitine. These results indicate that peroxisomal carnitine palmitoyltransferase played a role in the modulation of the intracellular CoA/long-chain acyl-CoA ratio at the hatching stage of chicken when long-chain fatty acids are actively oxidized in peroxisomes.     

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.     

Importance of albumin binding in the assay for carnitine palmitoyltransferase.

Alterations in the long-chain acyl-CoA binding to albumin in the carnitine palmitoyltransferase (CPT) assay appreciably affect the reaction at commonly used substrate concentrations. Since in the CPT assay the latter are typically well below saturation or Vmax. values, the measured enzyme activity depends on both the absolute quantity of albumin in the CPT assay and any biochemical modification of its binding. The present study verifies the striking dependence of the K0.5 for palmitoyl-CoA on albumin and the misleading 'activation' of the enzyme by compounds that also avidly bind to albumin. In assessing the intracellular physiological relevance of any modifier of CPT, the effects of protein binding in the assay assume particular importance. Indeed, any compound that alters CPT activity may do so, not directly, but as an assay artifact changing the free or unbound substrate concentrations.     

The localization of palmitoyl-CoA: Carnitine palmitoyltransferase in rat liver

1. The distribution of palmitoyl-CoA:carnitine palmitoyltransferase has been studied in subcellular fractions of rat liver. By using two different estimations for the enzyme activity and by differential centrifugation and linear sucrose density gradient centrifugation, the enzyme is shown to be localized both in mitochondria and microsomes.

2. The mitochondrial palmitoyl-CoA: carnitine palmitoyltransferase is localized in the inner membrane plus matrix fraction.

3. During palmitate oxidation by isolated mitochondria, in the presence of a physiological concentration of carnitine, palmitoylcarnitine accumulates. From this and experiments with sonicated mitochondria, it is concluded that the capacities of long-chain fatty acid activation and of palmitoyl-CoA:carnitine palmitoyltransferase in vitro by far exceed the capacity of fatty acid oxidation.     

Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its co-enzyme A and carnitine esters

1. The CoA and carnitine esters of 2-bromopalmitate are extremely powerful and specific inhibitors of mitochondrial fatty acid oxidation. 2. 2-Bromopalmitoyl-CoA, added as such or formed from 2-bromopalmitate, inhibits the carnitine-dependent oxidation of palmitate or palmitoyl-CoA, but not the oxidation of palmitoylcarnitine, by intact liver mitochondria. 3. 2-Bromopalmitoylcarnitine inhibits the oxidation of palmitoylcarnitine as well as that of palmitate or palmitoyl-CoA. It has no effect on succinate oxidation, but inhibits that of pyruvate, 2-oxoglutarate or hexanoate; however, the oxidation of these substrates (but not of palmitate, palmitoyl-CoA or palmitoyl-carnitine) is restored by carnitine. 4. In damaged mitochondria, added 2-bromopalmitoyl-CoA does inhibit palmitoylcarnitine oxidation; pyruvate oxidation is unaffected by the inhibitor alone, but is impaired if palmitoylcarnitine is subsequently added. 5. The findings have been interpreted as follows. 2-Bromopalmitoyl-CoA inactivates (in a carnitine-dependent manner) a pool of carnitine palmitoyltransferase which is accessible to external acyl-CoA. This results in inhibition of palmitate or palmitoyl-CoA oxidation. A second pool of carnitine palmitoyltransferase, inaccessible to added acyl-CoA in intact mitochondria, can generate bromopalmitoyl-CoA within the matrix from external 2-bromopalmitoylcarnitine; this reaction is reversible. Such internal 2-bromopalmitoyl-CoA inactivates long-chain beta-oxidation (as does added 2-bromopalmitoyl-CoA if the mitochondria are damaged) and its formation also sequesters intramitochondrial CoA. Since this CoA is shared by pyruvate and 2-oxoglutarate dehydrogenases, the oxidation of their substrates is depressed by 2-bromopalmitoylcarnitine, unless free carnitine is available to act as a ;sink' for long-chain acyl groups. 6. These effects are compared with those reported for other inhibitors of fatty acid oxidation.     

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.     

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.     

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.     

Correlation between the cellular level of long-chain acyl-CoA, peroxisomal beta-oxidation, and palmitoyl-CoA hydrolase activity in rat liver. Are the two enzyme systems regulated by a substrate-induced mechanism?

Data obtained in earlier studies with rats fed diets containing high doses of peroxisome proliferators (niadenate, tiadenol, clofibrate, or nitotinic acid) are used to look for a quantitative relationship between peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities, and the cellular concentration of their substrate and reaction products. The order of the hyperlipidemic drugs with regard to their effect on CoA derivatives and enzyme activities was niadenate greater than tiadenol greater than clofibrate greater than nicotinic acid. Linear regression analysis of long-chain acyl-CoA content versus palmitoyl-CoA hydrolase and peroxisomal beta-oxidation activity showed highly significant linear correlations both in the total liver homogenate and in the peroxisome-enriched fractions. A dose-response curve of tiadenol showed that carnitine palmitoyltransferase and palmitoyl-CoA synthetase activities and the ratio of long-chain acyl-CoA to free CoASH in total homogenate rose at low doses before detectable changes occurred in the peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. A plot of this ratio parallelled the palmitoyl-CoA synthetase activity. The specific activity of microsomally localized carnitine palmitoyl-transferase was low and unchanged up to a dose where no enhanced peroxisomal beta-oxidation was observed, but over this dose the activity increased considerably so that the specific of the enzyme in the mitochondrial and microsomal fractions became comparable. The mitochondrial palmitoyl-CoA synthetase activity decreased gradually. The correlations may be interpreted as reflecting a common regulation mechanism for palmitoyl-CoA hydrolase and peroxisomal beta-oxidation enzymes, i.e., the cellular level of long-chain acyl-CoA acting as the metabolic message for peroxisomal proliferation resulting in induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The findings are discussed with regard to their possible consequences for mitochondrial fatty acid oxidation and the conversion of long-chain acyl-L-carnitine to acyl-CoA derivatives.     

The effect of palmitoyl-CoA binding to albumin on the apparent kinetic behavior of carnitine palmitoyltransferase I

Substrate saturation plots of carnitine palmitoyltransferase I activity from isolated rat liver mitochondria vs. palmitoyl-CoA concentration in the presence of bovine serum albumin have been reported to yield sigmoidal kinetics. Under identical assay conditions we have confirmed these observations as reflected by nonlinear Lineweaver-Burke plots (1/ν₁ vs. 1|S|) and an average Hill coefficient of n_app. = 1.98 ± 0.09 (Mean±S.E. from four separate experiments). For these determinations the enzyme activity was plotted against the total [palmitoyl-CoA] in the presence of 0.13% bovine serum albumin. Utilizing the total [palmitoyl-CoA] to determine the kinetic properties of carnitine palmitoyltransferase I would be valid only if the relationship between total and free [palmitoyl-CoA] was linear, which is not the case as we have previously shown. When carnitine palmitoyltransferase I substrate saturation kinetics were reanalyzed using the previously determined free [palmitoyl-CoA]'s, the plots revealed a shift to standard hyperbolic kinetics. This observation was confirmed by an average Hill coefficient of n_app. = 1.04 ± 0.10 (Mean±S.E.) and linear Lineweaver-Burke plots. The double-reciprocal plots from these analyses yielded an average S_0.5 of 2.55 ± 0.82 μM(Mean±S.E.) palmitoyl-CoA and V_max of 19.69 ± 5.48 nmol/min per mg protein. These studies clearly demonstrate the importance of defining the free [palmitoyl-CoA] when analyzing the kinetics of carnitine palmitoyltransferase I in the presence of bovine serum albumin.     

Preventive effect of clofibrate on carnitine palmitoyltransferase I inhibition and mitochondrial membrane phospholipid depletion induced by galactosamine

We have previously reported that a D-galactosamine injection induces a decrease of carnitine palmitoyltransferase I activity correlated with a depletion of total phospholipid content in the mitochondrial membrane. The impact of a short-term clofibrate treatment on these membrane alterations is investigated, i.e., the kinetic properties of carnitine palmitoyltransferase I, including its sensitivity to malonyl-CoA and mitochondrial membrane content of the various phospholipids. A 4-day clofibrate treatment increases by 42% the apparent Km value of carnitine palmitoyltransferase I for palmitoyl-CoA, while the sensitivity of the enzyme to malonyl-CoA appears slightly decreased. Simultaneously, the cardiolipin content is increased by 70% in the mitochondrial membrane, whereas the phosphatidylethanolamine and phosphatidylcholine contents remain almost unaffected. This 4-day clofibrate treatment prevents the inhibition of carnitine palmitoyltransferase I activity subsequent to galactosamine administration but induces an increase in the apparent Km value for palmitoyl-CoA and a decrease of the sensitivity of the enzyme to malonyl-CoA. The contents of phospholipids which are decreased by galactosamine (phosphatidylcholine, -21%; phosphatidylethanolamine, -29%; cardiolipin, -40%) regain the control values when galactosamine administration is preceded by a clofibrate treatment. The data suggest that the clofibrate treatment counteracts the inhibition of activity of carnitine palmitoyltransferase I through the maintenance of mitochondrial membrane integrity.