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Effects of pH on the interaction of substrates and malonyl-CoA with mitochondrial carnitine palmitoyltransferase I.

ABSTRACT: The kinetics of carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) were examined in mitochondria from rat liver, heart and skeletal muscle as a function of pH over the range 6.8-7.6. In all three tissues raising the pH resulted in a fall in the Km for carnitine, no change in the Km for palmitoyl-CoA or Octanoyl-CoA, and a marked decrease in the inhibitory potency of malonyl-CoA. Studies with skeletal-muscle mitochondria established that increasing pH was accompanied by an increase in the Kd of the malonyl-CoA binding site for this ligand, coupled with a decrease in the Kd for fatty acyl-CoA species to compete for malonyl-CoA binding. Three principal conclusions are drawn. (1) The pH-induced shift in malonyl-CoA sensitivity of CPT I is not a phenomenon restricted to liver mitochondria. (2) At any given pH within the range tested, the ability of malonyl-CoA (and closely related compounds) to inhibit enzyme activity is governed by the efficiency of their binding to the malonyl-CoA site. (3) The competitive interaction between fatty acyl-CoA substrates and malonyl-CoA as regards CPT I activity is exerted at the malonyl-CoA binding site. Finally, the possibility is strengthened that the malonyl-CoA binding site is distinct from the active site of CPT I.

SUBMITTER: Mills SE

PROVIDER: S-EPMC1153518 | biostudies-other | 1984 Apr

REPOSITORIES: biostudies-other

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Effect of pH on malonyl-CoA inhibition of carnitine palmitoyltransferase I.

Project description:Malonyl-CoA inhibition of carnitine palmitoyltransferase I was found to be very pH-dependent. Malonyl-CoA concentrations causing 50% inhibition (I50) at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were 0.04, 1, 9, 40 and 200 microM respectively. It is suggested that a lowering of intracellular pH, such as might occur in ketoacidosis, may attenuate hepatic fatty acid oxidation by increasing malonyl-CoA sensitivity of carnitine palmitoyltransferase I.

| S-EPMC1152076 | biostudies-other

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

Project description: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.

| S-EPMC1152599 | biostudies-other

Characterization of a solubilized malonyl-CoA-sensitive carnitine palmitoyltransferase from the mitochondrial outer membrane as a protein distinct from the malonyl-CoA-insensitive carnitine palmitoyltransferase of the inner membrane.

Project description:By using octyl glucoside in the presence of glycerol, it is possible to obtain a solubilized malonyl-CoA-sensitive carnitine palmitoyltransferase (CPTo) from the outer membranes of rat liver mitochondria. H.p.l.c. on hydroxyapatite column has now allowed a clear separation of the CPTo from the malonyl-CoA-insensitive CPT activity of the inner membranes (CPTi). The separated CPTo activity showed inhibition by low micromolar concentrations of malonyl-CoA, 2-tetradecylglycidyl-CoA and etomoxir-CoA. On solubilization and fractionation, the CPTo rapidly lost activity, unlike the relatively stable CPTi activity. Reconstitution into asolectin liposomes enhanced the activity and the malonyl-CoA-sensitivity of the CPTo fractions, whereas it had no such effect on the activity or malonyl-CoA insensitivity of the CPTi fractions. A polyclonal antibody raised against the malonyl-CoA-insensitive enzyme, purified from the inner membranes, precipitated the CPTi activity, but showed no reactivity with the CPTo fractions. In Western blots, the above antibody did not react with any polypeptide of the CPTo fractions. Incubation of the outer-membrane preparations with [3H]etomoxir, in the presence of ATP and CoA, led to labelling of a 90 kDa polypeptide that in the above hydroxyapatite chromatography was eluted in the same region as the CPTo. No such polypeptide labelling was seen in the CPTi fractions. With heart and skeletal-muscle mitochondria, the correspondingly labelled polypeptide was of about 86 kDa. These results show that the CPTo and CPTi are distinct proteins, that a subunit of 90 kDa for liver and 86 kDa for muscle constitutes a component of their respective CPTo systems, and that the 66 kDa subunit of the CPTi does not constitute a part of the CPTo system.

| S-EPMC1131480 | biostudies-other

Involvement of hysteretic effects in the inhibition of carnitine palmitoyltransferase by malonyl-CoA.

Project description:Carnitine palmitoyltransferase in its normal mitochondrial environment behaves as a hysteretic enzyme, exhibiting slow changes in reaction rate after the addition of oleoyl-CoA or malonyl-CoA. Reaction rates become constant after a short time, but the sensitivity of the enzyme from fed rats to the inhibition by malonyl-CoA remains much greater than that of starved rats.

| S-EPMC1144541 | biostudies-other

Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes.

Project description:1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

| S-EPMC1131248 | biostudies-other

The relationship of rat liver overt carnitine palmitoyltransferase to the mitochondrial malonyl-CoA binding entity and to the latent palmitoyltransferase.

Project description:1. Confirming previous work [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPT1) from rat liver was found to be localized in outer rather than in inner mitochondrial membranes. 2. Antisera were raised against a liver mitochondrial CPT of Mr 68,000, which was presumed to be the latent from of the enzyme (CPT2). These antisera cross-reacted with solubilized CPT extracted from liver inner mitochondrial membranes and with polypeptides of Mr 68,000 and 60,000 in immunoblots of both inner and outer mitochondrial membranes. The antisera also precipitated CPT activity from detergent-treated total membrane and outer-membrane preparations. 3. The antisera did not precipitate [14C]malonyl-CoA binding material obtained either from total membranes or from outer membranes. 4. It was concluded that liver CPT1 and CPT2 have some epitopes in common and may have a similar subunit size. In addition, CPT1 and the entity that binds malonyl-CoA must be separated polypeptides.

| S-EPMC1131802 | biostudies-other

Carnitine palmitoyltransferase in human erythrocyte membrane. Properties and malonyl-CoA sensitivity.

Project description:Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids.

| S-EPMC1150109 | biostudies-other

Hysteretic behaviour of carnitine palmitoyltransferase. The effect of preincubation with malonyl-CoA.

Project description: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.

| S-EPMC1146927 | biostudies-other

Sensitivity of brown-adipose-tissue carnitine palmitoyltransferase to inhibition by malonyl-CoA.

Project description:Overt carnitine palmitoyltransferase in mitochondria isolated from interscapular brown adipose tissue of cold-adapted rats or rats maintained at normal temperature is extremely sensitive to inhibition by malonyl-CoA.

| S-EPMC1158357 | biostudies-other

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.

Project description: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.

| S-EPMC1152600 | biostudies-other

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