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

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.     

A proportion of rat liver mitochondrial carnitine palmitoyltransferase can be made activatable by malonyl-CoA

Treatment of rat liver mitochondrial membranes with cholate yields a soluble extract containing carnitine palmitoyltransferase (CPT) activity that is insensitive to malonyl-CoA. As found previously (I. Ghadiminejad and D. Saggerson (1990) FEBS Lett. 269, 406-408), addition of polyethylenen glycol 6000 (PEG 6000) to this extract conferred sensitivity to malonyl-CoA on the CPT. It is now shown that a sub-population of the CPT activity which is sedimentable at 7000 x g after addition of PEG 6000 is activated by malonyl-CoA, whereas the remainder is inhibited by malonyl-CoA. The presence of KCl increases the proportion of the activatable form of CPT. Possible physiological significance of this finding is discussed.     

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.     

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

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.     

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

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.     

Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition

Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ari?o, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.     

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.     

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

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.     

Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity.

In the fed state, hyperthyroidism increased glucose utilization indices (GUIs) of skeletal muscles containing a lower proportion of oxidative fibres. Glycogen concentrations were unchanged, but active pyruvate dehydrogenase (PDHa) activities were decreased. Hyperthyroidism attenuated the effects of 48 h of starvation to decrease muscle GUI. Glycogen concentrations and PDHa activities after 48 h of starvation were low and similar in euthyroid and hyperthyroid rats. The increase in glucose uptake and phosphorylation relative to oxidation and storage in skeletal muscle induced by hyperthyroidism may contribute to increased glucose re-cycling in the fed hyperthyroid state and to glucose turnover in the starved hyperthyroid state.     

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.     

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

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.     

Target size analysis by radiation inactivation of carnitine palmitoyltransferase activity and malonyl-CoA binding in outer membranes from rat liver mitochondria.

The functional molecular sizes of the protein(s) mediating the carnitine palmitoyltransferase I (CPT I) activity and the [14C]malonyl-CoA binding in purified outer-membrane preparations from rat liver mitochondria were determined by radiation-inactivation analysis. In all preparations tested the dose-dependent decay in [14C]malonyl-CoA binding was less steep than that for CPT I activity, suggesting that the protein involved in malonyl-CoA binding may be smaller than that catalysing the CPT I activity. The respective sizes computed from simultaneous analysis for molecular-size standards exposed under identical conditions were 60,000 and 83,000 DA for malonyl-CoA binding and CPT I activity respectively. In irradiated membranes the sensitivity of CPT activity to malonyl-CoA inhibition was increased, as judged by malonyl-CoA inhibition curves for the activity in control and in irradiated membranes that had received 20 Mrad radiation and in which CPT activity had decayed by 60%. Possible correlations between these data and other recent observations on the CPT system are discussed.     

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.     

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.     

Pig liver carnitine palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.     

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.     

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.     

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.     

The extreme C terminus of rat liver carnitine palmitoyltransferase I is not involved in malonyl-CoA sensitivity but in initial protein folding

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.     

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.