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Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.

Carnitine palmitoyltransferase I (CPT-I) and II (CPT-II) enzymes are components of the carnitine palmitoyltransferase shuttle system which allows entry of long-chain fatty acids into the mitochondrial matrix for subsequent oxidation. This system is tightly regulated by malonyl-CoA levels since this metabolite is a strong reversible inhibitor of the CPT-I enzyme. There are two distinct CPT-I isotypes (CPT-Ialpha and CPT-Ibeta), that exhibit different sensitivity to malonyl-CoA inhibition. Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels. Paradoxically it also activates long-chain fatty acid oxidation. To identify the exact target of C75 within the CPT system, we expressed individually the different components of the system in the yeast Pichia pastoris. We show here that C75 acts on recombinant CPT-Ialpha, but also on the other CPT-I isotype (CPT-Ibeta) and the malonyl-CoA insensitive component of the CPT system, CPT-II.

The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.

Muscle carnitine palmitoyltransferase I (M-CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine. To determine the role of the C-terminal region of M-CPTI in enzyme activity, we constructed a series of deletion and substitution mutants. The mutants were expressed in the yeast Pichia pastoris, and the effect of the mutations on M-CPTI activity and malonyl-CoA sensitivity was determined in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. Deletion of the last 210, 113, 44, 20, 10, and 9 C-terminal amino-acid residues resulted in an inactive M-CPTI, but deletion of the last 8, 7, 6, and 3 C-terminal residues had no effect on activity, demonstrating that leucine-764 (L764) is essential for catalysis. Substitution of L764 with alanine caused a 40% loss in catalytic activity, but replacement of L764 with arginine resulted in an 84% loss of activity; substitution of L764 with valine had no effect on catalytic activity. The catalytic efficiency for the L764R mutant decreased by 80% for both substrates. Secondary structure prediction of the M-CPTI sequence identified a 21-amino-acid residue, 744-764, predicted to fold into a coiled-coil alpha-helix in the extreme C-terminal region of M-CPTI that may be important for native folding and activity. In summary, our data demonstrate that deletion of L764 or substitution with arginine inactivates the enzyme, suggesting that L764 may be important for proper folding of M-CPTI and optimal activity.

Mitochondrial carnitine palmitoyltransferases I and II (CPTI and CPTII), together with the carnitine carrier, transport long-chain fatty acyl-CoA from the cytosol to the mitochondrial matrix for beta-oxidation. Recent progress in the expression of CPTI and CPTII cDNA clones in Pichia pastoris, a yeast with no endogenous CPT activity, has greatly facilitated the characterization of these important enzymes in fatty acid oxidation. It is now well established that yeast-expressed CPTI is a catalytically active, malonyl CoA-sensitive, distinct enzyme that is reversibly inactivated by detergents. CPTII is a catalytically active, malonyl CoA-insensitive, distinct enzyme that is detergent stable. Reconstitution studies with yeast-expressed CPTI have established for the first time that detergent inactivation of CPTI is reversible, suggesting that CPTI is active only in a membrane environment. By constructing a series of deletion mutants of the N-terminus of liver CPTI, we have mapped the residues essential for malonyl CoA inhibition and binding to the conserved first six N-terminal amino acid residues. Mutation of glutamic acid 3 to alanine abolished malonyl CoA inhibition and high affinity malonyl CoA binding, but not catalytic activity, whereas mutation of histidine 5 to alanine caused partial loss in malonyl CoA inhibition. Our mutagenesis studies demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl CoA inhibition and binding to liver CPTI, but not catalytic activity.

Long-chain fatty acids are the primary source of energy production in the heart. Carnitine palmitoyltransferase I (CPT-I) catalyzes the first reaction in the transport of long-chain fatty acids from the cytoplasm to the mitochondrion, a rate-limiting step in beta-oxidation. In this study, we report the functional expression of the human heart/skeletal muscle isoform of CPT-I (M-CPT-I) in the yeast Pichia pastoris. Screening of a human heart cDNA library with cDNA fragments encoding the rat heart M-CPT-I resulted in the isolation of a single full-length human heart M-CPT-I cDNA clone. The clone has an open reading frame of 2316 bp with a 5' untranslated region of 38 bp and a 256-bp 3' untranslated region with the poly(A)+ addition sequence AATAAA. The predicted protein has 772 amino acids and a molecular mass of 88 kDa. Northern blot analysis of mRNAs from different human tissues using the human M-CPT-I cDNA as a probe revealed an abundant transcript of approximately 3.1 kb that was only present in human heart and skeletal muscle tissue. Expression of the human M-CPT-I cDNA in P. pastoris, a yeast with no endogenous CPT activity, produced an 80-kDa protein that was located in the mitochondria. Isolated mitochondria from the M-CPT-I expression strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity that was detergent labile. The I50 for malonyl-CoA inhibition of the yeast-expressed M-CPT-I was 69 nM, and the Kms for carnitine and palmitoyl-CoA were 666 and 42 microM, respectively. The I50 for malonyl-CoA inhibition of the heart enzyme is 30 times lower than that of the yeast-expressed liver CPT-I, and the Km for carnitine is more than 20 times higher than that of the liver CPT-I. This is the first report of the expression of a heart CPT-I in a system devoid of endogenous CPT activity and the functional characterization of a human heart M-CPT-I in the absence of the liver isoform and CPT-II., (Copyright 1997 Academic Press. Copyright 1997Academic Press)

The human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene was expressed at high levels from a strain of the methylotrophic yeast Pichia pastoris containing approximately 24 copies of the expression vector. Levels of M-CPTI were more than ten-fold higher than previously reported by our group with a single-copy strain (Arch. Biochem. Biophys., in press) and were sufficient to perform reconstitution studies on the membrane protein, a key step in purification and structural analysis of the enzyme. Solubilization of yeast mitochondria containing M-CPTI in 5% Triton X-100 abolished M-CPTI activity. The detergent-inactivated M-CPTI was then reconstituted by removal of the detergent in the presence of phospholipids. The reconstituted proteoliposomes exhibited M-CPTI activity of 2.4 nmol palmitoylcarnitine formed/mg protein/min, a recovery of 23% of the activity present in the starting mitochondrial preparation. The malonyl-CoA sensitivity of the reconstituted reactivated M-CPTI was 88%. This is the first demonstration of direct reactivation of malonyl-CoA-sensitive M-CPTI activity from solubilized materials from any organism. Previously, M-CPTI was presumed to be irreversibly inactivated by detergents., (Copyright 1997 Academic Press.)

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.

A monoclonal antibody raised against beef heart mitochondria elicited a strong reaction on Western Blot with a 16 kD protein in preparations of beef heart mitochondria, ammonia particles, oligomycin sensitive ATPase and Complex V, in addition to showing a lesser affinity for the partially purified 30 kD ADP/ATP carrier. The antibody also reacted with a 17 kD protein in rat liver mitochondria and an enriched membrane vesicle fraction. The N-terminal sequence of the first twenty amino acids of both the beef heart and rat liver proteins contained significant homology. Comparison with results in the literature indicate that the proteins represent the delta subunit of the ATP synthetase complex. Further evidence suggests that the epitope for the antibody may reside at the C-terminal 30-40 amino acid residues of both proteins.

We have recently synthesized an azido [125I] CoA photolabel, N-(3-iodo-4-azidophenyl propionamide) cysteinyl-5-(2'thiopyridyl cysteine) CoA that specifically labeled the ADP/ATP carrier in beef heart mitochondria. In this study brown adipose tissue mitochondria were photolabeled with the azido [125I] ACT-CoA derivative with or without inhibitors. SDS gel electrophoresis and autoradiography of the separated proteins revealed exclusive photolabeling of two polypeptides corresponding to the ADP/ATP carrier and uncoupling protein. In the presence of carboxyatracytloside only the 32 kD UCP was labeled by [125I] ACT-CoA, whereas preincubation with GDP resulted in exclusive photolabeling of the 30 kD ADP/ATP carrier. Palmitoyl CoA but not palmitic acid inhibited photolabeling of both polypeptides.

A rapid counter-exchange between ATP and phosphoenolpyruvate (PEP) has been demonstrated in pea and maize mesophyll chloroplasts. Chloroplasts preloaded with either [14C] ATP or [14C] PEP readily exchange the radioactive compound with the externally added anions, ATP or PEP, whereas, cold external Pi counter-transports only with internal [14C] PEP. Flooding the system with cold Pi, however, will significantly reduce the counter-transport of external cold PEP with internal [14C] ATP. This ATP-PEP exchange is also markedly decreased by lowering the incubation temperature. The results indicate that the ATP-PEP counter-exchange could represent a key transport system in plant chloroplasts and may be particularly important in the photosynthesis of C4 plants. Furthermore, they provide information required to elucidate the mechanism of the ATP-PEP counter-transport system.

Long-chain acyl-CoA esters were extracted from freeze-clamped livers of fed and fasted rats according to the method of Mancha et al. [M. Mancha, G. B. Stokes, and P. K. Stumpf (1975) Anal. Biochem. 68, 600-608] and analyzed on a radially compressed C18, 5 microns, reverse-phase column using a gradient system consisting of acetonitrile and 25 mM KH2PO4, pH 5.3, at 254 nm. Total analysis time was 25 min. Eight peaks in the extract with carbon chain lengths of 12 to 18, which subsequently disappeared on alkaline hydrolysis, were identified. The major acyl-CoA peaks in the extract in order of increasing retention times were 14:0, 16:1, 18:2, 16:0, 18:1, and 18:0. Total liver long-chain acyl-CoA esters were 108 +/- 11 and 248 +/- 19 nmol/g protein for fed and fasted rats, respectively. On fasting (48 h) the levels of 18:2, 16:0, and 18:1 increased two-to threefold and that of 18:0 sixfold. The advantages of this method are that it not only provides a more direct determination of total tissue long-chain acyl-CoA esters, in that no decomposition of the CoA ester is involved, but it also detects the constituent molecular species.