Carnitine palmitoyltransferase: separation of enzyme activity and malonyl-CoA binding in rat liver mitochondria.

Carnitine palmitoyltransferase activity and malonyl-CoA binding capacity have been studied in Triton X-100 extracts and membrane residues of rat liver mitochondria. Rat liver mitochondria extracted twice with 0.5% Triton X-100 in a salt-free medium showed increased specific binding of [2-14C]malonyl-CoA when compared with intact mitochondria. High malonyl-CoA binding required the presence of salts and was inhibited by albumin. Further solubilization of the membrane residues in the Triton/KCl medium and subsequent hydroxylapatite chromatography gave a complete separation of carnitine palmitoyltransferase and malonyl-CoA binding. The results show that malonyl-CoA binds to mitochondrial component(s) which is different from and more difficult to extract from the mitochondrial membrane than most of the carnitine palmitoyltransferase.

Substrate inhibition of carnitine palmitoyltransferase by palmitoyl-CoA and activation by phospholipids and proteins.

When carnitine palmitoyltransferase is purified it shows increasing substrate inhibition by palmitoyl-CoA as the protein content of the assay mixture is decreased. The purified enzyme is stimulated by addition of phospholipids (phosphatidylcholine, cardiolipin) and proteins (albumin, fatty acid-binding protein, lambda-globulin) to the reaction mixture. The effects of phospholipid and protein are more than additive, particularly with relatively high concentrations of palmitoyl-CoA. It is suggested that the enzyme contains hydrophobic sites which require phospholipid to prevent spurious binding of palmitoyl-CoA and which normally anchor the enzyme to the mitochondrial membrane.

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.

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts.

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.

Photoaffinity labeling of mitochondrial proteins with 2-azido [32P]palmitoyl CoA.

A long-chain fatty acyl CoA photolabel, 2-azido [32P]palmitoyl CoA, was synthesized and its covalent interaction with mitochondrial membrane proteins examined. On binding of 2-azido [32P]palmitoyl CoA to beef heart mitochondria, two polypeptides were primarily labeled, the 30 kDa ADP/ATP carrier and a 41 kDa protein of unknown identity. Carboxyatractyloside and palmitoyl CoA completely protected against labeling of the 30 kDa protein indicating that it was the ADP/ATP carrier. With inverted submitochondrial particles, only the 30 kDa polypeptide was labeled by 2-azido [32P]palmitoyl CoA. The labeling was inhibited by bongkrekic acid and palmitoyl CoA but not carboxyatractyloside, providing evidence that the ADP/ATP carrier was covalently bound from the matrix side of the membrane. In brown adipose tissue mitochondria, 2-azido [32P]palmitoyl CoA photolabeled the ADP/ATP carrier and the 32 kDa uncoupling protein with some minor labeling of 36 and 68 kDa polypeptides. The results indicated that this physiological photolabeling reagent with the azido group on the CoA portion of the molecule interacts like 2-azido ADP with nucleotide binding sites of a number of important enzymes in cell metabolism. Moreover, the evidence strongly supports the hypothesis that long chain fatty acyl CoA esters are natural ligands for key nucleotide binding proteins.

Fatty acyl CoA esters as regulators of cell metabolism.

Long chain fatty acyl CoA esters have the ability to interact with certain proteins and thereby serve as effectors in cell metabolism. In particular, they can displace nucleotides from specific nucleotide dependent or binding proteins and interfere with their action. The ADP/ATP carrier and uncoupling protein are two examples where the interplay of nucleotide and acyl CoA binding to the proteins regulate their function. Other proteins such as glucokinase can be considered in this group. In certain tissues like liver they are affected during fasting and insulin deficiency, and when serum fatty acids and liver acyl CoA levels are elevated. More recently, an acyl CoA binding protein in E. coli has been found to be a transcription factor for gene regulation of fatty acid metabolism enzymes. There appears to be some consensus in the amino acid sequence for acyl CoA binding sites on these proteins which serve a variety of important roles in cellular metabolism.

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system.

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.

Characterization of a low molecular weight protein of the ATP synthetase complex from beef heart and rat liver mitochondria with a high affinity monoclonal antibody.

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.

Specific labeling of beef heart mitochondrial ADP/ATP carrier with N-(3-iodo-4-azidophenylpropionamido)cysteinyl- 5-(2'-thiopyridyl)cysteine-coenzyme A (ACT-CoA), a newly synthesized 125I-coenzyme A derivative photolabel.

An azido-125I-CoA photolabel was synthesized from N-(3-iodo-[125I]4-azidophenylpropionamido)cysteinyl- 5-(2'thiopyridyl) cysteine ([125I]ACTP) and CoASH, separated by chromatography on a silica gel TLC, and identified by autoradiography. Synthesis of [125I]ACT-CoA from [125I]ACTP and CoA was further confirmed by monitoring the release of one of the products, thiopyridone, at 343 nm and concurrent formation of [125I] ACT-CoA. Beef heart mitochondria were incubated in the presence of the 125I-CoA derivative with or without specific inhibitors and/or substrates of the ADP/ATP carrier, and immediately photolyzed for 5 s. Sodium dodecyl sulfate-gel electrophoresis and autoradiography of the separated proteins revealed exclusive photolabeling of a 30-kDa protein in the absence of inhibitors of the carrier. This specific photolabeling of the 30-kDa protein was prevented in a concentration-dependent manner by either carboxyatractylate or palmitoyl-CoA (0.1-5 microM), two known inhibitors of the ADP/ATP carrier. ADP reduced the extent of photolabeling of the 30-kDa protein, but palmitic acid, free CoASH, and dinitrophenol were ineffective, indicating the specificity of the reaction. CoA photolabels may be useful in probing ligand and/or substrate binding sites and in determining the structure-function relationship of the ADP/ATP carrier.

Fatty acyl coenzyme A-sensitive adenine nucleotide transport in a reconstituted liposome system.

The adenine nucleotide translocase was purified from bovine heart mitochondria and incorporated into membranes of phospholipid liposomes. The rate of transport of the adenine nucleotides was competitively inhibited by oleoyl coenzyme A with an approximate Ki of 1.0 microM. Significant inhibition was limited to those fatty acyl coenzyme A esters which are carnitine dependent for their oxidation in isolated mitochondria. Octanoyl coenzyme A was almost completely inactive as was palmitic acid and palmitoyl carnitine. By comparing the inhibitory characteristics of carboxyatractylate and bongkrekic acid with those of oleoyl-CoA, it was determined that the fatty acyl-CoA esters could produce inhibition whether the carrier was inserted into the liposome in either the conventional (65%) or reverse (30%) orientation. The results demonstrate that the interaction of long chain fatty acyl-CoA esters with the ADP/ATP carrier in a purified reconstituted system mimics their effects with isolated mitochondria and inverted submitochondrial particles. In general, these findings are consistent with the role of acyl-CoA esters acting as natural ligands and biological effectors of the translocator.

Functional characterization of mammalian mitochondrial carnitine palmitoyltransferases I and II expressed in the yeast Pichia pastoris.

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.

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.

Reconstitution of highly expressed human heart muscle carnitine palmitoyltransferase I.

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.

A single amino acid change (substitution of glutamate 3 with alanine) in the N-terminal region of rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA inhibition and high affinity binding.

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.

The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding.

Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.

Identification by mutagenesis of conserved arginine and tryptophan residues in rat liver carnitine palmitoyltransferase I important for catalytic activity.

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.

An adenine nucleotide-phosphoenolpyruvate counter-transport system in C3 and C4 plant chloroplasts.

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.

Photoaffinity labeling of hamster brown adipose tissue mitochondria by an [125I] coenzyme A derivative: differential interaction with the uncoupling protein and ADP/ATP carrier.

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.

Extraction of tissue long-chain acyl-CoA esters and measurement by reverse-phase high-performance liquid chromatography.

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.