Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.     

Acetyl-l-carnitine as a precursor of acetylcholine

Synthesis of [³H]acetylcholine from [³H]acetyl-l-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [³H] and [¹⁴C] labeled acetylcholine were produced when [³H]acetyl-l-carnitine andd-[U-¹⁴C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-l-carnitine to acetylcholine was dependent on concentration of acetyl-l-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-l-carnitine in the treatment of age-related cholinergic deficits.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

The localization of some coenzyme A-dependant enzymes in rat liver mitochondria

1. CoA, acetyl-CoA, l-carnitine and acetyl-l-carnitine when added to rat liver mitochondria equilibrate with approximately two-thirds of the total intramitochondrial water. The mitochondrial space calculated to be freely permeable to these solutes was identical with that obtained for sucrose. 2. Acetyl-CoA is rapidly deacylated by rat liver mitochondria at 0 degrees C, and special precautions are required to measure its mitochondrial permeation. 3. Rat liver mitochondria were separated into fractions that correspond to the inner membrane, the outer membrane, and the soluble proteins of the matrix and intermembrane compartment. Soluble enzymes considered to be located in the matrix were citrate synthase (EC 4.1.3.7), palmitoyl-CoA dehydrogenase (EC 1.3.2.2), electron-transferring flavoprotein, medium-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.2), l-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35) and 3-keto-acyl-CoA thiolase (EC 2.3.1.16). Carnitine palmitoyltransferase (EC 2.3.1.-) is largely associated with the inner-membrane fraction. A long-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.3) is associated with the outer-membrane fraction.     

Mechanism of action of beta-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA.

Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and -4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.     

Isolation and characterization of carnitine acetyltransferase from S. cerevisiae

Carnitine acetyltransferase was isolated from yeast Saccharomyces cerevisiae with an apparent molecular weight of 400,000. The enzyme contains identical subunits of 65,000 Da. The Km values of the isolated enzyme for acetyl-CoA and for carnitine were 17.7 microM and 180 microM, respectively. Carnitine acetyltransferase is an inducible enzyme, a 15-fold increase in the enzyme activity was found when the cells were grown on glycerol instead of glucose. Carnitine acetyltransferase, similarly to citrate synthase, has a double localization (approx. 80% of the enzyme is mitochondrial), while acetyl-CoA synthetase was found only in the cytosol. In the mitochondria carnitine acetyltransferase is located in the matrix space. The incorporation of 14C into CO2 and in lipids showed a similar ratio, 2.9 and 2.6, when the substrate was [1-14C]acetate and [1-14C]acetylcarnitine, respectively. Based on these results carnitine acetyltransferase can be considered as an enzyme necessary for acetate metabolism by transporting the activated acetyl group from the cytosol into the mitochondrial matrix.     

Modulation of rat-liver mitochondrial acetyl-CoA acetyltransferase activity by a reversible chemical modification with coenzyme A

The mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase, EC 2.3.1.9) is involved in ketone body biosynthesis. In its unmodified state, referred to as transferase B in former publications (Huth, W. (1981) Eur. J. Biochem. 120, 557-562), the enzyme is characterized by the highest specific activity of 21.65 mumol/min per mg protein (direction of acetoacetyl-CoA synthesis); several forms of the enzyme with lower specific activities result from chemical modification by an apparent covalent binding of CoASH. The chemical modification results in an inactivation of the enzyme: a 2 h incubation with 0.2 mM CoASH at pH 8.1 at 30 degrees C inactivates up to 95%. Both processes, the CoASH-binding and the resulting inactivation, can be simultaneously reversed by treatment with glutathione. The specificity of inactivation is limited to CoASH and the intact sulfhydryl group is a prerequisite for this process. The enzyme exhibits a limited number (n = 3.2) of high-affinity (Ka = 26.7 microM) specific binding sites for CoASH. The inactivation-reactivation cycle of acetyl-CoA acetyltransferase by CoASH and glutathione may involve a protein disulfide-thiol exchange and represents a mode of control in modulating the amount of active enzyme.     

Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase

In an attempt to clarify why the brain oxidizes fatty acids poorly or not at all, the activities of beta-oxidation enzymes present in rat brain and rat heart mitochondria were measured and compared with each other. Although the apparent Km values and chain-length specificities of the brain and heart enzymes are similar, the specific activities of all but one brain enzyme are between 4 and 50% of those observed in heart mitochondria. The exception is 3-ketoacyl-CoA thiolase (EC 2.3.1.16) whose specific activity in brain mitochondria is 125 times lower than in heart mitochondria. The partially purified brain 3-ketoacyl-CoA thiolase was shown to be catalytically and immunologically identical with the heart enzyme. The low rate of fatty acid oxidation in brain mitochondria, estimated on the basis of palmitoylcarnitine-supported respiration and [1-14C]palmitoylcarnitine degradation to be less than 0.5 nmol/min/mg of protein, may be the consequence of the low activity of 3-ketoacyl-CoA thiolase. Inhibition of [1-14C]palmitoylcarnitine oxidation by 4-bromocrotonic acid proves the observed oxidation of fatty acids in brain to be dependent on 3-ketoacyl-CoA thiolase and thus to occur via beta-oxidation. Since the reactions catalyzed by carnitine palmitoyltransferase (EC 2.3.1.21) and acyl-CoA synthetase (EC 6.2.1.3) do not seem to restrict fatty acid oxidation in brain, it is concluded that the oxidation of fatty acids in rat brain is limited by the activity of the mitochondrial 3-keto-acyl-CoA thiolase.     

Rat liver peroxisomes catalyze the initial step in cholesterol synthesis. The condensation of acetyl-CoA units into acetoacetyl-CoA

In the last few years, it has been demonstrated by this group and others that rat liver peroxisomes participate in cholesterol synthesis. It has been shown that the key regulatory enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase, is present in liver cells not only in the endoplasmic reticulum but also within peroxisomes. It has been also demonstrated that rat liver peroxisomes in the presence of cytosolic proteins in vitro are able to convert [14C]mevalonic acid to [14C]cholesterol. In addition, a recent study demonstrated that the largest cellular concentration of sterol carrier protein-2 is inside peroxisomes. It is of interest, therefore, to inquire whether other proteins known to be involved in cholesterol biogenesis are also present in peroxisomes. In this study we investigated the first step in cholesterol synthesis, the condensation of two acetyl-CoA units to acetoacetyl-CoA. It was demonstrated that peroxisomal thiolase, purified by DEAE-phosphocellulose chromatography from gemfibrozil-treated rats, is active not only toward acetoacetyl-CoA and 3-ketoacyl-CoA, consistent with literature reports, but is also capable of converting acetyl-CoA units to acetoacetyl-CoA. This is the first demonstration of condensation activity in rat liver peroxisomes.     

Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine.

Methanococcus voltae is a methanogenic bacterium which requires leucine, isoleucine, and acetate for growth. However, it also can synthesize these amino acids, and it is capable of low levels of autotrophic acetyl coenzyme A (acetyl-CoA) biosynthesis. When cells were grown in the presence of 14CO2, as well as in the presence of compounds required for growth, the alanine found in the cellular protein was radiolabeled. The percentages of radiolabel in the C-1, C-2, and C-3 positions of alanine were 64, 24, and 16%, respectively. The incorporation of radiolabel into the C-2 and C-3 positions of alanine demonstrated the autotrophic acetyl-CoA biosynthetic pathway in this bacterium. Additional evidence was obtained in cell extracts in which autotrophically synthesized acetyl-CoA was trapped into lactate. In these extracts, both CO and CH2O stimulated acetyl-CoA synthesis. 14CH2O was specifically incorporated into the C-3 of lactate. Cell extracts of M. voltae also contained low levels of CO dehydrogenase, 13 nmol min-1 mg of protein-1. These results further confirmed the presence of the autotrophic acetyl-CoA biosynthetic pathway in M. voltae. Likewise, 14CO2 and [U-14C]acetate were also incorporated into leucine and isoleucine during growth. During growth with [U-14C]leucine or [U-14C]isoleucine, the specific radioactivity of these amino acids in the culture medium declined, and the specific radioactivities of these amino acids recovered from the cellular protein were 32 to 40% lower than the initial specific radioactivities in the medium.Cell extracts of M. voltae also contained levels of isopropyl malate synthase, an enzyme that is specific to the leucine biosynthetic pathway, of 0.8 nmol min-1 mg of protein-1. Thus, M. voltae is capable of autotrophic CO2 fixation and leucine and isoleucine biosynthesis.     

Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate

The synthesis of high specific radioactivity [14C]-acetyl-Coenzyme A from [14C]sodium acetate, 2,6-dichlorobenzoic acid, 1,1'-carbonyldiimidazole, and CoA is reported. Starting with 1 mumol of [14C]sodium acetate, this method yields pure [14C]acetyl-CoA in yields approaching 40%. Chromatography on a reversed-phase ODS column was used to separate acetyl-CoA from Coenzyme A and side products. The acetylating agent is apparently a reaction intermediate, acetylimidazole.     

Pseudoketogenesis in the perfused rat heart

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.     

Selective inhibition of cholesterol synthesis by cell-free preparations of rat liver by using inhibitors of cytoplasmic acetoacetyl-coenzyme A thiolase.

Cytoplasmic acetoacetyl-CoA thiolase (acetyl-CoA C-acetyltransferase, EC 2.3.1.9) was partially purified from rat liver. The enzyme was irreversibly inactivated by 4-bromocrotonyl-CoA, but-3-ynoyl-CoA, pent-3-ynoyl-CoA and dec-3-ynoyl-CoA. In the case of the alk-3-ynoyl-CoA esters the potency as alkylating agents of acetoacetyl-CoA thiolase decreased with increased chain length of the alk-3-ynoyl moiety. Advantage was taken of the specific action of alk-3-ynoyl-CoA esters on acetoacetyl-CoA thiolase to show that in a postmitochondrial fraction from rat liver they are effective inhibitors of cholesterol synthesis from sodium [2-14C]acetate under conditions when mevalonate conversion into cholesterol and fatty acid synthesis are unafffected. Short-chain alk-3-ynoic acids have little effect on sterol synthesis, although dec-3-ynoic acid is an effective inhibitor owing to its conversion into the CoA ester by the microsomal fatty acyl-CoA synthetase.     

Acetyl coenzyme A and coenzyme A contents of growing Clostridium kluyveri as determined by isotope assays

CoASH and some of its acyl derivatives, especially acetyl-SCoA, occupy a central position in the energy metabolism of the anaerobic Clostridium kluyveri, both as intermediates and as regulatory effectors. The steady state concentrations of these compounds were determined in growing cultures of this organism using an anaerobic and fast deproteinization technique and radio isotope assays. Acetyl-SCoA was determined as [1-¹⁴C]citrate formed in the presence of [4-¹⁴C]oxaloacetate and citrate synthase; 0.49 mol/g cell wet wt. were found CoASH, CoAS-SCoA after borohydride reduction, and total acyl derivatives of coenzyme A after hydrolysis of the thiol esters were converted to thioethers with [2,3-¹⁴C]N-ethylmaleimide and brought to radiochemical purity by chromatographic methods. While disulfides of coenzyme A were undetectable, 0.13 mol CoASH and 1.17 mol of total acyl-SCoA per g wet wt. were found. These data are consistent with the regulatory scheme of the energy metabolism of C. kluyveri previously proposed.Abbreviations DTE dithioerythritol - NEM N-ethylmaleimide - NES N-ethylsuccinimide Enzymes (EC 2.7.2.1) Acetate kinase, ATP: acetate phosphotransferase - (EC 3.1.3.1) Alkaline phosphatase, orthophosphoric monoester phosphohydrolase - (GOT) Aspartate aminotransferase - (EC 2.6.1.1) L-aspartate:2-oxoglutarate aminotransferase - (CS) Citrate synthase - (EC 4.1.3.7) citrate oxaloacetate-lyase (pro 3S-CH₂COOacetyl-CoA) - (EC 2.8.3.8) CoA-transferase, acyl-CoA:acetate CoA-transferase - (EC 1.1.1.37) Malate dehydrogenase, L-malate:NAD⁺ oxidoreductase - (EC 1.18.1.3) NADH:ferredoxin reductase, ferredoxin:NAD⁺ oxidoreductase - (EC 3.1.4.1) Phosphodiesterase (snake venom), orthophosphoric diester phosphohydrolase - (EC 2.3.1.8) Phosphotransacetylase, acetyl-CoA:orthophosphate acetyltransferase - (EC 2.3.1.9) Thiolase, acetyl-CoA:acetyl-CoA C-acetyltransferase A preliminary account of this work has been given (Decker et al. 1976)     

Evidence for a homogeneous pool of acetyl-CoA in rat-liver mitochondria

Rat-liver mitochondria oxidized [1-14C]palmitate or [U-14C]palmitate and unlabelled pyruvate in a medium containing fluorocitrate and L-carnitine. The oxidation products (acetyl-L-carnitine, ketone bodies and citrate) were separated by anion-exchange chromatography and their specific activities were determined. The distribution of radioactivity over the two halves of the ketone bodies was essayed. Significant differences between the specific activities of citrate, acetyl-L-carnitine and the carboxylhalf of the ketone bodies were not observed; this was consistently the case, even when pyruvate contributed for more than 80% to the acetyl-CoA pool. Our results argue against compartition of mitochondrial acetyl-CoA. Instead, they strongly suggest that the acetyl-CoA originating from the simultaneous oxidation of pyruvate and palmitate equilibrates before being distributed over the various pathways of further metabolism.     

Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

The enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) was purified from rat spleen approx. 1500-fold in 1.6% yield. The specific activity of the purified enzyme was 0.317 +/- 0.089 mumol/min per mg of protein (mean +/- S.D., n = 6). The Km for the substrate acetyl-CoA was 137 +/- 13 microM and the pH optimum was about 8. Incubation of the purified enzyme was 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine followed by electrophoresis resulted in the incorporation of radioactivity into a protein of Mr 29,000. The enzyme was most active towards 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine as substrate, 1-palmitoyl-2-lyso-glycero-3-phosphocholine being a poor substrate. In addition, the enzyme preferred acetyl-CoA to palmitoyl-CoA or oleoyl-CoA as substrate.     

A study on the precursors of the acetyl moiety of acetylcholine in brain slices. Observations on the compartmentalization of the acetyl-coenzyme A pool

1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various (14)C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-(14)C]acetate, [(14)C]bicarbonate, [1,5-(14)C]citrate, dl-[1- or 5-(14)C]glutamate or [1- or 2-(14)C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-(14)C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-(14)C]acetate was only 1.25, 5.3 and 19.7% of that from [2-(14)C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-(14)C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-(14)C]pyruvate was used compared with that obtained when [2-(14)C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.     

Carnitine acetyltransferase in pea cotyledon mitochondria

Purified pea cotyledon mitochondria did not oxidise acetyl-CoA in the presence of carnitine. However, acetylcarnitine was oxidised. It was concluded that acetylcarnitine passed through the mitochondrial membrane barrier but acetyl-CoA did not. Only a sensitive radioactive assay detected carnitine acetyltransferase in intact mitochondrion or intact mitoplast preparations. When the mitochondria or mitoplasts were burst, acetyl-CoA substrate was available to the matrix carnitine acetyltransferase and a high activity of the enzyme was measured. The inner mitochondrial membrane is there-fore the membrane barrier to acetyl-CoA but acetylcarnitine is suggested to be transported through this membrane via an integral carnitine: acylcarnitine translocator. Evidence is presented to indicate that when the cotyledons from 48-h-grown peas are oxidising pyruvate, acetylcarnitine formed in the mitochondrial matrix by the action of matrix carnitine acetyltransferase may be transported to extra-mitochondrial sites via the membrane translocator.     

Biosynthetic thiolase from Zoogloea ramigera. II. Inactivation with haloacetyl CoA analogs

The thiolase involved in biosynthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera generates an acetyl-enzyme species during catalysis. Up to 0.86 [14C] acetyl eq/subunit of this homotetrameric enzyme is accumulated by acid precipitation in the presence of [14C]acetyl-CoA. Gel filtration of the same solutions produced only 7% acetyl-enzyme suggesting hydrolytic lability of the acetyl-enzyme during the 10-min isolation at 4 degrees C. In an effort to identify active site residues which may function as basic groups to deprotonate at C-2 of acetyl-CoA to generate the required nucleophilic equivalent in carbon-carbon bond formation, we have prepared and tested haloacetyl-thioesters, oxoesters, and amides in the panthetheine pivalate series (Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., and Walsh, C. T. (1987) J. Biol. Chem. 262, 82-89). The [14C]bromoacetyl-oxoester alkylatively inactivates thiolase irreversibly with stoichiometric incorporation of four labels/tetramer. Determination of amino acid composition of the radiolabeled tryptic peptide indicated trapping of Cys-89 (Peoples, O. P., Masamune, S., Walsh, C. T., and Sinskey, A. J. (1987) J. Biol. Chem. 262, 97-102), the same residue modified by iodoacetamide. When the bromoacetyl-thioester was used, inactivation was pH-dependent. The data are consistent with the competition of two processes, acylation, and alkylation. Direct (rather than secondary) alkylation of thiolase by the inactivator accounts for the significant 14C incorporation into thiolase with the thioester labeled with [14C] in the pantetheine pivalate moiety. It appears likely that the haloacetyl analogs described herein should be generally useful for affinity labeling other enzymes using acetyl-CoA as a substrate.