Kinetic characterization of long chain fatty acyl coenzyme A ligase from rat liver mitochondria.

Long chain fatty acyl coenzyme A ligase (EC 6.2.1.3) purified from rat liver mitochondria has been characterized with respect to several kinetic parameters. Many of the kinetic properties of the mitochondrial enzyme are similar to those of the purified microsomal enzyme with respect to palmitoyl-CoA formation, but there are distinct differences. The fatty acid and nucleotide specificities of the mitochondrial enzyme are similar to those of the microsomal enzyme, as are the apparent Km values for ATP and coenzyme A. On the other hand, the mitochondrial enzyme differs from the microsomal enzyme in that it has a lower pH optimum, is different in molecular weight, and does not show simple saturation kinetics with palmitate as substrate. Of particular interest is the evidence presented which indicates that the mitochondrial long chain fatty acyl-CoA ligase, unlike short and medium chain ligases, does not utilize an acyladenylate as an intermediate in the formation of fatty acyl-CoA.     

A Dictyostelium long chain fatty acyl coenzyme A-synthetase mediates fatty acid retrieval from endosomes

We have identified a subset of Dictyostelium endosomes that carry a long chain fatty acyl coenzyme A-synthetase (LC-FACS 1) on their cytosolic surface. Immunofluorescence studies and observations using GFP-fusion proteins collectively suggest that LC-FACS 1 associates with endosomes a few minutes after their formation, remains bound through the acidic phase of endocytic maturation and dissociates early in the phase where the endosomal content is neutralised prior to exocytosis. Mutants in the fcsA gene, encoding the LC-FACS 1 protein, were constructed by homologous recombination. These cells show a strong defect in the intracellular accumulation of fatty acids, either taken up together with the liquid medium or bound to the surface of particles. Because the mutant cells are otherwise fully competent for macropinocytosis and phagocytosis, we conclude that the LC-FACS 1 protein mediates the retrieval of fatty acids from the lumen of endosomes into the cytoplasm.     

The purification and some properties of electron transfer flavoprotein and general fatty acyl coenzyme A dehydrogenase from pig liver mitochondria.

Electron-transferring flavoprotein (ETF) and acyl dehydrogenases of pig liver mitochondria have been isolated in good yield by a new procedure. ETF and general acyl dehydrogenase appear homogenous, are free of reciprocal contamination, react with neither pyridine nucleotides not cytochrome c, and are completely dependent upon each other for reduction of dichlorophenol indophenol by acyl-CaA substrates. The properties of the present preparation (some of which differ significantly from those previously described) are presented. Sedimentation of ETF in 0.02 M KP-i yields a M-r for the native ETF of 58,00 plus or minus 3,000, whereas sedimentation of reduced and alkylated ETF in guanidine HCl yields a M-r of 26,000. Electrophoresis on sodium dodecyl sulfate gels in the presence or absence of mercaptoethanol gives a M-r of about 27,000 and flavin analysis gives a minimum molecular weight of about the same figure. Thus, ETF appears to contain one flavin (at least 90% FAD, by chromatographic and fluorescence characteristics) per 26,000 M-r, and therefore may be composed of two subunits with one flavin each. Sodium dodecyl sulfate gel electrophoresis of general acyl dehydrogenase in the absence of mercaptoethanol gives a band corresponding to a M-r of 84,000; in the presence of mercaptoethanol a band corresponding to a M-r of 42,000 is found. The minimum molecular weight based on flavin content is 40,500. These data considered in conjunction with previous reports from other laboratories, suggest a structure of four subunits per mol with one flavin per subunit..     

A DNA ligase from mitochondria of rat liver.

An ATP-dependent DNA ligase has been demonstrated in extracts of rat liver mitochondria. The activity may be released from the mitochondria by treatment with hypotonic solutions or a detergent, indicating an intramitochondrial localization. The properties of the partially purified enzyme are similar to those of the nuclear DNA ligase from rat liver.     

Distinct long chain and very long chain fatty acyl CoA synthetases in rat liver peroxisomes and microsomes

Mitochondria, peroxisomes, and microsomes were isolated from rat liver homogenates, and stearic acid and lignoceric acid beta-oxidation, as well as stearoyl CoA synthetase and lignoceroyl CoA synthetase activities in the three organelles, were compared. Stearic acid beta-oxidation in peroxisomes was sixfold greater compared to the oxidation in mitochondria. Lignoceric acid beta-oxidation, observed only in peroxisomes, was fivefold lower compared to stearic acid beta-oxidation. Stearoyl CoA synthetase was present whereas lignoceroyl CoA synthetase was absent in mitochondria. Stearoyl CoA synthetase and lignoceroyl CoA synthetase activities were present in microsomes and peroxisomes, but the activity of stearoyl CoA synthetase was several-fold greater compared to lignoceroyl CoA synthetase in both organelles. The differing responses to detergents and phospholipids of stearoyl CoA and lignoceroyl CoA synthetase activities in microsomes as well as peroxisomes indicated that each activity was catalyzed by a separate enzyme. Differences in detergent and phospholipid response were also noted when either stearoyl CoA or lignoceroyl CoA synthetase activity in one organelle was compared with the corresponding activity in the other organelle, suggesting that the same activity in different organelles may be catalyzed by separate enzyme proteins.     

Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes

Crude mitochondrial fractions were isolated by differential centrifugation of rat liver homogenates. Subfractionation of these fractions on self-generating continuous Percoll gradients resulted in clearcut separation of peroxisomes from mitochondria. Hexacosanoic acid beta-oxidation was present mainly in peroxisomal fractions whereas hexacosanoyl CoA oxidation was present in the mitochondrial as well as in the peroxisomal fractions. The presence of much greater hexacosanoyl CoA synthetase activity in the purified preparations of microsomes and peroxisomes compared to mitochondria, suggests that the synthesis of coenzyme A derivatives of very long chain fatty acids (VLCFA) is limited in mitochondria. We postulate that a specific VLCFA CoA synthetase may be required to effectively convert VLCFA to VLCFA CoA in the cell. This specific synthetase activity is absent from the mitochondrial membrane, but present in the peroxisomal and the microsomal membranes. We postulate that substrate specificity and the subcellular localization of the specific VLCFA CoA synthetase directs and regulates VLCFA oxidation in the cell.     

Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria

2-Methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both with and without 2-mercaptoethanol, the enzyme showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size. Its isoelectric point was 5.50 +/- 0.2, and A1%280 nm was 12.5. This enzyme contained protein-bound FAD. The purified enzyme dehydrogenated S-2-methylbutyryl-CoA and isobutyryl-CoA with equal activity. The activities with each of these compounds were co-purified throughout the entire purification procedure. This enzyme also dehydrogenated R-2-methylbutyryl-CoA, but the specific activity was considerably lower (22%) than that for the S-enantiomer. The enzyme did not dehydrogenate other acyl-CoAs, including isovaleryl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA, at any significant rate. Apparent Km and Vmax values for S-2-methylbutyryl-CoA were 20 microM and 2.2 mumol min-1 mg-1, respectively, while those for isobutyryl-CoA were 89 microM and 2.0 mumol min-1 mg-1 using phenazine methosulfate as an artificial electron acceptor. The enzyme was also active with electron transfer flavoprotein. Tiglyl-CoA and methacrylyl-CoA were identified as the reaction products from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced from R-2-methylbutyryl-CoA. Tiglyl-CoA competitively inhibited the activity with both S-2-methylbutyryl-CoA and isobutyryl-CoA with a similar Ki. The enzyme activity was also severely inhibited by several organic sulfhydryl reagents such as N-ethylmaleimide, p-hydroxymercuribenzoate, and methyl mercury iodide. The pattern and degree of inhibition were essentially identical for both substrates. The purified 2-methyl-branched chain acyl-CoA dehydrogenase was immunologically distinct from isovaleryl-CoA-, short chain acyl-CoA-, medium chain acyl-CoA-, or long chain acyl-CoA dehydrogenase.