Search PubMed⌕ Search

Biomedical subjects

F E Frerman

Publications and source records attributed to F E Frerman.

At least 73 records · Page 4Linked to original sources
PubMed1983

Leucine catabolism during the differentiation of 3T3-L1 cells. Expression of a mitochondrial enzyme system.

Leucine can be utilized efficiently as a precursor for lipid biosynthesis by adipose tissue, especially in the presence of glucose or glucose and insulin. During the differentiation of 3T3-L1 fibroblasts to adipocytes, the rate of lipid biosynthesis from L-[U-14C]leucine increases at least 30-fold and lipogenesis, with [U-14C] acetate as the precursor, increases by 10- to 15-fold. The specific activities of two mitochondrial dehydrogenases in the leucine oxidative pathway, the branched chain alpha-ketoacid dehydrogenase and isovaleryl-CoA dehydrogenase, as well as of leucine:alpha-ketoglutarate transaminase, increase at least 20-fold during the adipose conversion. Isovaleryl-CoA dehydrogenase was assayed in crude extracts using a specific fluorimetric method employing electron transfer flavoprotein as the electron acceptor for the flavoprotein dehydrogenase. The specific activity of 3-hydroxy-3-methylglutaryl-CoA lyase, the mitochondrial enzyme catalyzing the terminal reaction in the leucine degradation pathway, increases 4-fold during differentiation. The increases in the specific activities of the mitochondrial enzymes occur without a change in the specific activity of cytochrome oxidase, indicating that the increases do not simply reflect proliferation of mitochondria. The biosynthesis of at least 20 soluble mitochondrial polypeptides is enhanced during the adipose conversion of the fibroblasts as determined by polyacrylamide gel electrophoresis following incubation of the cells with [35S] methionine. The results provide a conservative estimate of the extent of changes in mitochondrial soluble proteins during the adipose conversion. They also establish that differentiated 3T3-L1 adipocytes metabolize leucine like mature adipose tissue and illustrate the roles of the branched chain alpha-ketoacid dehydrogenase and isovaleryl-CoA dehydrogenase in lipogenesis.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1983

Subunit structure of electron transfer flavoprotein.

The electron transfer flavoprotein from pig liver mitochondria is a 57,000-dalton electron transferase which links several primary flavoprotein dehydrogenases with the mitochondrial electron transport system. The protein was previously reported to be a dimer of apparently identical subunits. There are conflicting estimates in the literature regarding the FAD content of the protein. The results presented here clearly show that the protein contains nonidentical subunits based on polyacrylamide gel electrophoresis in the presence of 8 M urea and sodium dodecyl sulfate. The molecular weights of the subunits are 31,000 and 27,000. Analysis of peptides generated by cleavage of the subunits with cyanogen bromide show that the subunits have different primary structures. This result and amino acid analyses of the protein and the purified subunits show that the heterogeneity cannot be due to proteolysis. Using an experimentally determined molar extinction coefficient for the protein-bound flavin, a minimum Mr = 55,000 was calculated, indicating that the protein contains 1 mol of FAD/mol of protein.

Amino Acids↗
PubMed1980

Enzyme-activated inhibitors, alternate substrates, and a dead end inhibitor of the general acyl-CoA dehydrogenase.

Aspects of the binding and dehydrogenation of acyl-CoA thiol esters by the general acyl-CoA dehydrogenase from pig liver were investigated using a dead-end inhibitor, S-octyl-CoA, several alternate substrates, and three active site-directed inhibitors. Experiments with S-octyl-CoA indicate that the carbonyl group of acyl-CoA thiol esters is not absolutely required for binding to the enzyme. However, the mode of binding of the 8-carbon thiol ether can be distinguished from the mode of binding of the enoyl-CoA product, octenoyl-CoA. Octanoyl pantetheine, octanoyl-etheno-CoA, and octanoyl-3'-dephospho-CoA are alternate substrates of the dehydrogenase. Steady state kinetic constants obtained with these alternate substrates indicate that the adenosine 5'-diphosphate, but not the 3'-phosphate, of the nucleotide moiety of acyl-CoA substrates contribute to the tight binding of the substrates. The substrate analogs 3'-butynoyl-CoA and 3-octynoyl-CoA are active site-directed, mechanism-based irreversible inhibitors of the dehydrogenase. These inhibitors covalently modify the apoprotein rather than the flavin. This finding and the fact that 2,3-octadienoyl-CoA also completely and irreversibly inhibits the enzyme indicate that th 3-acetylenic thiol esters inhibit the enzyme by a mechanism involving: (1) base-catalyzed abstraction of a protein at C-2 followed by isomerization to the allene carbanion, (2) protonation of the carbanion, and (3) attack of a nucleophile in the enzyme-active site on C-3 of the 2,3-dienoyl-CoA. The data show that the alkynoyl-CoA's are activated and bound at the active site of the enzyme. The results suggest that abstraction of a proton at C-2 of acyl-CoA substrates is the initial step in the catalytic pathway of dehydrogenation of substrates by the enzyme.

Acyl Coenzyme A↗
PubMed1980

Properties of the general acyl-CoA dehydrogenase from pig liver.

The properties of the general acyl coenzyme A dehydrogenase from pig liver and the stable reduced dehydrogenase . product and oxidized dehydrogenase . acetoacetyl-CoA complexes of the dehydrogenase were investigated. The enzyme has a molecular weight of 178,000 to 183,000 determined by gel filtration chromatography and by gel electrophoresis at different acrylamide concentrations. The subunit molecular weight is 45,000 based on acrylamide gel electrophoresis in the presence of dodecyl sulfate which agrees with the minimum molecular weight calculated from the flavin:protein ratio and the amino acid analysis. The subunits are identical, or very similar, as judged by quantitative NH2-terminal analysis and mapping of tryptic peptides. Immunochemical analyses by the complement fixation technique show that the structure of the oxidized enzyme is different from the structure of enzyme . acyl-CoA complexes whether the flavin in these complexes is in the oxidized or reduced state. The amino acid analysis, isoelectric point, and a procedure for crystallizing the dehydrogenase are also reported.

Acyl-CoA Dehydrogenases↗
PubMed1980

The effect of temperature and membrane lipid composition on the rate of beta-oxidation by Escherichia coli.

Escherichia coli, strain D-1ML, was grown to mid-exponential phase at 14, 30 or 43 degrees C. Membrane fractions were prepared from cells grown at each temperature and analyzed with respect to the unsaturated to saturated fatty acid ratio and the effect of temperature on the rate of catalysis by succinate dichlorophenol reductase and the membrane-bound acyl-CoA synthetase. The ratio of unsaturated to saturated fatty acids increased from 0.72 : 1.00 in cells grown at 43 degrees C to 1.46 : 1.00 in cells grown at 14 degrees C. The specific activity of succinate dichlorophenol reductase was increased by greater than two-fold in cells grown at 43 degrees C relative to the specific activity of the reductase from cells grown at 14 degrees C. In addition the activity of succinate dichlorophenol reductase responded to thermal phase transitions that were characteristic of membranes isolated from cells grown at the three different temperatures. In contrast the specific activity of acyl-CoA synthetase exhibited little or no variation as a function of the fatty acid composition of the membrane and the activity of the synthetase was not subject to thermal phase transitions. Comparison of Arrhenius plots of the oxidation of [U-14C]palmitoleate by whole cells and acyl-CoA synthetase activity with palmitoleate as substrate demonstrated that the two exhibited virtually identical Arrhenius activation energies and temperature optima. The Arrhenius plots of other soluble beta-oxidation enzymes do not resemble the provile of beta-oxidation. These data strongly suggest that the acyl-CoA synthetase which catalyzes the transport of fatty acids into E. coli by group translocation mechanism is the rate-limiting step in beta-oxidation in E. coli.

Chlorophenols↗
PubMed1980

Characteristics of an acid active thiamine diphosphatase from beef brain.

That thiamine has a role in nerve conduction as well as synaptic transmission is suggested by the following observations. (1) Thiamine phosphate esters are hydrolyzed and released from nerve membranes during nerve conduction. (2) Ultraviolet radiation of single nerve fibers at the wavelength specific for thiamine destroys the ability of that nerve to conduct an impulse. (3) Thiamine diphosphatase (TDPase) is present on synaptosomes. Previous articles have characterized an alkaline active TDPase in brain; this report characterizes a PH 5 active TDPase and compares it properties to the pH 9 enzyme. Both enzymes require a divalent cation for optimal activity. The pH 5 enzyme is more sensitive to ATP. Myelin fractions of brain have the highest specific activity for the acid TDPase, and the nerve ending particles the highest total activity. No PO4 3-inhibition was observed. Kinetic constants of this enzyme activity are reported.

Adenosine Diphosphate↗
PubMed1979

Studies on the subunits of Escherichia coli coenzyme A transferase. Reconstitution of an active enzyme.

The alpha and beta subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified beta subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of alpha subunit. The presence of the substrate,acetoacetyl-CoA, is required to recover the catalytic activity of the beta subunit and mixtures containing purified alpha and beta subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native alpha2beta2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated alpha subunit. The results indicate that the alpha subunit serves as a structural subunit, or possible a maturation subunit, imposing a conformation on the beta subunit that is catalytically more competent.

Acetyl-CoA C-Acetyltransferase↗
PubMed1977

Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization.

The enzymes for beta-oxidation of fatty acids in inducible and constitutive strains of Escherichia coli were assayed in soluble and membrane fractions of disrupted cells by using fatty acid and acyl-coenzyme A (CoA) substrates containing either 4 or 16 carbon atoms in the acyl moieties. Cell fractionation was monitored, using succinic dehydrogenase as a membrane marker and glucose 6-phosphate dehydrogenase as a soluble marker. Acyl-CoA synthetase activity was detected exclusively in the membrane fraction, whereas acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities that utilized both C4 and C16 acyl-CoA substrates were isolated from the soluble fraction. 3-Hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities assayed with both C4 and C16 acyl-CoA substrates co-chromatographed on gel filtration and ion-exchange columns and cosedimented in glycerol gradients. The data show that these three enzyme activities of the fad regulon can be isolated as a multienzyme complex. This complex dissociates in very dilute preparations; however, in those preparations where the three activities are separated, the fractionated species retain activity with both C4 and C16 acyl-CoA substrates.

3-Hydroxyacyl CoA Dehydrogenases↗