scholarly journals Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method

1980 ◽  
Vol 191 (1) ◽  
pp. 147-154 ◽  
Author(s):  
C J Stanley ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Sodium Dodecyl ◽  
Tween 80 ◽  
New Method ◽  
Acid Dehydrogenase ◽  
Multienzyme Complexes ◽  
Oxoglutarate Dehydrogenase ◽  
Triton X ◽  
Dehydrogenase Complex

A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.

scholarly journals Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes

1985 ◽  
Vol 226 (2) ◽  
pp. 509-517 ◽  
Author(s):  
O L De Marcucci ◽  
A Hunter ◽  
J G Lindsay
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Sodium Dodecyl ◽  
Normal Component ◽  
Proteolytic Fragment ◽  
Multienzyme Complexes ◽  
The Common ◽  
Oxoglutarate Dehydrogenase ◽  
Lipoamide Dehydrogenase

The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.

scholarly journals Induction of the branched-chain 2-oxo acid dehydrogenase complex in 3T3-L1 adipocytes during differentiation

1983 ◽  
Vol 214 (1) ◽  
pp. 177-181 ◽  
Author(s):  
D T Chuang ◽  
C W C Hu ◽  
M S Patel
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Common Mechanism ◽  
Acid Dehydrogenase ◽  
Oxoglutarate Dehydrogenase ◽  
Branched Chain ◽  
Kinetics Of ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The activities of 2-oxo acid dehydrogenase complexes were measured during hormone-mediated differentiation of 3T3-L1 preadipocytes into adipocytes. Specific activity of leucine-activated branched-chain 2-oxo acid dehydrogenase complex increased approx. 10-fold in 3T3-L1 adipocytes compared with 3T3-L1 preadipocytes. In contrast, specific activity of the 2-oxoglutarate dehydrogenase complex increased by only 3-fold in 3T3-L1 adipocytes. The three catalytic component enzymes of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex showed concomitant increases in their specific activities. A close similarity in kinetics of induction of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex in 3T3-L1 adipocytes suggests that a common mechanism may be involved in hormone-dependent increases in the activities of the catalytic components of these two complexes in 3T3-L1 adipocytes during differentiation.

scholarly journals Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin

1980 ◽  
Vol 192 (2) ◽  
pp. 469-481 ◽  
Author(s):  
W A Hughes ◽  
R W Brownsey ◽  
R M Denton
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subcellular Fractionation ◽  
Alpha Subunit ◽  
Fat Cells ◽  
Phosphorylated Proteins ◽  
Alpha Subunits ◽  
Dehydrogenase Complex

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35–45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.

scholarly journals Partial purification and characterization of a pyruvate dehydrogenase-complex-inactivating enzyme from rat liver

1978 ◽  
Vol 169 (2) ◽  
pp. 321-328 ◽  
Author(s):  
A Lynen ◽  
E Sedlaczek ◽  
O H Wieland
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Potassium Phosphate ◽  
Partial Purification ◽  
Acid Extraction ◽  
High Dilutions ◽  
Dehydrogenase Complex

An enzyme inactivating the pyruvate dehydrogenase complex (inactivase) was purified about 8000-fold from rat liver by differential centrifugation, acid extraction of a lysosomerich 25000 g pellet, acetone fractionation, and adsorption on calcium phosphate gel. By exclusion chromatography on Sephadex G-100 a molecular weight of 21 000 was estimated. The purified enzyme was most stable at pH 5.8 in potassium phosphate buffer, and at pH 4.5 in McIlvaine buffer. At high dilutions the enzyme was very labile and was remarkably stabilized by high salt concentrations. Enzyme activity is inhibited by native rat blood serum, iodoacetamide and leupeptin, but not by phenylmethanesulphonyl fluoride, suggesting that it belongs to the class of thiol proteinases. Among various enzymes tested, only 2-oxoglutarate dehydrogenase was attacked by the inactivase to a similar extent to the pyruvate dehydrogenase complex. Studies on the inactivation mechanism indicate that although the overall reaction is completely lost after treatment with inactivase, each individual step of the multienzyme complex retains full catalytic activity. As judged from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the transacetylase subunit appears to be degraded into several smaller fractions.

scholarly journals Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex

1985 ◽  
Vol 225 (3) ◽  
pp. 731-735 ◽  
Author(s):  
K G Cook ◽  
A P Bradford ◽  
S J Yeaman
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Gel Filtration ◽  
Original Activity ◽  
Acid Dehydrogenase ◽  
Specific Activities ◽  
Tight Association ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaC1. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of E1 and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active E1 has an approx. 6-fold higher affinity for E2 than does phosphorylated E1. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.

scholarly journals Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides

1980 ◽  
Vol 189 (1) ◽  
pp. 161-172 ◽  
Author(s):  
C E Henderson ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Icosahedral Symmetry ◽  
Enzyme Complex ◽  
Molecular Masses ◽  
Dehydrogenase Complex

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.

scholarly journals Oxidative decarboxylation of 4-methylthio-2-oxobutyrate by branched-chain 2-oxo acid dehydrogenase complex

1986 ◽  
Vol 237 (2) ◽  
pp. 621-623 ◽  
Author(s):  
S M A Jones ◽  
S J Yeaman
Keyword(s):  
Pyruvate Dehydrogenase Complex ◽  
Oxidative Decarboxylation ◽  
Maximal Inhibition ◽  
Acid Dehydrogenase ◽  
Isolated Adipocytes ◽  
Oxoglutarate Dehydrogenase ◽  
Branched Chain ◽  
Kinase Phosphorylation ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

Highly purified branched-chain 2-oxo acid dehydrogenase complex (BCOADC) oxidizes 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with Km values of 67 microM and 18 microM respectively. The Vmax. for oxidation of these substrates is 27% and 53% respectively of that for 3-methyl-2-oxobutyrate. Highly purified pyruvate dehydrogenase complex (PDC) oxidizes 2-oxobutyrate (Km 100 microM; Vmax. 49% of that for pyruvate) but not 4-methylthio-2-oxobutyrate, whereas 2-oxoglutarate dehydrogenase complex will not utilize either 2-oxo acid as substrate. BCOADC kinase is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, with half-maximal inhibition by 45 microM and 50 microM respectively. Phosphorylation of BCOADC in isolated adipocytes is inhibited by both 4-methylthio-2-oxobutyrate and 2-oxobutyrate, consistent with their inhibitory action of BCOADC kinase. Phosphorylation of PDC is decreased by 2-oxobutyrate, but not by 4-methylthio-2-oxobutyrate.

scholarly journals Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis

1983 ◽  
Vol 215 (1) ◽  
pp. 133-140 ◽  
Author(s):  
P N Lowe ◽  
J A Hodgson ◽  
R N Perham
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Oxidative Decarboxylation ◽  
Multienzyme Complex ◽  
Complex Activity ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.

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