scholarly journals Insulin regulation of the activity and phosphorylation of branched-chain 2-oxo acid dehydrogenase in adipose tissue

1989 ◽  
Vol 258 (1) ◽  
pp. 229-235 ◽  
Author(s):  
G P Frick ◽  
H M Goodman
Keyword(s):  
Enzyme Activity ◽  
Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Phosphorylation Sites ◽  
Acid Dehydrogenase ◽  
Insulin Regulation ◽  
Phosphorylated Form ◽  
32P Incorporation ◽  
Branched Chain

The activity of the intramitochondrial branched-chain 2-oxo acid dehydrogenase (BCDH), like that of pyruvate dehydrogenase, is regulated, at least in part, by interconversion between the active dephosphorylated enzyme and its inactive phosphorylated form. The stimulatory effect of insulin on BCDH activity was compared with its effect on phosphorylation of the enzyme. Intact tissues were incubated in the presence or the absence of insulin, and then mitochondria were isolated and disrupted before assaying for enzyme activity or estimating the extent of enzyme phosphorylation. Tissues were incubated in either the presence or the absence of leucine, which also stimulated BCDH activity up to 10-fold. Insulin (1 munit/ml) doubled the activity of BCDH in the absence and in the presence of leucine. Together, 1 mM-leucine and insulin appeared to stimulate BCDH activity fully. Phosphorylation of BCDH was estimated indirectly by measuring the incorporation of 32P into phosphorylation sites that remained unesterified after preparing mitochondrial extracts under conditions that preserved the effect of insulin on BCDH activity. Increased incorporation of 32P in these experiments implies decreased phosphorylation in situ when tissues were incubated with insulin and leucine. In the absence of leucine, little incorporation of 32P into BCDH was detected. In the presence of leucine, however, incorporation of 32P into BCDH was markedly increased, and insulin increased 32P incorporation still further. The results support the hypothesis that leucine and insulin both stimulate the activity of BCDH by promoting its dephosphorylation.

scholarly journals The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation

1981 ◽  
Vol 196 (2) ◽  
pp. 459-469 ◽  
Author(s):  
W A Hughes ◽  
A P Halestrap
Keyword(s):  
Pyruvate Dehydrogenase ◽  
High Speed ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylated Protein ◽  
Acid Dehydrogenase ◽  
Liver And Kidney ◽  
32P Incorporation ◽  
Branched Chain ◽  
Osmotic Treatment ◽  
Dehydrogenase Complex

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.

scholarly journals Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ. Further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase

1986 ◽  
Vol 238 (1) ◽  
pp. 93-101 ◽  
Author(s):  
A P Thomas ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Phosphatase Activity ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Kinase ◽  
Epididymal Adipose Tissue ◽  
Ionophore A23187 ◽  
Dilute Suspensions ◽  
Dehydrogenase System ◽  
Pyruvate Dehydrogenase Phosphatase

Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.

scholarly journals Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism

1986 ◽  
Vol 234 (2) ◽  
pp. 295-303 ◽  
Author(s):  
R Paxton ◽  
P W Scislowski ◽  
E J Davis ◽  
R A Harris
Keyword(s):  
Skeletal Muscle ◽  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Oxidative Decarboxylation ◽  
Physiological Concentration ◽  
Phosphorylation State ◽  
Acid Dehydrogenase ◽  
Relative Contribution ◽  
Branched Chain ◽  
Two Peaks

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.

scholarly journals Branched-chain 2-oxo acid dehydrogenase interferes with the measurement of the activity and activity state of hepatic pyruvate dehydrogenase

1986 ◽  
Vol 236 (1) ◽  
pp. 111-114 ◽  
Author(s):  
G W Goodwin ◽  
R Paxton ◽  
S E Gillim ◽  
R A Harris
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Total Activity ◽  
Oxidative Decarboxylation ◽  
Specific Assay ◽  
Acid Dehydrogenase ◽  
Activity State ◽  
Branched Chain ◽  
Indicator Enzyme

Oxidative decarboxylation of pyruvate by branched-chain 2-oxo acid dehydrogenase can result in overestimation of the expressed and total activity of hepatic pyruvate dehydrogenase. Pyruvate is a poor substrate for branched-chain 2-oxo acid dehydrogenase relative to the branched-chain oxo acids; however, the comparable total activities of the two complexes in liver, the much greater activity state of branched-chain 2-oxo acid dehydrogenase compared with pyruvate dehydrogenase in most physiological states, and the use of high pyruvate concentrations, explain the interference that can occur in conventional radiochemical or indicator-enzyme linked assays of pyruvate dehydrogenase. Goat antibody that specifically inhibited branched-chain 2-oxo acid dehydrogenase was used in this study to provide a more specific assay for pyruvate dehydrogenase.

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 Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver

1978 ◽  
Vol 171 (3) ◽  
pp. 751-757 ◽  
Author(s):  
P J Parker ◽  
P J Randle
Keyword(s):  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Substrate Inhibition ◽  
Liver Mitochondria ◽  
Partial Purification ◽  
Oxidative Decarboxylation ◽  
Acid Dehydrogenase ◽  
Purification And Properties ◽  
Single Complex ◽  
Branched Chain

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.

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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