scholarly journals Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay

1986 ◽  
Vol 235 (2) ◽  
pp. 429-434 ◽  
Author(s):  
P A Patston ◽  
J Espinal ◽  
J M Shaw ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Chain Complex ◽  
Liver Mitochondria ◽  
Spectrophotometric Assay ◽  
Normal Diet ◽  
Casein Diet ◽  
Active Complex ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex

A rabbit polyclonal antibody to purified ox kidney branched-chain oxo acid dehydrogenase complex was shown by a variety of techniques to be an antibody to the E2 (acyltransferase) component. Rocket immunoelectrophoresis showed that the antibody does not discriminate between phosphorylated (inactive) or dephosphorylated (active) complex, and the same technique is used to assay total branched-chain complex (sum of active and inactive forms) in rat liver and heart mitochondrial extracts. The values obtained in normal rats fed on normal diet were comparable with those obtained by spectrophotometric assay of the holocomplex reaction after conversion of inactive complex into active complex. The values obtained in liver mitochondria from rats fed on 0%-casein diet or starved for 48 h were comparable with those in rats fed on normal diet, whereas earlier studies using spectrophotometric assay had shown substantial decreases in rats fed on 0%-casein diet or starved for 48 h. It has been shown that conversion of inactive complex into active complex requires prolonged incubation (120 min) in the presence of ketoleucine (4-methyl-2-oxopentanoate; to inhibit branched-chain oxo acid dehydrogenase kinase) to effect complete conversion in mitochondria from rats fed on 0%-casein diet, or starved for 48 h, or made diabetic with alloxan. By this technique, total activity of the complex in rat liver mitochondria was unaffected by diet or diabetes. The effects of diet and diabetes to decrease the activity of branched-chain complex in rat liver are therefore apparently mediated wholly through inactivation of the complex by phosphorylation.

scholarly journals Effects of low-protein diet and starvation on the activity of branched-chain 2-oxo acid dehydrogenase kinase in rat liver and heart

1986 ◽  
Vol 237 (1) ◽  
pp. 285-288 ◽  
Author(s):  
J Espinal ◽  
M Beggs ◽  
H Patel ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Kinase Activity ◽  
Liver Mitochondria ◽  
Normal Diet ◽  
Heart Mitochondria ◽  
Casein Diet ◽  
Acid Dehydrogenase ◽  
Low Protein ◽  
Branched Chain ◽  
Dehydrogenase Complex

The activity of branched-chain 2-oxo acid dehydrogenase kinase was 3-fold greater in extracts of heart mitochondria than in extracts of liver mitochondria from rats fed on normal diet. Feeding rats on a 0%-casein diet for 10 days increased the activity of branched-chain kinase 4-fold in extracts of liver mitochondria and in branched-chain dehydrogenase complex purified from such extracts; starvation (48 h) was without effect. In extract of heart mitochondria, kinase activity was increased 2-fold by feeding on 0%-casein diet and 1.5-fold by 48 h of starvation.

scholarly journals Purification and properties of a protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex

1985 ◽  
Vol 225 (2) ◽  
pp. 509-516 ◽  
Author(s):  
J Espinal ◽  
P A Patston ◽  
H R Fatania ◽  
K S Lau ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Chain Complex ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Activator Protein ◽  
Protein Activator ◽  
Liver And Kidney ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.

scholarly journals Activity of branched-chain 2-oxo acid dehydrogenase complex in rat liver mitochondria and in rat liver

1988 ◽  
Vol 256 (3) ◽  
pp. 929-934 ◽  
Author(s):  
M Beggs ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Active Form ◽  
Rat Liver Mitochondria ◽  
Acid Dehydrogenase ◽  
Liver Concentration ◽  
Branched Chain ◽  
Protein Free Diet ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

Four mitochondrial marker enzymes were used to show that: (1) high-protein (24%) diet increased the rat liver concentration and content of total branched-chain 2-oxo acid dehydrogenase complex (BCDC) by 31% by increasing mitochondrial specific activity of BCDC; (2) starvation increased the liver concentration of BCDC by 25% by decreasing liver weight; the liver content of mitochondria and the mitochondrial specific activity of BCDC were unchanged; (3) protein-free diet decreased rat liver BCDC concentration and content by 20%, by decreasing the liver concentration and content of mitochondria. Protein-free diet increased liver mitochondrial specific activities of L-glutamate, 2-oxoglutarate and NAD-isocitrate dehydrogenases. The validity of a mitochondrial method for the determination of the liver concentration of BCDC and the percentage in the active form in vivo is confirmed, and improvements are described. The experimental basis of criticisms of its use in this regard by Zhang, Paxton, Goodwin, Shimomura & Harris [(1987) Biochem. J. 246, 625-631] was not confirmed. The finding by Harris, Powell, Paxton, Gillim & Nagae [(1985) Arch. Biochem. Biophys. 243, 542-555], that starvation has no effect on the percentage of BCDC in the active form in rat liver, is confirmed.

Regulation by induction of branched-chain 2-oxo acid dehydrogenase complex in clofibrate-fed rat liver

1990 ◽  
Vol 172 (1) ◽  
pp. 243-248 ◽  
Author(s):  
Kazuo Ono ◽  
Hiroshi Shioya ◽  
Masatoshi Hakozaki ◽  
Kazuyuki Honda ◽  
Tsutomu Mori ◽  
...  
Keyword(s):  
Rat Liver ◽  
Acid Dehydrogenase ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex
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