Activation of branched-chain alpha-ketoacid dehydrogenase in isolated hepatocytes by branched-chain alpha-ketoacids.
The effects of branched-chain alpha-ketoacids on flux through and activity state of the branched-chain alpha-ketoacid dehydrogenase complex were studied in hepatocytes prepared from chow-fed, starved, and low-protein-diet-fed rats. Very low concentrations of alpha-ketoisocaproate caused a dramatic stimulation (50% activation at 20 microM) of alpha-ketoisovalerate decarboxylation in hepatocytes from low-protein-fed rats. alpha-Keto-beta-methylvalerate was also effective, but less so than alpha-ketoisocaproate. alpha-Ketoisocaproate did not stimulate alpha-ketoisovalerate decarboxylation by hepatocytes from chow-fed or starved rats. To a smaller degree, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate stimulated alpha-ketoisocaproate decarboxylation by hepatocytes from low-protein-fed rats. The implied order of potency of stimulation of flux through branched-chain alpha-ketoacid dehydrogenase was alpha-ketoisocaproate greater than alpha-keto-beta-methylvalerate greater than alpha-ketoisovalerate, i.e., the same order of potency of these compounds as branched-chain alpha-ketoacid dehydrogenase kinase inhibitors. Fluoride, known to inhibit branched-chain alpha-ketoacid dehydrogenase phosphatase, largely prevented alpha-ketoisocaproate and alpha-chloroisocaproate activation of flux through the branched-chain alpha-ketoacid dehydrogenase. Assay of the branched-chain alpha-ketoacid complex in cell-free extracts of hepatocytes isolated from low-protein-diet-fed rats confirmed that alpha-ketoacids affected the activity state of the complex. Branched-chain alpha-ketoacids failed to activate flux in hepatocytes prepared from chow-fed and starved rats because essentially all of the complex was already in the dephosphorylated, active state. These findings indicate that inhibition of branched-chain alpha-ketoacid dehydrogenase kinase activity by branched-chain alpha-ketoacids is important for regulation of the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase.
Han
A C
AC
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
Goodwin
G W
GW
Paxton
R
R
Harris
R A
RA
D020155:3-Hydroxybutyric Acid; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D002208:Caproates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D045782:Hemiterpenes; D006843:Hydrocarbons, Chlorinated; D006885:Hydroxybutyrates / Q000494:pharmacology; D007651:Keto Acids / Q000494:pharmacology*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology
D002208:Caproates; D045782:Hemiterpenes; D006843:Hydrocarbons, Chlorinated; D006885:Hydroxybutyrates; D007651:Keto Acids; D009097:Multienzyme Complexes; C016211:alpha-keto-beta-methylvaleric acid; C036533:2-chloroisocaproic acid; C001505:alpha-ketoisovalerate; C013082:alpha-ketoisocaproic acid; D012969:Sodium Fluoride; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D020155:3-Hydroxybutyric Acid