Phosphorylation affects the mobility of the E1 α-subunit of branched-chain 2-oxo acid dehydrogenase on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis

Abstract

heat-stable protein kinse inhibitor (50 units/ml), therefore ruling out the possibility that the protein kinase represents the free catalytic subunit of cyclic AMP-dependent protein kinase. Heparin, at a concentration of IOpg/ml, which inhibits the phosphorylation of acetyl-CoA carboxylase by casein kinase I and I1 (Tipper et al., 1983; Munday & Hardie, 1984) also had no effect on the phosphorylation reaction. The activity of acetyl-CoA carboxylase decreased dramatically in parallel with the phosphorylation by the protein kinase. At a subsaturating concentration ( 0 . 5 m ~ ) of the allosteric activator, citrate, the activity of acetyl-CoA carboxylase decreases from 0.54 pmol/min per mg to 0.03 pmol/min per mg, while at IOrnM-citrate (a saturating concentration) the acetyl-CoA carboxylase activity decreases from 2pmol/min per mg to 0.45 pmol/min per mg (Fig. 1). Rapid reversal of the phosphorylation and inhibition of acetyl-CoA carboxylase is achieved by the addition of the partially purified catalytic subunit of protein phosphatase2A (not shown). The physiological importance of this protein kinase in the control of hepatic lipogenesis is currently being investigated. It will be of interest to determine whether the protein kinase we have isolated can account for the high basal phosphorylation and low activity of acetyl-CoA carboxylase that is observed when the enzyme is isolated from rat liver in the presence of sodium fluoride (Holland e t al., 1984).