Abstract
Phosphorylation of the alpha-subunits of the branched chain alpha-keto acid and the pyruvate dehydrogenases was measured in mitochondria isolated from rat hearts perfused in the presence of [32P]phosphate and various substrates. Mitochondrial isolations were accomplished rapidly under conditions which would preclude the interconversion of these two enzyme complexes between their active (dephosphorylated) and inactive (phosphorylated) forms. Inactivation of the branched chain complex and the concomitant incorporation of [32P]phosphate into the alpha-chain of the dehydrogenase subcomponent were observed in hearts perfused with glucose (10 mM) or pyruvate (10 mM). alpha-Ketoisocaproate infusion caused a 15-fold activation and dephosphorylation of the branched chain dehydrogenase, while pyruvate dehydrogenase remained inactive and phosphorylated under these conditions. Both dehydrogenase components were partially activated, and most of the [32P]phosphate was removed from their respective alpha-subunits during substrate-free perfusion. Both enzyme complexes were activated and dephosphorylated completely during infusion of dichloroacetate (1 mM) and glucose (10 mM).
Highlights
Several laboratories have reported that thebranched chain The results of the present study suggest that changes in the a-keto acid dehydrogenase complex of mammalian tissues is phosphorylation state of the a-subunits of the pyruvate and regulated by a phosphorylation/dephosphorylation mecha- the branched chain dehydrogenases can be monitored in the nism analogous to that documented for the structurally and rat heart perfused in the presence of [32P]phosphateunder functionally similar pyruvate dehydrogenase complex
In order to document alterations in the phosphorylation state and activation state of the pyruvate and the branched chain a-ketoacid dehydrogenases in the isolated perfused rat heart, it was necessary to develop a procedure for isolating mitochondria from the perfused organ in which the interconversion of the enzyme complexes between their active and inactive forms was prevented
Extracts derived from mitochondria preof the pyruvatedehydrogenase complex following infusion of a-ketoisocaproate has been reported previously [28]
Summary
In order to document alterations in the phosphorylation state and activation state of the pyruvate and the branched chain a-ketoacid dehydrogenases in the isolated perfused rat heart, it was necessary to develop a procedure for isolating mitochondria from the perfused organ in which the interconversion of the enzyme complexes between their active and inactive forms was prevented. It should be noted that the enzymaticactivities of the pyruvate and branched chain a-keatcoid dehydrogenasecomplexes measuredinmitochondriaisolated from rathearts perfused under the conditions noted in Table I are in very good general agreement with the activities of these two enzymes measured in whole tissue extracts from perfused hearts. This agreemenint dicates that themitochondrial preparation which included the kinase and phosphataseinhibitors was successful in preventing the interconversion of the two enzyme complexes under scrutiny. Using this heartperfusion system andhaving measuredthe pared from hearts perfused with glucose or pyruvate(10 mM) changes in the extractablenzymatic activitiesof the pyruvate exhibitedvery low branchedchaindehydrogenaseactivity and branched chain complexes, we attempted tomonitor the [17].Perfusion with a-ketoisocaproate (2 m ~ )a,substrate for the branched chaincomplex, caused a substantial increase in branched chain a-keto acid dehydrogenase activity to 3.2 f
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