Abstract
Evidence for a reversible process resulting in stable activated and inactivated states of the mitochondrial branched chain alpha-keto acid dehydrogenase complex in isolated perfused rat heart is presented. The inactivation process is mediated by pyruvate infusion, while activation (up to 18-fold) is facilitated by branched chain alpha-keto acid substrates. The low activity state of the branched chain complex characteristic of freshly excised rat hearts could be maintained by infusion of either pyruvate or glucose. Activation of the complex in the perfused rat heart was achieved slowly by substrate-free perfusion, while rapid activation was accomplished by infusion of branched chain alpha-keto acids. The fully activated enzyme complex resulting from branched chain alpha-keto acid infusion subsequently could be inactivated maximally by infusion of pyruvate alone or intermediate degrees of inactivation could be produced by certain ratios of co-infused pyruvate and branched chain alpha-keto acid. alpha-Ketoisocaproate was an order of magnitude more effective than alpha-keto isovalerate either in preventing inactivation or in stimulating the opposing activation process when co-infused with pyruvate. The mitochondrial pyruvate transport inhibitor, alpha-cyanocinnamate, effectively prevented inactivation of the complex by infused pyruvate. Differential changes in the activation states of the branched chain alpha-keto acid dehydrogenase and pyruvate dehydrogenase complexes were evident when the two complexes were compared in apparently similar flux-inhibited (via octanoate infusion) and flux-stimulated (via dichloroacetate infusion) metabolic conditions. The differential effect of pyruvate concentration on the activity states of the two complexes was also well-defined. The results of the present study suggest distinct systems for the regulation of the activity of the two multienzyme complexes of interest. While our results argue neither for nor against an inactivation of the branched chain alpha-keto acid dehydrogenase complex by a protein kinase, the regulatory properties of such an intramitochondrial protein kinase may not be similar to the pyruvate dehydrogenase kinase. The mechanistic nature of the suggested novel regulatory system concerned with the pyruvate-mediated inactivation of the branched chain alpha-keto acid activation cannot be inferred at the present time.
Highlights
The opposing activation process when co-infused with The products of the oxidative decarboxylation of branched pyruvate
Regulation of thekinase-phosphatase interconversion chain a-keto acid dehydrogenase complex bya protein system for pyruvate dehydrogenase is effected by a variety of kinase, the regulatory properties of such an intrami- interacting factors, including NADH, NAD’ [20,21,22,23], acetyltochondrial protein kinase may not be similar to the CoA, CoASH [20,21,22,23], ATP, ADP [24,25,26], monovalent and pyruvate dehydrogenase kinase
Our previous study of the regulation of the branched chain that the level of branched chain a-keto acid dehydrogenase a-keto acid dehydrogenase in the perfused rat heart indicated activity observed in the presence of even 0.05 mM a-ketoisothat the metabolifclux through thisenzyme complex changed caproate exceeds the control level seen at 6 min of perfusion markedlyinresponse to the metabolic conditions imposed (Fig. 1) by a factor of 2
Summary
Hydrogenase complex presumably due to an inhibitoryeffect of dichloroacetate on the pyruvate dehydrogenkainsaese [36]. The extractable activity of the branched chain a-keto acid dehydrogenase complex inthe perfused hearts at various time points during the perfusion period and as established by the various defined perfusion conditions was determined by freeze-clampingthe beating hearts with a large gridded chaina-ketoacidsin varioustissues has beeninfluenced aluminum clamp precooled in liquid nitrogeTnh. e frozen hearts were strongly by comparative studies performed in the rat. The initial objec- paredfrom[l-14C]leucineand[I-"Clvaline,respectively,using the tive in our study was to developareliable assay for the determination of branched chain a-keto acid dehydrogenase activity inrat heart tissue. This would allow an investigation of alterations in the activityof the branched chain a-keatocid preparation and purification methodsof Rudiger et al [42]. At this point during thperfusion experiment, a stable, maximal metabolic flux through the branched chaincomplex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
