The purification and regulatory properties of α-oxoglutarate dehydrogenase from Acinetobacter lwoffi

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Abstract
The α-oxoglutarate dehydrogenase multienzyme complex was purified from Acinetobacter lwoffi to a high degree of homogeneity as shown by gel electrophoresis and analytical ultracentrifugation. Sedimentation-velocity analyses gave s20,w values which increased with increasing protein concentration, suggesting dissociation of the complex in dilute solution. The maximum s20,w value thereby obtained and the value determined by active enzyme centrifugation were both in the range 28–29S. Electron micrographs of the complex indicated a molecular diameter of 20–22nm (200–220Å). The overall activity of the complex was inhibited by NADH, and kinetic studies indicated sites of action on the first and third enzyme components. AMP and ADP relieved this inhibition and also stimulated enzyme activity. Assays specific for the first enzyme component showed this to be the site of action of the adenylates. The activity of the complex varied with energy charge in a manner consistent with its role in energy metabolism.