Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase

Abstract

Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits α, β, γ with M_r 65000, 34000 and 12000, respectively. On dodecylsulfate polyacrylamide gels the smallest of these subunits was heavily stained with silver but poorly with Coomassie brilliant blue. All three subunits were resolved and clearly detectable by high‐performance liquid chromatography in a dodecylsulfate‐containing buffer. Biotin was localized exclusively in the α chain. Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the α chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin‐Sepharose. No β or γ chain were detectable in this α chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated α chain, however, was a catalytically active carboxyltransferase as evidenced from the isotopic exchange between [1‐¹⁴C]pyruvate and oxaloacetate. The rate of this exchange reaction was about 9 U/mg protein and was completely independent of the presence of Na⁺ ions. The ease with which the α chain was released from the membrane characterize this subunit as a peripheral membrane protein. The β and γ chain, on the other hand, stick so firmly in the membrane that they are only released by detergents, thus indicating that these are integral membrane proteins. Limited tryptic digestion of oxaloacetate decarboxylase led to a rapid cleavage of the α chain, yielding a polypeptide of M_r 51000 which was devoid of biotin. Degradation of the β chain required prolonged incubation periods and was markedly influenced by Na⁺ ions which had a protective effect against proteolysis. A proton is required in the decarboxylation of oxaloacetate and CO₂ arises as primary product. The other alternative, i. e. generation of HCO⁻₃ with H₂O as substrate, has been excluded.