Subunit composition of oxaloacetate decarboxylase and characterization of the alpha chain as carboxyltransferase.
Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits alpha, beta, gamma with Mr 65 000, 34 000 and 12 000, 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 alpha chain. Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the alpha chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin-Sepharose. No beta or gamma chain were detectable in this alpha chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated alpha 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 alpha chain was released from the membrane characterize this subunit as a peripheral membrane protein. The beta and gamma 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 alpha chain, yielding a polypeptide of Mr 51 000 which was devoid of biotin. Degradation of the beta 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 HCO3- with H2O as substrate, has been excluded.
D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002262:Carboxy-Lyases / Q000378:metabolism*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D012964:Sodium; D014357:Trypsin
D001639:Bicarbonates; D010446:Peptide Fragments; D002245:Carbon Dioxide; D012964:Sodium; D014357:Trypsin; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase