Fat Metabolism in Higher Plants: XXXVIII. PROPERTIES OF WHEAT GERM ACETYL COENZYME A CARBOXYLASE

Abstract

Abstract Wheat germ acetyl coenzyme A carboxylase has been purified over 1000-fold. Ultracentrifugal analysis showed two protein components (7.3 S and 9.4 S); only one of these binds avidin, as evidenced by an increase in the sedimentation coefficient of the 9.4 S component to 11.5 S. However, only one protein peak was observed upon chromatography on molecular sieving columns. The leading edge of this protein showed the highest specific activity, contained the highest amount of biotin per mg of protein, and was the major site for ATP-32Pi exchange (avidin-sensitive), whereas the trailing edge of the protein peak was found to be specific for the binding of the 3H-acetyl moiety of 3H-acetyl-CoA and catalyzed malonyl-CoA-14C-acetyl-CoA exchange. A synergistic effect was observed upon addition of fractions from the leading edge and the trailing edge of the protein peak. Disc gel electrophoresis established that five components are associated with the single protein peak obtained from molecular sieve columns. Two of the five bands catalyzed ATP-32Pi exchange, two bands catalyzed malonyl-CoA-14C-acetyl-CoA exchange, and the fifth band catalyzed both exchange reactions as well as H14CO3- fixation. Biotin content, molecular weights, and the activities of the five individual bands suggest that one protein occurs as either a monomer or a dimer which contains biotin and catalyzes ATP-32Pi exchange, and that another protein occurs as both a monomer and a tetramer which do not contain biotin but catalyze malonyl-CoA-14C-acetyl-CoA exchange and bind acetyl moieties to the protein. The fifth band appears to be the active enzyme consisting of one carboxylating component (9.4 S) and one or more transcarboxylating components (7.3 S).