Biochemical Properties of Cytochrome &dependent Microsomal Fatty Acid Elongation and Identification

Recently, our laboratory reported the involvement of cytochrome be in the elongation of hepatic microsomal fatty acids (Keyes, S. R., Alfano, J. A., Jansson, I., and Cinti, D. L. (1979) J. Biol. Chem. 254,7778-7784). In this paper, a correlation between the rates of ba reoxidation in the presence of malonyl-CoA and malonyl-CoA incorporation measured under various conditions was demonstrated, thus providing further evidence in support of our hypothesis. The additional data in this paper detail the biochemical characteristics of the microsomal fatty acid elongation reaction. The rates of ba reoxidation and malonyl-CoA incorporation were linear with protein concentration up to 2 mg/ml and were optimal between pH 7.2 and 7.4. The rates were linear with malonyl-CoA concentration up to 3 PM and were maximal at 20 CM with a K,,, of 8 PM. The bg reoxidation rate was always 10 to 15% faster than the incorporation rate. The products formed during fatty acid elongation were 18:O (15%), 18:1(65%), and 20:1(20%) using either NADH or NADPH as the reducing agent. The rate of incorporation was 30% faster with NADPH than with NADH, although the extent of incorporation was nearly identical. The apparent K,,, was 7 PM for NADH and 8 PM for NADPH. The 18:l formed during malonyl-CoA incorporation was 60% oleic acid and 40% vaccenic acid as demonstrated by ozonolysis. Both palmitic and palmitoleic acids were substrates for the malonyl-CoA incorporation reaction and the elongation rates of the two fatty acids were 60% and 40%, respectively, of the rate of elongation utilizing the endogenous microsomal fatty acids. The formation of 18:l using endogenous fatty acids was partially, but not completely, blocked by 1 m~ KCN as would be expected from the composition of the 18:l formed during malonyl-CoA incorporation. Finally, the rates of bs reoxidation and malonyl-CoA incorporation and product formation were unaffected by carbon monoxide.