Biosynthesis of fatty acids in cell-free preparations. 3. Coenzyme A dependent reactions in a soluble enzyme system of mammary gland

Abstract

A soluble enzyme system (MGE), prepared from the mammary gland of lactating rats, which synthesizes fatty acids from acetate on addition of ATP, was shown to contain about 50 [mu]g of CoA/ml. Activation of acetate and other n-fatty acids with CoA in the MGE was inferred from the formation of hydroxamic acids, some of which were identified by paper chromatography. Activation of acetate predominated. ADP could replace ATP in the activation reaction. The MGE contained "endogenous" substrates from which acetyl-CoA and higher acyl-CoA compounds were formed under appropriate conditions. The presence of a pyruvic oxidase system in the MGE was inferred from the formation of acethydroxamic acid from pyruvate in the presence of ATP. The MGE could act as acetyl-donor for acetylation of sulfanilamide when it was coupled with the aryl-amine-acetylating enzyme (A60) of Chou and Lipmann (1952). alpha-Oxoglutarate and malonate, which stimulated fatty acid synthesis from acetate in the MGE, had no effect on the formation of acetyl-CoA. Hg2+ inhibited fatty acid synthesis from acetate by virtue of abolishing formation of acetyl-CoA. Arsenate caused only a moderate inhibition of acetyl-CoA formation, but had a strong inhibitory effect on fatty acid synthesis. Removal of CoA and DPN from the MGE by treatment with Dowex-1 ion-exchange resin abolished fatty acid synthesis from acetate. The system was fully reactivated by addition of boiled MGE or of CoA and DPN. Addition of excess CoA and DPN to fresh MGE, or to MGE treated with Dowex-1 resin, considerably enhanced the incorporation of acetate into fatty acids. The bearing of the results on the mechanism of biosynthesis of fatty acids is discussed.