Biosynthesis of fatty acids in cell-free preparations. 2. Synthesis of fatty acids from acetate by a soluble enzyme system prepared from rat mammary gland

Abstract

A soluble enzyme system, synthesizing short- and long-chain fatty acids from acetate, was prepared by high-speed centrifugal fractionation of homogenates of mammary gland of lactating rats. Neither of the "mitochondrial" nor the "microsomal" fractions of the mammary-gland homogenates were required for fatty acid synthesis. The ability of the soluble supernatant fluid to synthesize fatty acids was usually greater than that of the entire homogenate. In the soluble enzyme preparations ATP alone was sufficient to activate synthesis from acetate, and the requirement for ATP was absolute. ADP could effectively replace ATP. Fatty acid synthesis was markedly stimulated in the presence of ATP by oxaloacetate, alpha-oxoglutarate, and to a lesser extent by succinate. A strong stimulation was caused by malonate; a combination of alpha-oxoglutarate with malonate elicited on occasion a 30-fold increase in the amount of [Cl4] acetate incorporated into fatty acids. Anaerobic incubation caused inhibition of synthesis only in the presence of oxaloace-tate; no inhibition was seen anaerobically in the presence of alpha-oxoglutarate plus malonate. Hg2+ and arsenate almost completely abolished fatty acid synthesis; cyanide and 2:4-dini-trophenol caused a moderate (30-70%) inhibition, while azide and fluoride were without effect. The implication of the results is discussed.