Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding

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Abstract
A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was .apprx. 2.5 .mu.mol of NADPH oxidized/min per mg of protein at 37.degree. C. The enzyme appeared homogeneous by sodium dodecyl sulfate/polacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (MW 480,000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0.degree. C for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approximately 2 mol of taurine were found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4''-phosphopantetheine in the native enzyme. Rat mammary gland fatty acid synthase required free CoA for activity.