Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding.
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 about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 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 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was 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.
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003065:Coenzyme A / Q000378:metabolism; D004952:Esters; D064429:Fatty Acid Synthases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008722:Methods; D009249:NADP / Q000378:metabolism; D010204:Pantetheine / Q000378:metabolism; D011247:Pregnancy; D051381:Rats
D000214:Acyl Coenzyme A; D000596:Amino Acids; D004952:Esters; D010204:Pantetheine; D009249:NADP; D064429:Fatty Acid Synthases; D003065:Coenzyme A