Acyl carrier protein. V. Identification of 4'-phosphopantetheine bound to a mammalian fatty acid synthetase preparation.

Previous investigations from several laboratories have established the sequence of reactions leading to de novo synthesis of fatty acids.'-8 Previous reports have also shown that all these reactions in E. coli occur with the substrates bound as thioesters to an acyl carrier protein (ACP).7-12 Recently, the substrate binding site of ACP has been shown to be the sulfhydryl group of a prosthetic group, 4'phosphopantetheine. 4'-Phosphopantetheine is bound to ACP through phosphodiester linkage with the hydroxyl group of a serine residue.'3 14 Evidence has been reported which suggests the involvement in plant fatty acid synthesis of a protein similar both functionally and physically to ACP.1" 16 Thus, in bacterial and plant fatty acid synthetase preparations which are soluble and can be fractionated, ACP has been shown to play a primary role in fatty acid synthesis. The fatty acid synthetase systems of yeast,", 2 pigeons,17 18and mammals,'9 on the other hand, occur as multienzyme complexes which have resisted fractionation into enzymatically active components, and demonstration of ACP in these complexes has not been achieved. This report presents evidence that a fatty acid synthetase partially purified from the adipose tissue of rats contains proteinbound 4'-phosphopantetheine, suggesting that a protein similar to ACP is present in the mammalian enzyme system. Materials and Methods.-Malonyl-CoA was synthesized by the method of Trams and Brady,20 acetyl-CoA by the method of Simon and Shemin,21 and C14-coenzyme A was synthesized enzymatically from 4'-phosphopantetheine which was synthesized as described by Moffatt and Khorana.22 Dephospho-CoA pyrophosphorylase and dephospho-CoA kinase were prepared by the method of Novelli23 through the calcium phosphate gel step. Aquacide II was purchased from Calbiochem; E. coli alkaline phosphatase from Worthington Biochemical Co.; CoA from P-L Biochemicals; and ATP, acetyl phosphate, and Crotalus adamenteus venom from Sigma Chemical Co. This venom was shown to contain a pyrophosphatase that cleaves CoA to form 4'-phosphopantetheine and an adenine-containing fragment.24 The calcium salt of 1-C'4-D-pantothenic acid (3 ,c/ ,umole) was a gift from Merck & Co., Inc., through the efforts of Drs. Charles Rosenblum and Harry Robinson. Mammalian fatty acid synthetase: The enzyme was prepared from rat epididymal adipose tissue by slight modifications of the published procedure.25 The alumina Cz gel step was omitted, and in one experiment the enzyme was purified only through the calcium phosphate gel step. Enzyme at this stage of purification can be stored in liquid nitrogen for several weeks without significant loss of activity. Fatty acid synthetase activity was assayed spectrophotometrically by measuring TPNH oxidation.25 Removal of CoA from enzyme preparation: Fatty acid synthetase, purified through the calcium phosphate gel step, is free of CoA (see Results). It is necessary to remove contaminating CoA in order to determine the amount of pantothenate associated with the synthetase in crude preparations. This was accomplished by filtration on Sephadex G-50. In a typical experiment, an aliquot of crude adipose tissue extract containing 57 mg of protein and 103 units of enzyme activity in 17 ml was concentrated with aquacide II to approximately 3.5 ml. The concentrate was ad-