James K. Stoops

Further evidence for the multifunctional enzyme characteristic of the fatty acid synthetases of animal tissues.

Physicochemical studies of chicken liver fatty acid synthetase have shown that the enzyme has been isolated in a high state of purity. Polyacrylamide gel electrophoresis of the enzyme preparations in SDS-phosphate, SDS-Tris-glycine, and Tris-glycine buffers indicate that 90% of the protein consists of one component. We have demonstrated that proteolysis of the synthetase may occur during the isolation of the enzyme. In addition, the purified enzyme undergoes proteolysis in the presence of denaturing agents, as is evident from the appearance of numerous bands on SDS gels. The proteolysis may be avoided by rapid purification of the enzyme and by heat treatment of the synthetase in the presence of denaturing agents. In addition, the nutritional state of the animal at the time of sacrifice may affect the banding pattern of the enzyme on SDS gels. Preparations obtained from starved-refed chickens generally gave over 90% of the stain in one band, while preparations obtained from starved animals exhibited several bands. The molecular weight of the native enzyme is 460,000–500,000, while in the presence of Tris-glycine buffer the enzyme rapidly dissociated into subunits of 230,000–250,000 molecular weight. The molecular weight has been estimated by sedimentation equilibrium in the presence of 6 m guanidinium chloride (214,000) and by SDS-polyacrylamide gel electrophoresis (220,000). It is proposed that the fatty acid synthetase consists of two subunits of the same or nearly equal size and, since the subunits contain the catalytic sites and the 4′-phosphopantetheine, the synthesis is a multifunctional enzyme. The amino acid analysis and the 4′-phosphopantetheine content have been determined. As reported earlier (Arslanian, Stoops, Oh, and Wakil, 1976, J. Biol. Chem.251, 3194–3196), the synthetase contains 1.6 mol of 4′-phosphopantetheine/mol of enzyme. In this study, the prosthetic group content was shown to be independent of the procedure used to isolate the 4′-phosphopantetheine for analysis.

Effect of estrogen on fatty acid synthetase in the chicken oviduct and liver.

Estrogen administered to one-month-old female chickens resulted in a 180-fold increase in the amount of fatty acid synthetase, a seven-fold increase in the enzyme activity per gram of tissue and a 25-fold increase in the weight of the oviduct. In contrast, the fatty acid synthetase content in liver increased three-fold; activity per gram of tissue increased two-fold and the weight increased two-fold. The alrge increase in the fatty acid synthetase activity in the oviduct was due to a corresponding increase in the amount of the fatty acid synthetase protein since the specific activities of highly purified preparations of oviduct and liver fatty acid synthetases were the same and the two enzymes had the same end point as determined by immunoprecipitation. That the increase in activity of the oviduct enzyme is not due to a modification was further supported by physicochemical comparison of the oviduct enzyme with the chicken liver enzyme. Thus, the synthetase complexes have similar size, their subunit composition and size appear to be the same, and both are multifunctional enzymes. Finally, kinetic studies and product analyses indicated no catalytic difference between the enzyme induced by estrogen in the oviduct and the liver enzyme.

The reaction of chicken liver fatty acid synthetase with 5,5'-dithiobis(2-nitrobenzoic acid).

Abstract Chicken liver fatty acid synthetase is rapidly inhibited by 5,5′-dithiobis(2-nitrobenzoi acid). The inhibition results from the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) with the cysteine-SH residue of the β-ketoacyl synthetase site. The adjacent pantetheine-SH of the other subunit displaces 2-nitro-5-thiobenzoic acid from the mixed disulfide resulting in the formation of a disulfide bond between the two residues and thereby cross-linking the two subunits. Scatchard analysis of the 5,5′-dithiobis(2-nitrobenzoic acid) inhibition indicated that there are two β-ketoacyl synthetase sites in the homodimer. The mixed disulfide formed between the pantetheine-SH and the cysteine-SH was reduced by 2-mercaptoethanol resulting in restoration of enzyme activity.

The isolation of the two subunits of yeast fatty acid synthetase

Abstract The two subunits that comprise the yeast fatty acid synthetase (designated α and β) have been isolated. The separation was performed using DEAE Biogel A chromatography after first treating yeast fatty acid synthetase with 3,4,5,6 tetrahydrophthalic anhydride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fractions eluted from the ion exchange column indicated that the separation of the subunits was essentially complete. It was possible to remove the 3,4,5,6 tetrahydrophthalate derivative from the subunits and regenerate certain of the partial activities. The α subunit was found to have the β-keto reductase activity as well as the acyl carrier protein component associated with it. The β subunit had the acetyl and malonyl transacylases and the palmitoyl transferase activity associated with it. The different extent to which the malonyl and acetyl transacylase activities were regained indicated that these two catalytic sites have separate domains in the β subunit.

Electron microscope studies of human α2-macroglobulin-chymotrypsin complex: Demonstration that the two structures assigned to native and proteolyzed α2-macroglobulin represent two views of the proteolyzed molecule

Electron microscope studies of native and protease-bound human alpha 2-macroglobulin have led to two contradictory models for these two structures. One viewpoint maintains that the native structure has the shape of )+(, which contracts on binding of the protease to the shape of ([). An opposing view proposes that the native structure has the shape of a padlock and that )+( and ([) are the side and end views of the proteolyzed molecule. In this investigation, electron microscope studies of the alpha-chymotrypsin-treated alpha 2-macroglobulin utilizing a tilt stage have shown that the two shapes [)+( and ([)] interconvert. This demonstrates that these two shapes represent the side and end views of the proteolyzed alpha 2-macroglobulin which are related by a 90 degree rotation of the prototype molecule.

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.

The development and application of a novel chromophoric substrate for investigation of the mechanism of yeast fatty acid synthase

The acetyl transacylase activity of the fatty acid synthase from yeast has been investigated using p-nitrophenylthiol acetate. The chromophoric nature of the nitrophenylthiol moiety affords a convenient spectrophotometric assay for the transacylase function as well as a means to investigate the kinetics and the mechanism of this process. A probable kinetic scheme for enzyme catalyzed transacetylation from p-nitrophenylthiol acetate to an acyl acceptor (CoA or N-acetylcysteamine) is proposed and the kinetic constants for acetylation of enzyme and for acetyl transfer to an acceptor were determined. It was also demonstrated that p-nitrophenylthiol acetate can replace acetyl-CoA as a substrate in fatty acid synthesis.

Fractionation and molecular-weight determination of large polypeptides by gel filtration in guanidiniun chloride☆

Abstract A Sepharose CL-2B column equilibrated with 6 m guanidinium chloride has been used to fractionate peptides ranging in size from 68,000 to 300,000 daltons and to generate a standard curve for the estimation of the molecular weights of proteins within this range. The utility of this chromatography system for the fractionation of even larger polypeptides is demonstrated by the estimation of the apparent molecular weight of the crosslinked yeast fatty acid synthetase polypeptides in the 106 molecular-weight range and its separation from its noncrosslinked components of 210,000 daltons.

Fatty Acid Synthetases of Eukaryotic Cells

The synthesis of long-chain fatty acids, such as palmitic, occurs in the living cell by head-to-tail condensation of C2 units as acetyl-CoA. The condensation occurs in two steps. First, acetyl-CoA is converted to malonyl-CoA by acetyl-CoA carboxylase, a multienzyme system containing biotin [Eq. (1); for a recent review see Wakil et al., 1983]. The second step (Eq. 2) is the conversion of acetyl-CoA and malonyl-CoA to palmitate, catalyzed by fatty acid synthetases (FAS) in the presence of NADPH.