Active‐site peptides of malonyl and palmitoyl transferase from yeast fatty acid synthetase were isolated and sequenced to try to prove the hypothesis [J. Ayling, R. Pirson & F. Lynen (1979) Biochemistry 11, 526–533] that both enzymes are identical. For this purpose synthetase modified with 5,5′‐dithiobis(2‐nitrobenzoic acid) was labelled with either [14C]malonyl or [14C]palmitoyl residues follwed by proteolytic digestion of the labelled protein. [14C]Malonyl‐peptides were isolated by conventional purification procedures; their structures were determined by a combination of methods. [14C]Palmitoyl‐peptide material was purified by high‐performance liquid chromatography and the structure determined by solid‐phase Edman degradation and other analytical methods. Serine was identified as the acyl acceptor group in both transferases. Comparison of the sequence data available shows that the sequence around the acyl acceptor group in both cases is identical. This proves the identity of malonyl and palmitoyl transferase.
The avian sarcoma virus src gene product, p6Osrc, has been purified 850-fold from cytoplasmic extracts of the rat tumor cell line RR1022 by using ammonium sulfate fractionation, hydrophobic chromatography on w-aminohexyl agarose, and ion exchange chromatography on phosphocellulose. Partially purified p6Osrc is a monomer, with a native molecular weight of about 60,000 and an apparent pI of 6.0.In immunoprecipitates, p6Osrc catalyzed phosphorylation of Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) in 20 mM Tris-HCI, pH 7.2/5 mM MgCl2 and, after 10 min at 370C, was stopped by washing the immunoprecipitates three times with 200 Al of 20 mM Tris-HCI, pH 7.2/5 mM MgCl2 and boiling for 2 min in 5% NaDodSO4/1% 2-mercaptoethanol. After centrifugation to remove protein A-Sepharose, the supernatant was brought to 10% in trichloroacetic acid and heated to 90'C for 15 min to hydrolyze ATP. After the precipitates were cooled, they were collected on Whatman glass fiber filters. The filters were washed with 0.1 M Na pyrophosphate/5% trichloroacetic acid anadwith methanol before drying. 32P was quantitated by liquid scintillation counting. This filter assay is a modification of the NaDodSO4/polyacrylamide gel assay developed by Collett and Erikson (5 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4 -5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA.Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found reactive towards acetyl-CoA.Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.Fatty acid synthetase from yeast is a stable multienzyme complex ( M , = 2.3 x lo6) which catalyzes the formation of long-chain fatty acyl-coenzyme A compounds according to Eqn (1) :( 1) where n = 7.8. As shown in this laboratory [l], an acetyl residue derived from acetyl-CoA in a recurrent reaction sequence is elongated by successive addition of C2 carbon units derived from malonyl-CoA. Intermediates of the biosynthetic process are never released * Deceased.
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