Document Detail


One-step purification and properties of a two-peptide fatty acid synthetase from the uropygial gland of the goose.
MedLine Citation:
PMID:  817736     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cell-free extracts from the uropygial gland of goose catalyzed the incorporation of malonyl-CoA into normal fatty acids and methylmalonyl-CoA into multimethyl branched acids with NADPH as the preferred reductant (J. S. Buckner and P.E. Kolattukudy (1975), Biochemistry 14, 1771). Purification of fatty acid synthetase from this extract was accomplished in one step by gel filtration with Sepharose 4B. Homogeneity of the fatty acid synthetase was shown by analytical ultracentrifugation, immunodiffusion assays, polyacrylamide disc gel electrophoresis, and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. At a pH of 7.0, apparent Km values of 3.6 X 10(-5) M and 1.5 X 10(-5) M were calculated for malonyl-C0A and NADPH, respectively. The major products synthesized by the enzyme from malonyl-CoA and methylmalonyl-C0A were free hexadecanoic acid and free 2, 4, 6, 8-tetramethyldecanoic acid, respectively, with acetyl-CoA as primer. A molecular weight value of 547 000 was determined for the goose fatty acid synthetase by sedimentation equilibrium centrifugation. The purified enzyme had an s20,w of 13.5S and was partially dissociated in low-ionic strength buffer into a 9.3S species, and this dissociation was accompanied by a corresponding partial inactivation of the enzymatic activity. Reassociation and reactivation of the partially dissociated fatty acid synthetase were accomplished in either 0.2 M KCl or 200 muM NADPH. These properties of the goose enzyme are similar to those of other animal fatty acid synthetases, as was the amino acid composition. Dissociation of the purified enzyme with sodium dodecyl sulfate resulted in only two equal molecular weight polypeptides (269 000), as determined by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Injection of labeled pantothenic acid into the uropygial gland resulted in the synthesis of labeled fatty acid synthetase in which the label appeared to be located exclusively in the 4'-phosphopantotheine moiety. Analysis of the labeled enzyme by gel filtration and polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate showed that the labeled pantothenate was contained exclusively in the half molecular weight moiety. The enzyme contained one 4'-phosphopantetheine residue per subunit (269 000), as determined by measurement of the taurine generated by hydrolysis of performic acid-treated enzyme. Sodium dodecyl sulfate-activated proteolytic activity was shown to be associated with goose fatty acid synthetase, and this proteolysis was shown to result in the formation of small-molecular-weight protein fragments (less than 200 000) during treatment of the enzyme with sodium dodecyl sulfate. This proteolysis could be prevented by diisopropyl fluorophosphate and p-chloromercuribenzoate. These results strongly suggest that the goose uropygial gland fatty acid synthetase consists of two multifunctional polypeptide subunits, each containing one covalently linked 4'-phosphopantetheine.
Authors:
J S Buckner; P E Kolattukudy
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  15     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1976 May 
Date Detail:
Created Date:  1976-08-02     Completed Date:  1976-08-02     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1948-57     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / analysis
Animals
Fatty Acid Synthetase Complex / isolation & purification*,  metabolism
Geese
Immunodiffusion
Molecular Weight
Pantothenic Acid / metabolism
Sebaceous Glands / enzymology*
Ultracentrifugation
Chemical
Reg. No./Substance:
0/Amino Acids; 79-83-4/Pantothenic Acid; EC 6.-/Fatty Acid Synthetase Complex

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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