Liberation, purification, and properties of thioesterase component of pigeon liver fatty acid synthetase complex.
Proteolysis of pigeon liver fatty acid synthetase with elastase results in the quantitative cleavage of the thioesterase component from the enzyme complex. This thioesterase component is two or three times more active catalytically in the isolated state than in the native fatty acid synthetase, and its activity is not affected by the presence or absence of reducing thiols. The proteolytically cleaved thioesterase is separated from the core enzyme in one step by size-exclusion chromatography on a Sephadex G-75 column. The peptide obtained by gel permeation is homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. Size-exclusion chromatography on Bio-Gel A 0.5 m and Sephadex G-75 columns, sucrose density gradient ultracentrifugation, and N-terminal amino acid analysis also indicate that the proteolytically cleaved thioesterase is homogeneous. The sedimentation coefficient of the thioesterase is approximately 2.9 S. Proteolytic cleavage with elastase also quantitatively releases the [1,3-14C]- or [1,3-3H]diisopropylphosphofluoridate-labeled thioesterase component from the correspondingly labeled fatty acid synthetase. Binding studies with 14C- or 3H-labelled diisopropylphosphofluoridate and fatty acid synthetase show that 2 mol of the label are bound per mol of the enzyme when complete loss of fatty acid-synthesizing activity occurs. The molecular weight of the thioesterase component is estimated to be 36000 by size-exclusion chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis.
D000596:Amino Acids; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D010856:Columbidae / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010196:Pancreatic Elastase; D013869:Thiolester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; D010196:Pancreatic Elastase