Isolation and characterization of an acyl carrier protein from pigeon liver fatty acid synthetase by controlled proteolysis with elastase.
Controlled proteolytic cleavage of 4'-phospho[14C]pantetheine-labeled pigeon liver fatty acid synthetase generates two 4'-phospho[14C]pantetheine-labeled peptides, Ec1 and Ec2. These are separated from each other and the core enzyme by gel permeation chromatography on a Sephadex G-75 column. The two radioactively labeled peptides constitute 50% of the radioactivity initially present in the 4'-phospho[14C]pantetheine-labeled fatty acid synthetase. The remaining label in the core enzyme is released quantitatively by proteolytic cleavage with trypsin. The molecular weights of Ec1 and Ec2 peptides, as determined by size exclusion chromatography and SDS-polyacrylamide gel electrophoresis, are 12000 and 6000, respectively. Both the higher and lower molecular weight peptides are homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. The higher molecular weight peptide, Ec1, is characterized as an acyl carrier protein by the transacylation reaction between the unlabeled Ec1 peptide and radioactively labeled acetyl- and malonyl-CoA. Since Ec2 peptide also contains the prosthetic group present in the Ec1 peptide, the Ec2 peptide appears to result from the proteolytic cleavage of the higher molecular weight peptide, Ec1. Amino acid composition of the acyl carrier protein shows the presence of 1 mol of 4'-phosphopantetheine per mol of protein. 2 mol of acyl carrier protein are present per mol of the fatty acid synthetase. The amino acid analysis is in good agreement with the molecular weight of the Ec1 peptide, as determined by gel filtration and SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid analysis of this peptide shows the presence of an arginine residue.
D000213:Acyl Carrier Protein / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D010856:Columbidae; D064429:Fatty Acid Synthases / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010196:Pancreatic Elastase
D000213:Acyl Carrier Protein; D000596:Amino Acids; D046911:Macromolecular Substances; D064429:Fatty Acid Synthases; D010196:Pancreatic Elastase