title: Isolation and characterization of the methionine aminopeptidase from porcine liver responsible for the co-translational processing of proteins.
abstract: A methionine aminopeptidase that specifically removes methionine residues from peptides with amino-terminal sequences of Met-Ala-, Met-Val-, Met-Ser-, Met-Gly-, and Met-Pro- but not Met-Leu- or Met-Lys- has been isolated to homogeneity from porcine liver by a procedure involving five chromatographic steps. The enzyme, whose specificity matches that predicted for the entity responsible for the co-translational amino-terminal processing of nascent polypeptide chains, has a measured molecular mass of 70,000 Da by SDS-polyacrylamide electrophoresis and 67,000 Da by gel chromatography (under nondenaturing conditions), suggesting the native molecule is a monomer. It is activated by Co2+ and inhibited by beta-mercaptoethanol and EDTA. With octapeptide substrates related to the amino-terminal portion of the beta-chain of human hemoglobin (with a histidine in position 3), the enzyme had a pH optimum of 6.0. With a synthetic peptide devoid of histidine, it showed no pH dependence from 6.0 to 8.0. This sensitivity may be due to the propensity of peptides with histidine in the third position to bind divalent cations such as Co2+. The measured Km and kappa cat values were affected by residues in the second position. The peptide corresponding to the natural sequence (Met-Val-His-) gave a kappa cat/Km value of 260 mM-1 s-1; substitution of alanine in the second position raised the kappa cat/Km to 1523 mM-1 s-1, but substitution of proline lowered the value to 130. The effects are primarily on the kappa cat. The substitution of proline (for histidine) in the third position, the mutation found in hemoglobin Long Island, prevents the removal of the methionine residue, as occurs with the mutant protein. The porcine liver enzyme is similar to methionine aminopeptidases isolated from Escherichia coli, Salmonella typhimurium, and yeast in that it also is stimulated by Co2+. However, it is much larger than these enzymes and differs somewhat in specificity, particularly with the yeast enzyme.
authors.affiliation: Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717-1700.
authors.forename: R L
authors.initials: RL
authors.lastname: Kendall
authors.forename: R A
authors.initials: RA
authors.lastname: Bradshaw
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D013552:Swine
chemical_list: D000596:Amino Acids; D002413:Cations, Divalent; D006882:Hydroxyapatites; D008623:Mercaptoethanol; D017886:Durapatite; D004492:Edetic Acid; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases