PMID:2851590
| Citation |
Yoshimoto, T, Murayama, N, Honda, T, Tone, H and Tsuru, D (1988) Cloning and expression of aminopeptidase P gene from Escherichia coli HB101 and characterization of expressed enzyme. J. Biochem. 104:93-7 |
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| Abstract |
The aminopeptidase P gene in Escherichia coli HB101 was cloned into the plasmid pBR322. Introduction of the hybrid plasmid, pAPP01, into the E. coli DH1 resulted in an 8-fold increase of aminopeptidase P activity as compared with that of the host. The enzyme was purified by series of chromatographies on DEAE-Sephadex, QAE-Sephadex, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc-gel and SDS-gel electrophoreses. the enzyme was inhibited strongly by EDTA and slightly by p-chloromercuribenzoate, but was not affected by diisopropyl phosphorofluoridate, E-64, or iodoacetic acid. The optimum pH of the enzyme was 8.5. The enzyme was stable at pH 8 to 9. After incubation for 30 min at pH 8.0, 50% remaining activity was observed at 50 degrees C. The enzyme was activated 3-fold by the addition of 5 microM Mn2+. The molecular weight of the enzyme was estimated to be 50,000 and 200,000 by SDS-PAGE and gel filtration, respectively. The amino terminal amino acid was identified to be serine by Edman degradation, indicating that the enzyme is composed of a homo-tetramer. The enzyme hydrolyzed X-Pro bonds (X = amino acid) of peptides. These characteristics suggest that cloned aminopeptidase P is identical to APP-II reported by Yoshimoto et al. (Agric. Biol. Chem. 52(8), in press (1988]. |
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| Keywords |
Amino Acid Sequence; Aminopeptidases/genetics; Aminopeptidases/isolation & purification; Aminopeptidases/metabolism; Cloning, Molecular; DNA Restriction Enzymes; Escherichia coli/enzymology; Escherichia coli/genetics; Genes; Genes, Bacterial; Kinetics; Molecular Weight; Plasmids; Species Specificity; Substrate Specificity |
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