Purification and characterization of Streptomyces griseus metalloendopeptidases I and II.
Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).
Tsuyuki
H
H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
Kajiwara
K
K
Fujita
A
A
Kumazaki
T
T
Ishii
S
S
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides; D011402:Pronase; D013305:Streptomyces griseus / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D007527:Isoenzymes; D009842:Oligopeptides; D008666:Metalloendopeptidases; D011402:Pronase