Novel extracellular alkaline metalloendopeptidases from Vibrio sp. NUF-BPP1: purification and characterization.
We found two types of novel alkaline metalloendopeptidases (AP1 and AP2) from a marine bacterium, isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) and identified as Vibrio sp. (NUF-BPP1). We studied the structure-function relationship of these marine bacterial proteases. The electrophoretically homogeneous proteases had a molecular mass of 48 kDa for AP1 and 36 kDa for AP2 on SDS-PAGE, and showed optimum activity at around pH 9.5-10.0 (casein as substrate). Calcium chloride (5 mM) stabilized the activities over pH 6-11, but o-phenanthroline and EDTA inhibited the activities of both AP1 and AP2. The EDTA-inactivated proteases were partly restored to activity by addition of either zinc or calcium. Sodium chloride (3.5 M) increased the activities toward Z-Gly-Leu-NH2. N-Terminal sites of hydrophobic amino acid residues (Leu, Ile, Phe, Tyr, and Trp) of the peptide substrates were cleaved by AP1 and by AP2. Autolysis of AP1 in the absence of calcium ion probably produced AP2 by releasing a fragment (molecular mass of about 12 kDa) from the C-terminal end of AP1.
Fukuda
K
K
Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Japan.
Hasuda
K
K
Oda
T
T
Yoshimura
H
H
Muramatsu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D004492:Edetic Acid / Q000737:chemistry / Q000494:pharmacology; D005399:Fishes / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000378:metabolism / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000737:chemistry / Q000145:classification / Q000201:enzymology*
D007202:Indicators and Reagents; D012965:Sodium Chloride; D004492:Edetic Acid; D008666:Metalloendopeptidases