Characterization of novel metallo-proteases released from ascidian hemocytes by treatment with calcium ionophore.
We have previously demonstrated that calcium ionophore induced the release of a novel metallo-protease from hemocytes of a solitary ascidian, Halocynthia roretzi. Here, we isolated the enzymes, PI and PII, from the culture media of H. roretzi hemocytes, which had been treated with calcium ionophore, A23187. The purification procedure included hydrophobic and anion-exchange chromatographies, and gel filtration. The molecular weights of the enzymes were estimated to be 11,000 by gel filtration, but the apparent sedimentation coefficients were 5.0 S, which suggests that the H. roretzi enzymes are of larger proteins with molecular weights of 80,000-90,000. The most susceptible substrate was succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, and the optimum pH was 8.0, in either case of PI or PII. The activities of PI and PII enzymes were strongly inhibited by metal-chelating agents and propioxatin A, but not by phosphoramidon, a typical metallo-protease inhibitor. Zinc and calcium ions were found to be essential for the maximum expression of protease activity in both enzymes. Thus, the isolated enzymes are characterized as phosphoramidon-insensitive metallo-proteases, which are inhibited by propioxatin A. Extracellular roles of these enzymes were also discussed.
Azumi
K
K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Yokosawa
H
H
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D006434:Hemocytes / Q000187:drug effects* / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014561:Urochordata
D003470:Culture Media; D008670:Metals; D011480:Protease Inhibitors; D000001:Calcimycin; D008666:Metalloendopeptidases