Abstract
A metalloprotease was purified from a culture supernatant of the fish pathogen, Listonella (Vibrio) anguillarum. The molecular weight of the enzyme was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was inhibited by EDTA and o-phenanthroline.Zn2+ and Mn2+ at 10μM reactivated the enzyme which had been inactivated with 0.1μM EDTA.Ca2+ at 1mM enhanced the enzyme activity by 170% and increased stability of the enzyme activity at high temperatures. One noteworthy finding is that the enzyme hydrolyzed only butyloxycarbonyl-leucyl-seryl-threonyl-arginyl-4-methylcoumaryl-7-amide (Boc-Leu-Ser-Thr-Arg-MCA, a substrate for plasma activated protein C (APC), an anticoagulation factor) among the 17 peptydyl-4-methylcoumaryl-7-amide. The enzyme extended the clotting time of rainbow trout plasma. The enzyme enhanced the hydrolyzing activity of Boc-Leu-Ser-Thr-Arg-MCA in trout plasma. These results indicate that the enzyme possesses an activity similar to APC and activates protein C of rainbow trout. The enzyme inhibited activities of Factor Xa and thrombin. From these findings, it was concluded that the metalloprotease produced by L. anguillarum plays a role as an anticoagulation factor similar to APC.
