Purification and Characterization of Proteinases Identified as Cathepsins L and L-like (58kDa) Proteinase from Mackerel (Scomber australasicus)

概要

論文の詳細を見る
Cathepsins L and L-like (58kDa) proteinase from mackerel were purified to electrophoretical homogeneity by Concanavalin A-Sepharose and Econo-Pac S chromatographies. The molecular weights of cathepsins L and L-like proteinase were 30,000 and 58,000, and the optimal pH for the hydrolysis of Z-Phe-Arg-MCA (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-[4-methyl] coumarylamide) were 5.0 and 5.5, respectively. The stability of both purified proteinases at various pHs was low, when the pH was above 7.0. According to the substrate specificity analysis, these proteinases hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, but did not hydrolyze Z-Arg-MCA and L-Arg-MCA. The activities of these two proteinases were effectively activated by cysteine and dithiothreitol. Their thiol-dependent proteolytic activity against Z-Phe-Arg-MCA was strongly inhibited by E-64 (trans-epoxysuccinyl-L-leucylamido[4-guanidino]butane), antipain, chymostatin, iodoacetic acid, and leupeptin, but not inhibited by pepstatin or phenylmethane sulfonyl floride. The inactivation rate constants (K_D) of cathepsins L and L-like proteinases at 50℃ were 5.1 × 10^<-5> and 6.9×10^<-4>s^<-1>, respectively. K^+ , Na^+ , Mg^+ , and Sr^+ did not affect them, while Zn^<2+>, Cd^<2+>, Co^<2+>, Ni^<2+>, Cu^<2+>, Hg^<2+>, Fe^<2+> , and Fe^<3+> inhibited the activity of the purified catepsins L and L-like proteinase.
社団法人日本農芸化学会の論文
1993-09-23