Purification and characterization or porcine kidney cathepsin B

Three types of cathepsin B were purified from porcine kidney by a procedure involving ammonium sulfate fractionation, acetone fractionation, pH-gradient elution from CM-cellulose, gel filtration, salt gradient elution chromatography of CM-cellulose and finally preparative isoelectric focusing. The isoelectric points for the three types of cathepsin B were at pH 5.5 (Type I), pH 5.9 (Type II), and pH 6.2 (Type III). The molecular weights were estimated at 24,000 (Types I and II), and 29,000 (Type III) by SDS-polyacrylamide gel electrophoresis. The preparations were all unstable above pH 7.0 when compared with the stability at their optimal pH of around pH 6.0. The isozymes cleaved benzyloxycarbonyl-phenylalanyl-arginine-4-methyl-7-coumarylamide at pH 6.0 and at 40 degrees C with Km values of 0.225 mM (Types I and III) and 0.204 mM (Type II) and with kcat values of 71 s-1 (Types I and II) and 63 s-1 (Type III). They were strongly inhibited by iodoacetate, leupeptin and antipain, but not by DFP, PMSF, or pepstatin. The values of apparent inhibition constant for leupeptin were 15 nM (Types I and II) and 17 nM (Type III).