Purification and Characterization of Porcine Kidney Cathepsin B1

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Abstract
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 elutiori 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 as 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 at pH 6.0 and at 40°C with Km values of 0.225 nmi (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).

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