Purification of Cat Submaxillary Kallikrein

Abstract

The cat submaxillary gland contains 1, 000-2, 400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6-7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930-1, 260 KU in the dog vasodilator assay, and hydrolyzed 15-25 and 9-12 μmol of N-α-benzoyl-L-arginine ethyl ester (BAEE) and N-α-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in I min at 25°C and pH 8.0. The K_m's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34mM, respectively. Hydrolysis of N-α-benzoyl-L-tyrosine ethyl ester (BTEE), N-α-benzoyl-arginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5×10⁴ by Sephadex G-100 gel filtration and 4.7×10⁴ by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.