PURIFICATION OF CAT SUB-MAXILLARY KALLIKREIN

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 pl'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 1 min at 25°C and pH 8·0. The Km'S of the purified kallikrein with BAEE and TAME were 0·67 and 0·34 mM 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 × l04 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. © 1979 By The Journal Of Biochemistry.