Purification and Properties of Bovine Serum Kallikrein Activated with Casein*

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Abstract
Bovine serum kallikrein [EC 3. 4. 4. 21] activated by treatment with casein was investigated. Casein-treatment induced the appearance of at least three principles, I, II and III, separable on a DEAE-Sephadex A-50 column, which showed both esterolytic and kinin releasing activity. Of these enzymes, Fraction I with the highest kinin releasing activity was purified. The purification procedures consisted of (NH4)2SO4 fractionation, batch-wise adsorption on, and elution from DEAE-Sephadex A-50 and CM-Sephadex C-50, chro-matography on a CM-Sephadex C-50 column, and gel-filtration on a Sephadex G-150 column. The enzyme recovery was found to be reproducible, and the total absorbancy at 280 mμ of the purified material from 4.7 liters of serum was about 20. The purified preparation showed an almost single band on cellulose acetate membrane electrophoresis at pH8.6. On disc gel electrophoresis at pH8.3, however, five protein components representing sharp bands could be detected in a narrow zone, suggesting that the enzyme was microheterogeneous. The apparent molecular weight of the purified enzyme was estimated to be about 95,000 by gel-filtration on a Sephadex G-200 column, differing from those of glandular and urinary kallikreins, estimated to range between 24,000 and 40,000. Serum kallikrein rapidly released kinin from bovine kininogen-I (high molecular weight kininogen), but scarcely from bovine kininogen-II (low molecular weight kini-nogen), indicating a significant difference from hog serum kallikrein which acts on kininogen-II. The enzyme specifically hydrolyzed N-substituted arginine and lyiine esters like thrombin [EC 3.4.4.13] and plasrain [EC 3.4. 4. 14]; however, unlike plasmin, neither caseinolytic nor fibrinolytic activities were detected in it. The Km. values were estimated toward N-cr-tosyl-L-arginine methylester (1.56× 10˜3u), N-a-tosyl-L-lysine mcthylester (6.02 × 10˜4 u), and N-or-benzoyl-L-arginine ethylester (9.62 × 10'm). The enzyme was moit stable at around pH9 and its optimum pH was found to be 8.8. Trasylol, soy bean trypsin inhibitor, DFP, benzamidine and TLCK strongly inhibited the enzyme activity, while egg white and lima bean trypsin inhibitors and ε-aminocaproic acid and heparin had no effects. These properties indicated that bovine serum kallikrein it an esterolytic enzyme which is functionally distinguished from thrombin, plasmin and the Hageman factor (factor-XII), enzymes in blood plasma known to have esterolytic activity.

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