Isolation and some properties of dog and rat pancreatic kallikreins

Dog and rat pancreatic kallikreins [EC 3.4.21.8] were isolated by a purification procedure involving acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-100 gel filtration, and Ampholine isoelectric focusing. Dog pancreatic kallikrein was separated into three forms with isoelectric points (pI's) of 4.1, 4.2, and 4.4. Rat pancreatic kallikrein was present in two forms, both with a pI of approximately 4.1. Vasodilator activities of the three dog kallikreins and the rat kallikrein were 610–1,350 kallikrein units (KU)/mg and 810–870 KU/mg, respectively. Their esterolytic activities with Nα-benzoyl-L-arginine ethyl ester (BAEE) and Nα-toluenesulfonyl-L-arginine methyl ester (TAME) were 14–23 and 2.4–4.8 units/mg (dog), respectively, and 300–390 and 33–43 units–mg (rat), respectively, at 25°C and pH 8.0. Rat kallikrein showed some activity toward Nα-benzoyl-DL-argrnine-p-nitroanilide (BApNA) (0.4–0.7 unit/mg). The Kin'S of the three dog kallikreins with BAEE and TAME were 0.27–0.33 and 0.14–0.26 mM, respectively, and those of the rat kallikrein were 0.065 and 0.058 mM, respectively. The molecular weights of the three dog kallikreins and the rat kallikrein were estimated by Sephadex G-l00 gel filtration to be 27,000–30,000 and 30,000, respectively. Dog kallikrein-III and rat kallikrein consisted of 240 and 277 amino acid residues, and the molecular weights calculatcd from their amino acid compositions were 27,000 and 31,600, respectively. The optimum pH's of the three dog kallikreins and the rat kallikrein in BAEE hydrolysis were 9.0–9.2 and 9 2, respectively. Both kallikreins were inhibited by heavy metal ions such as Zn2+, Hg2+, or Cd2+. The dog kallikreins were inhibited by Trasylol (weakly) and two potato kallikrein inhibitors (PKI-56 and -64) as well as by diisopropylfluorophosphate (DFP).