The isolation and properties of pig submandibular kallikrein.
The kallikrein from pig submandibular glands was highly purified, with an overall yield of 31%. Affinity chromatography on bovine basic pancreatic trypsin inhibitor linked to Sepharose 4B was an especially effective step in the purification procedure, giving a purification factor of 80. The enzyme is a single-chain molecule, occurring, as does pig urinary kallikrein, as a major B-form of apparent mol.wt. 39600 and minor amounts of an A-form of apparent mol.wt. 35900; the two forms can be separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid composition of pig submandibular kallikrein is very similar to, but not quite identical with, that of the two-chain beta-kallikrein isolated from pig pancreatic autolysates. Submandibular kallikrein contains notably more glucosamine and hexoses than does pancreatic beta-kallikrein. Submandibular kallikrein, and also urinary kallikrein, exhibit an unusual biphasic hydrolysis of substrate esters that is not shared by pancreatic beta-kallikrein. For the submandibular enzyme, the K(m) for the initial reaction phase of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester is 0.15+/-0.01mm (mean+/-s.e.m.), but rises to 0.69+/-0.04mm (mean+/-s.e.m.) in the stationary reaction phase; the V(max.) does not differ significantly between the two phases. The esterolytic activities of submandibular and urinary kallikreins on a number of esters of different amino acids resemble each other much more closely than those of pancreatic beta-kallikrein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004952:Esters; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007610:Kallikreins / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine; D007700:Kinetics; D008970:Molecular Weight; D009439:Neuraminidase; D010179:Pancreas / Q000201:enzymology; D013363:Submandibular Gland / Q000201:enzymology*; D013552:Swine
D000596:Amino Acids; D004952:Esters; D009439:Neuraminidase; D007610:Kallikreins