Purification and characterization of a serine protease (esterase B) from rat submandibular glands.
A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005779:Immunodiffusion; D007610:Kallikreins / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012697:Serine Endopeptidases; D013363:Submandibular Gland / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D010450:Endopeptidases; D007610:Kallikreins; D012697:Serine Endopeptidases; C049262:esterase B