A novel serine protease with vasoconstrictor activity coded by the kallikrein gene S3.
A fraction separated from rat submandibular gland homogenates was found to contain a potent vasoconstrictor when tested on isolated rabbit aortic rings. The vasoconstrictor was purified by a series of chromatographic steps. The purified compound (2.77 x 10(-9) M) induced 40% of the maximum contractile response to 60 mM KCl. Constriction was slow in onset, long-lasting, rinse-resistant, and unchanged by de-endothelialization; in addition, it was dose-related and inhibited by both EGTA and verapamil, but it was not affected by DUP753, an angiotensin II receptor antagonist. The compound was found to be a protein having a pI of 7.36 and a molecular weight of approximately 29,000 and exhibiting partial immunologic identity to rat glandular kallikrein and rat tonin. After 2-mercaptoethanol treatment, it separated into heavy (approximately 19,900) and light (approximately 10,700) chains having amino-terminal sequences of AY(X)HNNDLMLL and VVGGYN(X)ETNSQ, respectively. We found that they correspond to the amino-terminal and internal sequence of a previously unidentified kallikrein-like serine protease whose mRNA, named S3, has been found in the rat submandibular gland and prostate. The vasoconstrictor is able to hydrolyze t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide (a thrombin substrate), although its Kcat/Km was only 0.02% that of rat thrombin. Both vasoconstrictor and enzymatic activity on t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide were completely suppressed by amidinophenylmethylsulfonyl fluoride and soybean trypsin inhibitor; however, they were unaffected by hirudin, a thrombin inhibitor. At pH 6.5, it released angiotensin II when incubated with sheep angiotensinogen, although it had approximately one-tenth the activity of tonin. The submandibular enzymatic vasoconstrictor is a kallikrein-like enzyme, having some properties of both tonin and thrombin. It directly contracts vascular smooth muscle, acting via a mechanism that requires intact enzymatic activity.
Yamaguchi
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Hypertension Research Division, Henry Ford Hospital, Detroit, Michigan 48202.
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Scicli
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D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D005779:Immunodiffusion; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D007610:Kallikreins / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats; D012697:Serine Endopeptidases / Q000235:genetics* / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects*
D007610:Kallikreins; D012697:Serine Endopeptidases; C067805:submandibular enzymatic vasoconstrictor