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Abstract
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.
Highlights
A fraction separated from rat submandibular gland it submandibular enzymatic vasoconstrictor (SEV).’The rat homogenates was found to contain a potent vasocon- submandibular gland is rich in serine proteases; most promistrictor when tested on isolated rabbit aortic rings. nent are the enzymes of the kallikrein family [2,3,4,5], some of
We recently found a potent proteinic vasoconstrictor in a ’ The abbreviations used are: SEV, submandibular enzymatic vasfraction separated from a rat submandibular gland homogenate
Molecular Weight and Isoelectric Point of SEV-The final SEV preparation showed a single band onSDS-PAGE, which migrated at the position corresponding to 28,900 k 2,300 daltons(mean f S.D.; n = 3) (Fig. 1A)
Summary
The isoelectric point of SEV was 7.36, as reconfirmed by analytical isoelectric focusing on precast polyacrylamide gel This was consistent with the values of7.07-7.45 obtained during purification with the Rotofor cell (Fig. S2,Miniprint). When nonreduced protein was sequenced, two amino acids were detected in each cycle (Fig. 2A). A single amino acid was detected per cycle in both heavy and light chains (Fig. 2, B and C). The 11th and 12th amino acids could not be determined because of the low yield; they were estimated to be Leu and His by excluding Ser and Gln (11th and 12th amino acids in the light chain) from the results obtained with nonreduced protein ( I l t h , Leu and Ser; 12th, His and Gln).
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