The submandibular gland of the rat contains several enzymes belonging to the kallikrein family. These include tissue kallikrein, antigen gamma (T-kininogenase), esterase B and tonin. In the present study, a new member of this family, which we have named KLP-S3, was identified and purified from the submandibular gland. KLP-S3 was classified as a kallikrein-like enzyme on the basis of its immunological similarity to other kallikrein-like enzymes and its showing 70% and 73% identity in partial amino acid sequence with tissue kallikrein and tonin respectively. Furthermore, the 44 sequenced amino acid residues showed complete correspondence to the mRNA S3 of the kallikrein gene family, which was the rationale for the name kallikrein-like protein (KLP) S3. KLP-S3 consisted of three isoenzymes with pI 6.75, 6.90 and 6.95, which significantly differed from those of other kallikrein-like enzymes. In conjunction with its immunological relationship to kallikrein, this parameter (pI) was considered robust enough to identify the enzyme during purification, since a specific physiological substrate for KLP-S3 has yet to be identified. In SDS/PAGE the three isoenzymes ran as one band with a molecular mass of 25,800 Da, which after reduction with 2-mercaptoethanol was split into two chains with molecular masses of 16,500 and 13,300 Da. In common with other kallikrein-like enzymes, KLP-S3 was inhibited by phenylmethanesulphonyl fluoride, and was thus classified as a serine protease. It was also inhibited by soya-bean trypsin inhibitor but not by aprotinin. It showed weak reactivity against the chromogenic substrates S2288, S2266, S2366 and S2302 (D-Ile-Pro-Arg 4-nitroanilide, D-Val-Leu-Arg 4-nitroanilide, Glu-Pro-Arg 4-nitroanilide and D-Pro-Phe-Arg 4-nitroanilide respectively) and did not cleave rat T-kininogen or dog high-molecular-mass/low-molecular-mass kininogen. Its specific angiotensin II-generating activity (angiotensin I as substrate) was 0.04% of that of rat tonin. KLP-S3 (1-100 nM) induced a statistically significant angiotensin-independent contraction of isolated rat aorta rings. The maximum contraction was 15% of the response to the alpha-adrenoceptor agonist phenylephrine (1 microM). The concentration of KLP-S3 in the rat submandibular gland was by single radial immunodiffusion estimated to be 47 +/- 3 micrograms/mg of protein.

The rat kallikrein family consists of multiple closely related proteins. A method for demonstration and identification of kallikrein-like proteins has been developed based on their differences in isoelectric point and their immunological similarity. The method, which involved separation in flat-bed isoelectro-focusing gels (pH range 3-9) and detection by immunoblotting using polyclonal antiserum against one of the family members, has been used in the present study to detect kallikrein-like proteins in the rat prostate. Nine immunoreactive kallikrein-like protein bands were detected with pI ranging from 5.30 to 8.35. Of these, six were completely purified and three were partially purified. Two proteins (pI 5.30 and 6.75-6.90) corresponded to protein bands in gels of rat submandibular-gland extracts, and were identified by partial amino acid sequence analysis as rK8 and rK9 respectively. In addition, sequence analysis revealed complete sequence similarity between rK9 and the immunoreactive prostate proteins with pI 7.15, 7.25, 7.50 and 8.27. On the basis of this finding and immunological and biochemical characterization, we concluded that all the kallikrein-like proteins detected, except for rK8, represented isoenzymes of rK9. The molecular masses of the prostate rK9 isoenzymes (24,600-29,300 Da) were close to that of submandibular-gland rK9 (24,600 Da), although differences were observed after reduction with mercaptoethanol. The prostate rK9 isoenzymes were, like submandibular-gland rK9, inhibited by soya-bean trypsin inhibitor but not by aprotinin, and were classified as serine proteases as they were inhibited by phenylmethanesulphonyl fluoride. rK8 (28,700 Da) showed no activity with any of the substrates tested, and its inhibitory profile could therefore not be studied. No other enzymes of the kallikrein family were found in the rat prostate.