ラット顎下腺顆粒管細胞に存在する 2 種類のトリプシン様プロテアーゼの精製, 性質及び同定
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概要
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Granular convoluted tubules of rat submandibular glands contain many kinds of physiological active peptides, namely NGF, EGF and some proteases, such as kallikrein, tonin, and so on. Recently, many kinds of trypsin-like proteases with different isoelectric points (pI) have been found in the homogenate of the rat submandibular gland, although they were only fractionated with electrofocusing. Two types of TAMEases which hydrolyze p-tosyl-arginine methyl ester (TAME) were thus purified by gel filtration, and DEAE ion-exchange chromatography and high-performance liquid chromatography and then their properties were investigated in this paper. The results obtained were as follows ; One TAMEase with pI 5.2 had a molecular weight of 28, 000 consisted of two subunits with molecular weights of 20, 000 and 8, 000, whereas the other TAMEase with pI 6.1 was of 32, 000. The former (pI 5.2) hydrolyzed TAME, TLME, BAPA and H-Pro-Phe-Arg-MCA, but not BTEE, whereas the latter (pI 6.1) hydrolyzed TAME and weakly TLME and BAPA but not H-Pro-Phe-Arg-MCA or BTEE. pH optima of the activities of two TAMEases for TAME were at 8.5 and temperature optima of the activities of two TAMEases for BAPA were around 50℃. TAMEase (pI 5.2) was strongly inhibited by soybean trypsin inhibitor, leupeptin, PMSF, TLCK, whereas TAMEase (pI 6.1) was weakly inhibited. Both TAMEases were not inhibited by PCMB at all. TAMEase (pI 6.1) was activated by 20μM of EGTA, although it was markedly inhibited by 10nM of Zn^<2+> ion. TAMEase (pI 5.2) was slightly activated by 1mM of Cd^<2+> and Mn^<2+> and cleaved 42K band, which resulted in a major band of 39k and minor bands of 37K, 34K and 32K. The 42K band was identified as actin by the method of Western blotting. TAMEase (pI 6.1) also cleaved 42K band without EDTA, and 52k band, still unidentified, in the presence of 20μM EDTA. The pH optimum of these cleavages was at 7.5. Anti TAMEase (pI 6.1) IgG did not form any precipitin line with TAMEase (pI 5.2), whereas anti TAMEase (pI 5.2) IgG did a slight precipitin line with TAMEase (pI 6.1) with a spur formation between anti TAMEase (pI 5.2) IgG and TAMEase (pI 5.2). By the indirect immunofluoresence method, these TAMEases were found to localize in the granular convoluted tubules of the submandibular glands of rats. TAMEase (pI 5.2) was nearly identical to PSR-V described by lkeno et al., although minor differences were observed between them, whereas TAMEase (pI 6.1) was partly similar to tonin, although there is no report on the esteroprotease activity of tonin inhibited by the very low concentration of Zn^<2+> ion. Both TAMEases might cleave a cytoskelton protein and actin, and TAMEase (pI 6.1) with EDTA might only cleave an unknown 52K band when the physiological active enzymes were secreted into the blood stream, although the physiological roles of the two types of TAMEase have not yet been fully clarified.
- 九州歯科学会の論文
- 1988-06-25
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