SHIMADA Junko The University of Tokushima, School of Dentistry, Administrative Staff, 歯学部, 教務員 (10170945)
YAMATO Kanako The University of Tokushima, School of Dentistry, Research Associate, 歯学部, 助手 (40243711)
ICHIMIYA Seiko The University of Tokushima, School of Dentistry, Research Associate, 歯学部, 助手 (30223845)
HAYASHI Hiroyuki The University of Tokushima, School of Dentistry, Assistant Professor, 歯学部・附属病院, 講師 (80243707)
NAKAMURA Ryo The University of Tokushima, School of Dentistry, Professor, 歯学部, 教授 (30034169)
|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1995 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1994 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Several kinds of trypsin-like enzymes were isolated from P.gingivalis and B.forsythus, and characterized their properties on the biological and antigenic distinction. Three trypsin-like enzymes (Pase-B,Pase-C,Pase-S) were from the culture supernatant of P.gingivalis. Pase-S resembled Pase-B in its hydrolytic specificity, cleaved only arginine residue of peptides, type IV and denatured type I collagen. Pase-C hydrolyzed BLpNA and showed the strongest capacity of degrading native type I collagen. This enzyme was the only one to possess hemagglutinating activity. Western blotting analysis of these three enzymes using polyclonal antibody against Pase-S,revealed immunological distinction from Pase-C.Pase-B reacted with the antibody and was found to contain a considerable amount of carbohydrates in its structure as compared to the other. The N-terminal amino acid sequence of Pase-S was identical with Pase-B from 7th to 12th, but 10th and 13th could not be detected. It suggests that Pase-S fr
om P.gingivalis is less active in its pathogenicity than Pase-C and that the enzyme may be an isoenzyme of Pase-B.
The another enzyme was isolated from the whole cells of B.forsythus by solubilizing with 0.8% octylglucopyranoside, and was purified by using ion exchange and gel filtration high performance liquid chromatography. By SDS-polyacrylamide gel electrophoresis, this enzyme was separated into three protein bands (91,76,72kDa). However, only one band corresponding to 91kDa of molecular weights showed enzyme activity. The enzyme was inhibited by TLCK,antipain, pepstatin. DTT and sulfhydryl reagents had no effect on this activity. The purified enzyme hydrolyzed synthetic substrate, more effective BLpNA than BApNA,it did not hydrolyze casein. But it acted on the carboxyl side of arginine in insulin B chain. Thus this enzyme is probably an endopeptidase.