Purification, characterization and structure analyzes of calcium binding protein of oral steptococci.

1997 Fiscal Year Final Research Report Summary

• Project/Area Number: 08672380
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 矯正・小児・社会系歯学
• Research Institution: KAGOSHIMA UNIVERSITY
• Principal Investigator: YAMAGUCHI Taihei KAGOSHIMA UNIVERSITY Dental Hospital Preventive Dentistry Research Associate, 歯学部附属病院, 助手 (80230358)
• Co-Investigator(Kenkyū-buntansha): INOUE Masakazu KAGOSHIMA UNIVERSITY Dental School Proventive Dentistry Professor, 歯学部, 教授 (30028740)
• Project Period (FY): 1996 – 1997
• Keywords: oral Streptococcus milleri / fimbriae / calcium dependence / saliva aggregation / calcium binding protein / saliva agglutinin / adhernce activity

Research Abstract

H.8.
A part of Streptococcus intermedius was recognized by human salivary agglutinin in the presence of calcium ion. In this study, a calcium binding protein has been purified from the bacterial sonicated extract of a isolate of, 1208-1, by Phenyl Sepharose HP hydrophobic column chromatography and Mono Q anion-exchange chromatography, followed by the second Mono Q column and Superose 12 gel filtration in the presence of biionic detergent, CHAPS.The purified material has a Mr of 51 kDa as homogeneic monomer meterial and approximately 200 kDa as tetramer by SDS-PAGE and by gel filtration. The isoelectric point was pH 4.7. The ^<45>Ca autoradiograph showed that only the monomer protein bound to Ca^<2+>.
H.9.
The saliva aggregation of 1208-1 cells was depended on amount of saliva and on calcium ion over pH 5.5. The saliva agglutinin was purified by Sephacrul S400HR gel filtration. SDS-PAGE indicated that the agglutinins were two high molecular weight proteins (-100kDa). Both saliva filtrate a nd the purified agglutinin boiled for 5 min showed no aggregation activity. Adsorption analyzes showed that the present agglutinins were related with aggregation of a S.intermedius K1K isolate and a Streptococcus mutans MT8148 strain also, but another mechanisim related with saliva aggregation of a S.intermedius K16-1K isolate. Adherence assay showed that the agglutinin on the surface of actinomyces cells and hydroxyapatite beads accounted for binding of streptococcus cell and that agglutinin effectively inhibited the adhernce to apatite beads coated with native saliva but not to actinomyces cells. Saliva filtrates from 10 donors showed different aggregating activity. Adherence activites of streptococcal cells to actinomyces cells and apatite beads coated with each native saliva were not related with aggregation activity.Each saliva filtrates as effector inhibited the adherence to coated apatite deads in proportion to aggregation titer. SDS-PAGE demonstrated that the depth of the agglutinin bands related with aggregative titer.