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

在钙离子存在下，人唾液凝集素能识别部分中间链球菌。本研究采用Phenyl SepharoseHP疏水柱层析和Mono-Q阴离子交换层析，经二次Mono-Q柱层析和Superose 12凝胶过滤，在双离子洗涤剂CHAPS存在下，从菌株1208-1的细菌超声提取液中分离纯化了一种钙结合蛋白。经SDS-PAGE和凝胶过滤，纯化后的产物均一单体分子量为51 kDa，四聚体分子量约为200 kDa。等电点为pH 4.7。~(45)Ca放射自显影显示，1208-1细胞只有与Ca~(2+)结合的单体蛋白。1208-1细胞的唾液聚集依赖于唾液量和pH 5.5以上的钙离子。唾液凝集素经Sephacrul S400HR凝胶过滤纯化。SDS-PAGE分析表明，该凝集素为两种高分子量蛋白质(-100 kDa)。两种唾液都过滤一种…此外，纯化后的凝集素煮沸5min后无凝集活性。吸附分析表明，这种凝集素与中间链球菌K1K株和变形链球菌MT8148菌株的聚集有关，但与中间链球菌K16-1K株的唾液聚集有关。粘附性分析表明，放线菌细胞和羟基磷灰石小球表面的凝集素是与链球菌细胞结合的主要原因，凝集素有效地抑制了天然唾液包裹的磷灰石小球的黏附，但对放线菌细胞的黏附无明显影响。10名献血者的唾液滤液具有不同的聚集活性。链球菌细胞对放线菌细胞的黏附活性和涂布每种天然唾液的磷灰石微珠的黏附活性与聚集活性无关，每种唾液滤液作为效应物抑制涂布的磷灰石颗粒的黏附与聚集效价成正比。SDS-PAGE显示凝集素条带的深度与凝集效价有关。较少

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 … More 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. Less