Venkitaraman A R, Vacca-Smith A M, Kopec L K, Bowen W H
Department of Dental Research, University of Rochester Medical Center, New York 14642, USA.
J Dent Res. 1995 Oct;74(10):1695-701. doi: 10.1177/00220345950740101101.
GlucosyltransferaseB, GtfC, and GtfD were purified by hydroxyapatite column chromatography, followed by ultrafiltration from the culture supernatant fluids of three Streptococcus milleri constructs (gift from Dr. H.K. Kuramitsu) which harbored individual gtf genes of Streptococcus mutans GS5. GtfB, GtfC, and GtfD were enzymatically active both in solution and in an experimental pellicle (HA-CWS-Gtf) formed by adsorbing Gtf onto the surface of clarified human whole saliva (CWS)-coated hydroxyapatite (HA). The Km values for sucrose for all three enzymes were lower when the enzyme was adsorbed to a surface, compared with when it was in solution. In solution phase assays, and in the absence of primer dextran, glucan production was enhanced 75% when both GtfB and GtfD were present in the reaction mixture, compared with the sum of the individual enzyme activities (p < 0.005). This enhancement did not occur when GtfC was additionally present, or when the GtfB+GtfD enzyme pair was adsorbed onto HA-CWS. In additional experiments, glucan formed by GtfB or GtfC, but not by GtfD, on a HA-CWS-Gtf surface increased adherence of Streptococcus mutans GS5 and Streptococcus sobrinus 6715 by seven- to nine-fold compared with adherence when no glucan was present on the pellicle surface (p < 0.001). Further, treatment of the HA-CWS-GtfB-glucan or HA-CWS-GtfC-glucan pellicle with alpha-1,6 dextranase significantly reduced adherence of both streptococcal strains (p < 0.001). These results show that GtfB, GtfC, and GtfD are enzymatically active in an adsorbed state and that the nature of their product glucan can influence the adherence of cariogenic oral streptococci to an experimental pellicle.
葡糖基转移酶B（GtfB）、GtfC和GtfD通过羟基磷灰石柱色谱法进行纯化，随后从三种米勒链球菌构建体（由H.K. Kuramitsu博士馈赠）的培养上清液中进行超滤，这些构建体含有变形链球菌GS5的单个gtf基因。GtfB、GtfC和GtfD在溶液中以及在通过将Gtf吸附到澄清的人全唾液（CWS）包被的羟基磷灰石（HA）表面形成的实验性薄膜（HA-CWS-Gtf）中均具有酶活性。与在溶液中时相比，当酶吸附到表面时，这三种酶对蔗糖的米氏常数（Km值）更低。在溶液相测定中，且在没有引物葡聚糖的情况下，当反应混合物中同时存在GtfB和GtfD时，葡聚糖的产生比单独酶活性的总和提高了75%（p < 0.005）。当额外存在GtfC时，或者当GtfB + GtfD酶对吸附到HA-CWS上时，这种增强并未发生。在额外的实验中，与薄膜表面不存在葡聚糖时的黏附相比，GtfB或GtfC（而非GtfD）在HA-CWS-Gtf表面形成的葡聚糖使变形链球菌GS5和远缘链球菌6715的黏附增加了7至9倍（p < 0.001）。此外，用α-1,6葡聚糖酶处理HA-CWS-GtfB-葡聚糖或HA-CWS-GtfC-葡聚糖薄膜可显著降低两种链球菌菌株的黏附（p < 0.001）。这些结果表明，GtfB、GtfC和GtfD在吸附状态下具有酶活性，并且它们产物葡聚糖 的性质可影响致龋性口腔链球菌对实验性薄膜的黏附。
