Actinobacillus actinomycetemcomitans が産生する酸性ホスファターゼのプロテインチロシン/セリンホスファターゼ様の性質に関する研究

Abstract

Actinobacillus actinomycetemcomitans has been implicated as one of the causative organisms of localized juvenile periodontitis and adult periodontitis. A. actinomycetemcomitans has been known to have a high acid phosphatase (ACP) activity, which is previously known to be one of the virulence factors in periodontopathic bacteria. Little is known about ACP from oral bacteria. In order to examine the potential role of ACP, the ACP of A. actinomycetemcomitans was purified to homogeneity and characterized. The ACP in the cell-associated form of A. actinomycetemcomitans was released from the membranes with Triton X-114, then purified by ion-exchange chromatography on DEAE Bio-Gel and Gel filtration, followed by affinity chromatography on Hi-Trap Blue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single protein band with a molecular weight of Mr ≈ 40, 000. The molecular weight of the native enzyme was estimated to be 80, 000 by gel filtration with a Superdex-200 column. The pH optimum for the purified enzyme was about 5.4 and the temperature optimum was 37℃. The purified enzyme was inhibited by sodium orthovanadate, which is one of the inhibitors of protein tyrosine phosphatase (PTP) activity. However, it was not inhibited by okadaic acid, which is a specific inhibitor of protein serine/threonine phosphatase (PP) activity. The purified enzyme dephosphorylated o-phospho-L-tyrosine, phosphotyrosine-containing peptides and Raytide, which are used as substrates for PTP. Also, the purified enzyme dephosphorylated O-phospho-L-serine and phosphoserine-containing peptides and casein, which is good substrate for PP. On the other hand, phosphorylase kinase, which is used as a substrate for PP, was not dephosphorylated by the purified enzyme. It turned out that ACP of A. actinomycetemcomitans is classified in the PTP family, while it is not an SH-enzyme because it does not completely need the thiol reducing agent. The purified enzyme exhibited substantial activity against both Tyr and Ser-containing substrates. Thus, these findings suggest for the first time the existence of a novel tyrosine/serine protein phosphatase in oral bacteria and that ACP of A. actinomycetemcomitans has unique biochemical properties compared with any known PTPs.