Prevotella intermedia が産生する酸性ホスファターゼに関する研究

Abstract

Prevotella intermedia (P. intermedia), an anaerobic gram-negative bacterium, has been implicated as a pathogen in adult periodontitis, in acute necrotizing ulcerative gingivitis and in pregnancy gingivitis. P. intermedia has been known to have a high acid phosphatase (ACP) activity, one of the potential virulence factors in periodontopathogenic bacteria. However, little is known about the structure and function of ACPs from oral bacteria. In this study, in order to examine the potential role of ACP, the ACP from P. intermedia ATCC 25611 was purified to homogeneity and characterized. The ACP from P. intermedia was solubilized by extraction with 1% Triton X-114, and the crude extract was purified by ion-exchange chromatography on DEAE Bio-Gel, CM Bio-Gel and cellulose phosphate, followed by hydrophobic chromatography on Phenyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme revealed a single protein band with a molecular weight of Mr &ap;30 kDa. The molecular weight of the native enzyme from P. intermedia was estimated to be &ap;30 kDa using a gel filtration column with TSK gel G3000SW in the HPLC system. These findings suggest that the purified ACP is a monomer. The optimum pH for the enzyme was about 4.9 and the optimum temperature was 46℃. The enzyme activity was inhibited by sodium orthovanadate, sodium molybdate, sodium fluoride and Zn^<2+>, which are known as the inhibitors of protein tyrosine phosphatase (PTP) activity. On the other hand, it was not inhibited by okadaic acid, which is known to be a specific inhibitor of protein serine/threonine phosphatase (PP) activity. The enzyme had high activities against o-phospho-L-tyrosine and phosphotyrosine-containing peptides. The purified ACP was found to dephosphorylate the tyrosine-phosphorylated protein of a molecular weight &ap;170 kDa in an A431 cell lysate. The N-terminal amino acid sequence was determined for the purified ACP, and significant homology (64.3% identical amino acid residues) was found using the Swiss Prot Protein database between the purified ACP and the Pho C, the acid phosphatase of Morganella morganii. Thus, these findings suggest the possibility that the ACP from P. intermedia constitutes a novel class of ACPs, which has a PTP-like activity.