Prevotella intermedia が産生するアルカリホスファターゼのプロテインチロシンホスファターゼ様の性質について

Abstract

Prevotella intermedia is frequently detected in the microflora of patients with several types of periodontitis, and thought to be one of the most important etiological agents. P. intermedia was found to have high Alkaline phosphatase (ALPase) activity. ALPases are previously known to be one of the virulence factors to periodontal organisms. However, many enzymological properties of ALPase are unknown. In this study ALPase of Prevotella intermedia ATCC25611 was solubilized by extraction with 1% Triton X-114, and the solubilized enzyme was purified 47.4-fold with 3.3% recovery by using ion-exchange chromatography, gel-filtration and affinity column chromatography. The purified enzyme was observed as a single protein band that corresponded to molecular weight of 54, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparations. The molecular weight of the native enzyme was estimated to be about 150, 000 by gel filtration with TSK-gel G3000SW_<XL>. These findings indicated which P. intermedia ALPase is a dimer or a trimer. The purified enzyme dephosphorylated phosphotyrosine-containing human gastrine, a peptide substrate for protein tyrosine phosphatases (PTPases), and was inhibited by sodium orthovanadate, sodium molybdate and Zn^<2+>, which are inhibitors of PTPases activity. The purified enzyme was indicated that the optimal pH for ALPase activity was about 9.5 and the optimal pH for PTPase activity was about 6.0. The PTPase activity was reduced by approximately 10% of maximum at pH7.0. This result suggests that the purified enzyme can have about 90% of the maximum activity even under physiologically relevant conditions. The N-terminal sequence of the purified enzyme showed about 50% homology with the sequence of amino acids sharing an active site signature motif, (I/V) HCXAGXGR (S/T)G, which all of PTPases conserve. Therefore P. intermedia ALPase was found to have protein tyrosine phosphataselike activity and characterization.