Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381

Y. Yamashita, K. Toyoshima, M. Yamazaki, N. Hanada, T. Takehara

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

Cell-associated alkaline phosphatase (ALPase) of Bacteroides gingivalis 381 was found in the outer part of the periplasmic space by using an ultracytochemical procedure. Cell-associated ALPase was solubulized by extraction with 1% Triton X-100, and the solubilized enzyme was purified 904-fold with 5.6% recovery by using affinity column chromatography for mammalian intestinal-form ALPase. The purified enzyme gave a single protein band that corresponded to the enzyme activity band on polyacrylamide gel electrophoresis preparations. A single protein band at a molecular weight of 61,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparations. The molecular weight of the native enzyme was estimated to be 130,000 by gel filtration with TSK-gel G3000SW. These findings indicate that B. gingivalis ALPase is a homodimer. The optimal pH of the enzyme was between 9.1 and 9.3 in the absence of divalent metal ions and was between 10.1 and 10.3 in the presence of manganese or zinc ions. The apparent K(m) for p-nitrophenylphosphate was 0.037 ± 0.003 mM (mean ± standard deviation) at pH 9.2 in the absence of divalent metal ions and 0.22 ± 0.02 mM at pH 10.2 in the presence of 1 mM manganese ions. Under both of the conditions described above, the purified enzyme was able to hydrolyze casein and O-phosphoserine, suggesting that B. gingivalis ALPase can act as a phosphoprotein phosphatase. ALPase that immunologically cross-reacted with the purified enzyme was found in the extracellular soluble fraction. This means that ALPase is released from the periplasmic space into the culture supernatant as a soluble form.

Original languageEnglish
Pages (from-to)2882-2887
Number of pages6
JournalInfection and Immunity
Volume58
Issue number9
Publication statusPublished - Jan 1 1990
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

Fingerprint

Dive into the research topics of 'Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381'. Together they form a unique fingerprint.

Cite this