This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yamashita, Y Articles by Takehara, T Search for Related Content PubMed PubMed Citation Articles by Yamashita, Y Articles by Takehara, T

 Previous Article  |  Next Article 

Infect Immun. 1990 September; 58(9): 2882-2887

Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381.

Department of Preventive Dentistry, Kyushu Dental College, Kitakyushu, Japan.

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 solubilized 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 km 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.

This article has been cited by other articles:

• Nagano, K., Read, E. K., Murakami, Y., Masuda, T., Noguchi, T., Yoshimura, F. (2005). Trimeric Structure of Major Outer Membrane Proteins Homologous to OmpA in Porphyromonas gingivalis. J. Bacteriol. 187: 902-911 [Abstract] [Full Text]  
• Masuda, K., Yoshioka, M., Hinode, D., Nakamura, R. (2002). Purification and Characterization of Arginine Carboxypeptidase Produced by Porphyromonas gingivalis. Infect. Immun. 70: 1807-1815 [Abstract] [Full Text]  
• Zappa, S., Rolland, J.-L., Flament, D., Gueguen, Y., Boudrant, J., Dietrich, J. (2001). Characterization of a Highly Thermostable Alkaline Phosphatase from the Euryarchaeon Pyrococcus abyssi. Appl. Environ. Microbiol. 67: 4504-4511 [Abstract] [Full Text]  
• Chen, X., Ansai, T., Awano, S., Iida, T., Barik, S., Takehara, T. (1999). Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from Prevotella intermedia. J. Bacteriol. 181: 7107-7114 [Abstract] [Full Text]