Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381.
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.
Yamashita
Y
Y
Department of Preventive Dentistry, Kyushu Dental College, Kitakyushu, Japan.
Toyoshima
K
K
Yamazaki
M
M
Hanada
N
N
Takehara
T
T
D000469:Alkaline Phosphatase / Q000032:analysis*; D000596:Amino Acids / Q000032:analysis; D001439:Bacteroides / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D012995:Solubility; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
D000596:Amino Acids; D008345:Manganese; D000469:Alkaline Phosphatase; D015032:Zinc