Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381

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

研究成果: ジャーナルへの寄稿記事

19 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)2882-2887
ページ数6
ジャーナルInfection and Immunity
58
発行部数9
出版物ステータス出版済み - 1 1 1990

Fingerprint

Porphyromonas gingivalis
Alkaline Phosphatase
Enzymes
Ions
Periplasm
Manganese
Polyacrylamide Gel Electrophoresis
Molecular Weight
Metals
Phosphoserine
Phosphoprotein Phosphatases
Octoxynol
Caseins
Affinity Chromatography
Sodium Dodecyl Sulfate
Gel Chromatography
Zinc
Proteins
Gels

All Science Journal Classification (ASJC) codes

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

これを引用

Yamashita, Y., Toyoshima, K., Yamazaki, M., Hanada, N., & Takehara, T. (1990). Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381. Infection and Immunity, 58(9), 2882-2887.

Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381. / Yamashita, Yoshihisa; Toyoshima, K.; Yamazaki, M.; Hanada, N.; Takehara, T.

:: Infection and Immunity, 巻 58, 番号 9, 01.01.1990, p. 2882-2887.

研究成果: ジャーナルへの寄稿記事

Yamashita, Y, Toyoshima, K, Yamazaki, M, Hanada, N & Takehara, T 1990, 'Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381', Infection and Immunity, 巻. 58, 番号 9, pp. 2882-2887.
Yamashita, Yoshihisa ; Toyoshima, K. ; Yamazaki, M. ; Hanada, N. ; Takehara, T. / Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381. :: Infection and Immunity. 1990 ; 巻 58, 番号 9. pp. 2882-2887.
@article{8f336e4594e04c80886fb929bcbb001d,
title = "Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381",
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.",
author = "Yoshihisa Yamashita and K. Toyoshima and M. Yamazaki and N. Hanada and T. Takehara",
year = "1990",
month = "1",
day = "1",
language = "English",
volume = "58",
pages = "2882--2887",
journal = "Infection and Immunity",
issn = "0019-9567",
publisher = "American Society for Microbiology",
number = "9",

}

TY - JOUR

T1 - Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381

AU - Yamashita, Yoshihisa

AU - Toyoshima, K.

AU - Yamazaki, M.

AU - Hanada, N.

AU - Takehara, T.

PY - 1990/1/1

Y1 - 1990/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0025182191&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025182191&partnerID=8YFLogxK

M3 - Article

C2 - 2117573

AN - SCOPUS:0025182191

VL - 58

SP - 2882

EP - 2887

JO - Infection and Immunity

JF - Infection and Immunity

SN - 0019-9567

IS - 9

ER -