徳島大学 教育・研究者情報データベース(EDB)

1. 青山学院大学.理工学部

1. (英) Mizutani Yukihito

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

2. (英) Narikawa Tatsuya

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

3. 佐藤 高則

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

4. (英) Sakurai Nobuhiko

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

5. (英) Kaji Hiroyuki

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

6. (英) Yamada Shingo

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

7. (英) Samejima Tatsuya

   役割	任意	共著
   貢献度	任意
   学籍番号	推奨

(英) A New UV Method for Serum γ-Glutamyltransferase Assay Using Recombinant 4-Aminobenzoate Hydroxylase as a Coupling Enzyme.

(英) 4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent. Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.

(日) 4-Aminobenzoate Hydroxylase (4-ABH)遺伝子をマッシュルームよりcDNAクローニングを行い，GSTとの融合タンパク質として大腸菌内で効率良く発現させた．この組換え体4-ABHを共役酵素として，血清中のγ-Glutamyltransferaseの測定系について検討を行った．その結果，4-ABHを用いた場合，従来法と良い相関を示し，今後臨床検査への応用が可能となった．

1. (英) Amino Acid Sequence
2. (英) Detergents
3. (英) Dose-Response Relationship, Drug
4. (英) Escherichia coli
5. (英) Hydrogen-Ion Concentration
6. (英) Hydroxylation
7. (英) Mixed Function Oxygenases
8. (英) Models, Chemical
9. (英) Molecular Sequence Data
10. (英) Multienzyme Complexes
11. (英) NAD
12. (英) NADH, NADPH Oxidoreductases
13. (英) Peroxidases
14. (英) Protein Engineering
15. (英) Recombinant Proteins
16. (英) Sequence Homology, Amino Acid
17. (英) Spectrophotometry
18. (英) Ultraviolet Rays
19. (英) gamma-Glutamyltransferase

1. (英) Article.PublicationTypeList.PublicationType: Journal Article

2. (英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't