A new UV method for serum gamma-glutamyltransferase assay using recombinant 4-aminobenzoate hydroxylase as a coupling enzyme.
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.
Mizutani
Y
Y
Department of Chemistry, College of Science & Engineering, Aoyama Gakuin University, Setagaya-ku, Tokyo, 157-8572, Japan.
Narikawa
T
T
Satoh
T
T
Sakurai
N
N
Kaji
H
H
Yamada
S
S
Samejima
T
T
D000595:Amino Acid Sequence; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation / Q000187:drug effects; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry / Q000379:methods*; D014466:Ultraviolet Rays; D005723:gamma-Glutamyltransferase / Q000097:blood*
D003902:Detergents; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D009243:NAD; D006899:Mixed Function Oxygenases; D010544:Peroxidases; C021845:NAD+ peroxidase; C050092:4-aminobenzoate hydroxylase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D005723:gamma-Glutamyltransferase