title: Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity.
abstract: Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k(cat) values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190±10, 840±30, and 500±10 s(-1), respectively. The k(cat) values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240±60, 1450±30, and 2250±80 s(-1), respectively, at 1.0M. On the other hand, the k(cat) values at 0.05-1.0M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s(-1). These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K(m) values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K(m) value at 1.0M diethanolamine (0.83±0.15 mM) was 12-fold higher than that at 0.05M (0.07±0.01 mM), and that at 1.0M N-methylethanolamine (2.53±0.20 mM) was 14-fold higher than that at 0.2M (0.18±0.02 mM), while that at 1.0M triethanolamine (0.31±0.01 mM) was similar as that at 0.2M (0.25±0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.
authors: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
authors: Satoshi
authors: S
authors: Sekiguchi
authors: Yasuhiko
authors: Y
authors: Hashida
authors: Kiyoshi
authors: K
authors: Yasukawa
authors: Kuniyo
authors: K
authors: Inouye
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000588:Amines / Q000737:chemistry / Q000494:pharmacology; D000605:Amino Alcohols / Q000737:chemistry / Q000494:pharmacology; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007124:Immunoenzyme Techniques / Q000379:methods; D066298:In Vitro Techniques; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism
chemical_list: D000588:Amines; D000605:Amino Alcohols; D000814:Aniline Compounds; D009943:Organophosphorus Compounds; C068798:4-aminophenylphosphate; D000469:Alkaline Phosphatase