title: Determination of specific activities and kinetic constants of biotinidase and lipoamidase in LEW rat and Lactobacillus casei (Shirota).
abstract: Enzyme kinetic parameters, such as K(m), V(max) (or V), k(cat)/K(m), and K(i) (by biotin or lipoic acid) for biotinidase and lipoamidase were determined in Lewis (LEW) rat and Lactobacillus casei (Shirota) using fluorimetric high-performance liquid chromatography (HPLC). It was found that the final protein concentration below 0.1mg/ml is sufficient to obtain linear hydrolytic reaction and to determine the Michaelis-Menten type kinetic parameters (K(m), V, K(i)). We applied this HPLC enzyme assay method onto the rat and some bacteria. The highest specific activities (Vs) for biotinidase were found in Lactobacillus casei (Shirota) and rat kidney. It was also found that the largest K(i) by product for biotinidase and lipoamidase were present in the Lactobacillus casei (Shirota). There has been found specie (between rat and mouse) differences and tissue (organ) differences, together with tissue region differences and sex differences in some tissues. Summary of the distributions of both enzymes in LEW rat was also presented. Therefore, this HPLC determination method for the enzyme kinetic parameters in tissues is expected to be an indispensable tool for the investigation of the various diseases in humans.
authors.affiliation: Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan. khayakawa@nch.go.jp <khayakawa@nch.go.jp>
authors.forename: Kou
authors.initials: K
authors.lastname: Hayakawa
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.forename: Elena A
authors.initials: EA
authors.lastname: Terentyeva
authors.forename: Xiao-Kang
authors.initials: XK
authors.lastname: Li
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Kimura
authors.forename: Masahiko
authors.initials: M
authors.lastname: Hirano
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Yoshikawa
authors.forename: Takeaki
authors.initials: T
authors.lastname: Nagamine
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Katsumata
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Ogata
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000581:Amidohydrolases / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D043523:Biotinidase / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D006801:Humans; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley
chemical_list: D000581:Amidohydrolases; C023940:lipoamidase; D043523:Biotinidase