title: Purification and identification of the essential ionizable groups of honeybee, Apis mellifera L., trehalase.
abstract: Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl beta-glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1% carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (Km) was 0.66 mM, and the molecular activity (k0) was 86.2 s(-1). The enzyme was an "inverting" type which produced beta-glucose from alpha, alpha-trehalose. Dependence of the V and Km values on pH gave values for the ionization constants, pKe1 and pKe2, of essential ionizable groups 1 and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe1, and pKe2 were shifted to higher values of + 0.2 and + 0.5 pH unit, respectively. The ionization heat (deltaH) of ionizable group 1 was estimated to be + 1.8 kcal/mol, and the deltaH value of group 2 was + 1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (-COO(-), ionizable group 1) and the other a protonated type (-COOH, ionizable group 2), although the pKe2 value is high.
authors.affiliation: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: M
authors.initials: M
authors.lastname: Tsuji
authors.forename: M
authors.initials: M
authors.lastname: Nakamura
authors.forename: M
authors.initials: M
authors.lastname: Nishimoto
authors.forename: M
authors.initials: M
authors.lastname: Okuyama
authors.forename: H
authors.initials: H
authors.lastname: Mori
authors.forename: A
authors.initials: A
authors.lastname: Kimura
authors.forename: H
authors.initials: H
authors.lastname: Matsui
authors.forename: S
authors.initials: S
authors.lastname: Chiba
mesh_terms: D000818:Animals; D001516:Bees; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity; D013696:Temperature; D014198:Trehalase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014199:Trehalose / Q000378:metabolism
chemical_list: D014199:Trehalose; D014198:Trehalase; D005947:Glucose