Purification and identification of the essential ionizable groups of honeybee, Apis mellifera L., trehalase.

Article abstract of DOI:10.1271/bbb.65.2657

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.

Full text of DOI:10.1271/bbb.65.2657

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Purification and Identification of the Essential
Ionizable Groups of Honeybee, Apis mellifera L.,
Trehalase
Jin-Ha LEEa, Masahisa TSUJIa, Mitsuru NAKAMURAa, Mamoru NISHIMOTOa, Masayuki
OKUYAMAa, Haruhide MORIa, Atsuo KIMURAa, Hirokazu MATSUIa & Seiya CHIBAa
a Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University
Published online: 22 May 2014.
To cite this article: Jin-Ha LEE, Masahisa TSUJI, Mitsuru NAKAMURA, Mamoru NISHIMOTO, Masayuki OKUYAMA, Haruhide
MORI, Atsuo KIMURA, Hirokazu MATSUI & Seiya CHIBA (2001) Purification and Identification of the Essential Ionizable
Groups of Honeybee, Apis mellifera L., Trehalase, Bioscience, Biotechnology, and Biochemistry, 65:12, 2657-2665, DOI:
10.1271/bbb.65.2657
To link to this article: http://dx.doi.org/10.1271/bbb.65.2657
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