Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA.
alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.
Wongchawalit
Jintanart
J
Graduate School of Agriculture, Hokkaido University, Sapporo.
Yamamoto
Takeshi
T
Nakai
Hiroyuki
H
Kim
Young-Min
YM
Sato
Natsuko
N
Nishimoto
Mamoru
M
Okuyama
Masayuki
M
Mori
Haruhide
H
Saji
Osamu
O
Chanchao
Chanpen
C
Wongsiri
Siriwat
S
Surarit
Rudee
R
Svasti
Jisnuson
J
Chiba
Seiya
S
Kimura
Atsuo
A
D000818:Animals; D001483:Base Sequence; D001516:Bees / Q000201:enzymology*; D050260:Carbohydrate Metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D000520:alpha-Glucosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; C042059:glucosidase I; D000520:alpha-Glucosidases