Purification and characterization of neutral alpha-mannosidase that is activated by Co2+ from Japanese quail oviduct.
An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.
Oku
H
H
Department of Chemistry, Osaka University College of Science.
Hase
S
S
Ikenaka
T
T
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003035:Cobalt / Q000494:pharmacology; D003370:Coturnix; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D066298:In Vitro Techniques; D008361:Mannosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010057:Oviducts / Q000201:enzymology*; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D009844:Oligosaccharides; D003035:Cobalt; D008361:Mannosidases; D043323:alpha-Mannosidase