Purification and characterization of neutral alpha-mannosidase from hen oviduct: studies on the activation mechanism of Co2+.
Neutral alpha-mannosidase was purified to homogeneity from hen oviduct. The molecular mass of the enzyme was 480 kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits. The activated enzyme hydrolyzed both p-nitrophenyl alpha-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral a-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GlcNAc (Km =0.44 mM) was hydrolyzed four times faster than Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GIcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)++ +Manbeta1-4GlcNAc was obtained as the end product from Man9GlcNAc on digestion with the activated alpha-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule. Since the Co2+-activated enzyme gradually lost its activity on incubation with EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation. The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain alpha-mannosidase seems not to be involved.
Yamashiro
K
K
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka.
Itoh
H
H
Yamagishi
M
M
Natsuka
S
S
Mega
T
T
Hase
S
S
D000818:Animals; D002240:Carbohydrate Sequence; D002413:Cations, Divalent; D002614:Chelating Agents / Q000494:pharmacology; D002645:Chickens; D003035:Cobalt / Q000378:metabolism* / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D006863:Hydrogen-Ion Concentration; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010057:Oviducts / Q000201:enzymology*; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D002413:Cations, Divalent; D002614:Chelating Agents; D009844:Oligosaccharides; D003035:Cobalt; D004492:Edetic Acid; D008361:Mannosidases; D043323:alpha-Mannosidase