Characterization of jack-bean alpha-D-mannosidase as a zinc metalloenzyme.
1. Two methods were used to obtain alpha-mannosidase free from unbound Zn2+, (a) by removal of excess of metal ion from preparations purified in the presence of Zn2+ and (b) by purification under conditions that eliminate the need to add Zn2+. 2. The purified enzyme is homogeneous on ultracentrifugation, polyacrylamide-gel electrophoresis and gel chromatography. 3. The molecular weight is estimated to be 230 000. 4. The enzyme contains between 470 and 565 mug of zinc/g of protein, corresponding to between 1.7 and 2 atoms of zinc/enzyme molecule. The contents of other metals are much lower. 5. The enzyme is inactivated by chelating agents and activity is restored by Zn2+. 6. No other metal ion was found to replace Zn2+ with retention of activity. Some bivalent metal ions, e.g. Cu2+, rapidly inactivate the enzyme. 7. The results indicate that jack-bean alpha-mannosidase exists naturally as a zinc-protein complex and may be considered as a metalloenzyme.
D002845:Chromatography; D003300:Copper; D004186:Disaccharidases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008361:Mannosidases / Q000302:isolation & purification*; D008667:Metalloproteins / Q000302:isolation & purification; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D014461:Ultracentrifugation; D015032:Zinc / Q000302:isolation & purification*
D008667:Metalloproteins; D003300:Copper; D004186:Disaccharidases; D008361:Mannosidases; D015032:Zinc