Purification and properties of alpha-D-mannosidase from rat epididymis.
1. alpha-d-Mannosidase from rat epididymis was purified 300-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. Mammalian alpha-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation and the action of a toxic cation can be prevented by Zn(2+) or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn(2+), EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn(2+) is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn(2+) reactivates the enzyme during the assay. 5. Evidence is presented that alpha-mannosidase is a dissociable Zn(2+)-protein complex, in which Zn(2+) is essential for enzyme activity.
D000096:Acetone; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004492:Edetic Acid; D004822:Epididymis / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008358:Mannose; D011725:Pyridines; D051381:Rats; D015032:Zinc
D011725:Pyridines; D002104:Cadmium; D000096:Acetone; D003035:Cobalt; D003300:Copper; D004492:Edetic Acid; D006026:Glycoside Hydrolases; D015032:Zinc; D008358:Mannose