Purification and properties of α-d-mannosidase from rat epididymis

Abstract

1. α-d-Mannosidase from rat epididymis was purified 300-fold. β-N-Acetyl-glucosaminidase and β-galactosidase were removed from the preparation by treatment with pyridine. Zn2+ was added during the purification to stabilize the α-mannosidase. 2. Mammalian α-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 α-mannosidase is accelerated by EDTA and reversed or prevented by Zn2+. Other cations, such as Co2+, Cd2+ and Cu2+, accelerate inactivation and the action of a toxic cation can be prevented by Zn2+ or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn2+, EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn2+ is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn2+ reactivates the enzyme during the assay. 5. Evidence is presented that α-mannosidase is a dissociable Zn2+–protein complex, in which Zn2+ is essential for enzyme activity.