1. alpha-Mannosidase from jack-bean meal was purified 150-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. At pH values below neutrality, alpha-mannosidase undergoes reversible spontaneous inactivation at a rate dependent on the temperature, the degree of dilution and the extent of purification. The enzyme is also subject to irreversible inactivation, which is prevented 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; an excess of Zn(2+) again exerts a protective action, and so does EDTA in suitable concentration. 4. Neither Zn(2+) nor EDTA has any marked effect in the assay of untreated enzyme. In an EDTA-treated preparation, however, Zn(2+) reactivates the enzyme during assay. 5. It is postulated that alpha-mannosidase is a dissociable Zn(2+)-protein complex in which Zn(2+) is essential for enzyme activity.

1. Four different types of alpha-mannosidase activity were shown to occur in several tissues from the rat. There is the Zn2+-dependent enzyme, active at acidic pH, and three enzymes that are active near to neutral pH. 2. The 'neutral' enzymes are activated by Fe2+, Co2+ or Mn2+. 3. Optimum activities for these three enzymes are shown at pH values of 5.2, 6.5 and 7.3. The activity at pH6.5 is the only one evident without metal-ion activation, but activity is enhanced by all three metal ions. The activity at pH 5.2 is seen only in the presence of Fe2+ or Co2+, and the activity at pH7.3 is seen only in the presence of Co2+ or Mn2+ and in a non-chelating buffer medium. 4. The pH6.5-active enzyme is inactivated by EDTA, but activity is restored by excess of metal ion. 5. The enzymes differ markedly in their stability. The pH6.5-active enzyme is very labile and the pH7.3-active enzyme is the most stable. 6. Tissue preparations vary widely in their activity at pH6.5, but where activity is low it can be increased by incubation with one of the activating metal cations. 7. All the enzymes active at neutral pH are inhibited by heavy-metal ions and stabilized to some extent by thiol groups.

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.

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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.

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1. alpha-Mannosidase from the limpet, Patella vulgata, was purified nearly 150-fold, with 40% recovery. beta-N-Acetylglucosaminidase was removed from the preparation by treatment with ethanol. The final product was virtually free from beta-galactosidase. 2. Limpet alpha-mannosidase was assayed at pH3.5 and at this pH it was necessary to add Zn(2+) for full activity. At pH5, the enzyme had the same activity in the presence or absence of added Zn(2+). 3. On incubation at acid pH, the enzyme underwent reversible inactivation, which was prevented by adding Zn(2+). 4. EDTA accelerated inactivation and the addition of Zn(2+) at once restored activity. No other cation was found to reactivate the enzyme. 5. Cl(-) had an unspecific effect on hydrolysis by limpet alpha-mannosidase. It increased the rate of reaction with substrate. The anion did not prevent or reverse inactivation by EDTA. 6. It is concluded that alpha-mannosidase is a metalloenzyme or enzyme-metal ion complex, dissociable at the pH of activity, and that it requires Zn(2+) specifically.