Activation by Ferrous Ions of an α-Mannosidase in Rat Testis and Liver

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.

Section: Purification Stage 1 Mealmentioning

confidence: 99%

Characterization of jack-bean α-D-mannosidase as a zinc metalloenzyme

Snaith¹

1975

“…A third type of activity with an intermediate pH optimum (Snaith, 1973;Phillips et al, 1974) has been purified from Golgi membranes (Tulsiani et al, 1977). This intermediate a-mannosidase activity has also been detected in body fluids such as cerebrospinal fluid and urine (Cheron et al, 1975), and in serum (Cheron et al, 1975;Hirani et al, 1977), where it is quantitatively the major form of a-mannosidase.…”

mentioning

confidence: 99%

Comparison of the α-mannosidases in fibroblast cultures from patients with mannosidosis and mucolipidosis II and from controls

Halley¹,

Winchester²,

Burditt³

et al. 1980

The intracellular and extracellular acidic alpha-mannosidase in cultures of fibroblasts from mucolipidosis-II patients has normal kinetics. The extracellular activity in cultures of cells from mannosidosis patients is normal, but a mutant enzyme is associated with the cell surface and intracellular fraction. The results support the involvement of membrane cycling and recognition markers in lysosomal enzyme localization.

“…An acidic a-mannosidase, derived from lysosomes, activated by Zn2 + and having maximal activity at pH around 4.5, has been demonstrated in several tissues, including epididymis (Snaith & Levvy, 1969, 1973Conchie & Findlay, 1959). A non-lysosomal neutral a-mannosidase, which is activated by Co2+ and is maximally active at around neutral pH, has been demonstrated in the cytosol of several tissues (Shoup & Touster, 1976;Marsh & Gourlay, 1971).…”

mentioning

confidence: 99%

“…A non-lysosomal neutral a-mannosidase, which is activated by Co2+ and is maximally active at around neutral pH, has been demonstrated in the cytosol of several tissues (Shoup & Touster, 1976;Marsh & Gourlay, 1971). Unlike ram epididymis (Bullock & Winchester, 1973), it was not possible to demonstrate the occurrence of neutral amannosidase activity in rat epididymis when the enzymic activity was measured in the crude tissue extract at various pH values (Snaith, 1973(Snaith, , 1977. More recently, however, it has been possible to demonstrate neutral a-mannosidase activity in rat epididymis by isolating an (NH4)2SO4 fraction of the enzyme that is largely devoid of the acidic enzyme (Snaith, 1977).…”

mentioning

confidence: 99%

Enzymic characteristics of the isoenzymes of rat epididymal neutral α-mannosidases and their changes during development in vivo

Dutta¹,

Majumder²

1984

Three isoenzymic species of neutral alpha-mannosidase (I, II and III) were partially purified from rat epididymis and characterized. Calcium phosphate gel preferentially adsorbed acidic alpha-mannosidase activity, thereby removing the acidic enzyme from the neutral mannosidases. The neutral enzymes, which are of cytosolic origin, require Co2+ for activity, and Mn2+ can substitute partially for Co2+. Mg2+ or Ca2+ had little effect on the activity of the isoenzymes, whereas Zn2+ (100 microM) was a potent inhibitor of the mannosidases. The Km values of mannosidases I, II and III for Co2+ were 10, 10 and 2.7 mM respectively. There was marked alteration of the specific activity of the neutral alpha-mannosidases during epididymal development in vivo. The specific activities of mannosidases I and II were relatively high in the young (24 days) rats, and during the subsequent development the specific activities decreased markedly (approx. 2-3-fold). On the contrary, mannosidase III, which showed relatively low specific activity during 24-45 days of age, increased markedly (approx. 2-fold) as the animals reached adulthood.