P-D-mannosidase

G. Legler and E. Jiilich, Synthesis of 5-amino-5-deoxy-D-mannopyranose and l,5-dideoxy-l,5-imino-D-mannitol, and inhibition of a- and P-D-mannosidases, Carbohydr. Res., 128 (1984) 61-72. 

Baker s yeast a-D-glucosidase Almond p-D-glucosIdase °Snail p-D-mannosidase... 

A highly purified form of the P-D-mannosidase (mol. wt. 6.4 x 10, pH optimum 2.4—3.4) from the fruiting bodies of the mushroom Polyporus sulfureus has been recommended for use in structural studies on glycoproteins. 

Results are expressed as fig of p-nitrophenol liberated per g of seed from 6 mM p-nitrophenyl a-D-mannoside in 1 hr at 37° and pH 5. Coarsely ground beans. Defatted sweet-almond meal. A commercial /3-D-glucosidase preparation purified from emulsin. a-D-Mannosidase activity is given per g of purified material. 

For the assay of a-D-mannosidase, the incubation mixture employed in our laboratory contained 0.5 ml of M acetate buffer at a pH appropriate for the particular enzyme preparation, 1.5 ml of 16 mM p-nitrophenyl a-D-mannopyranoside, 1.5 ml of water (which could be replaced by other additives as required), and 0.5 ml of suitably diluted, enzyme preparation. After 1 hour at 37°, the reaction was terminated, and the color was developed by adding 4 ml of 0.4 M glycine-sodium hydroxide buffer, pH 10.5. The mixture was centrifuged for 15 minutes at 1500 g, and the color intensity of the liberated p-nitrophenol (25-150 fig) in the supernatant liquor was measured on a Spekker photoelectric absorptiometer, with use of Ilford No. 601 violet filters having maximal transmission at 430 nm, and a 1-cm light path. Separate control-experiments for enzyme and sub-... 

With a-D-mannosidase preparations in a high state of purity, the addition of 0.01% of bovine albumin to the assay mixture may lead to a small increase in activity (not more than 10%), probably by lessening denaturation of the enzyme. a-D-Mannosidase from marine-molluscan sources is, to a considerable extent, activated in the assay by chloride ion, and, to some extent, by certain other anions. Maximum activity is displayed by the enzyme from the limpet, P. vulgata, when 0.1 M sodium chloride is included in the incubation mixture46 (see Section 11,5 p. 412). Chloride ion has no effect on the activity of jack-bean or rat-epididymal a-D-mannosidase. 

One unit of a-D-mannosidase liberates 1 /xg of p-nitrophenol from 6 mM p-nitro-phenyl a-D-mannoside in 1 hr at 37° and pH 5. Protein was determined by the method of Lowry and coworkers,59 with bovine albumin as the standard. Zn2+ was added to the preparation after Stage 3. 

At pH values above neutrality, jack-bean a-D-mannosidase, unlike the enzyme from the other two sources, is stable, and the native, protein-metal complex does not dissociate. By working at pH 8, it is possible to purify the enzyme from jack-bean meal without addition of zinc.27 The procedure shown in Table V was followed with only slight modification (see Table IX, Section III,5 p. 433). 

With the exception of the enzyme from the limpet, P. vulgata, a-D-mannosidase from most of the important sources shows optimal activity at pH values lying between 4 and 5. For the enzyme from jack-bean meal39 and that from rat epididymis,80 we employed a pH of 5 for routine assays. If this is not the actual optimum, it is close to it on the broad pH-activity curves, and, at this pH, the addition of Zn2+ and other cations has relatively little effect in the assay, thus simplifying the study of the various metal complexes that can be formed by the enzyme protein. 

Fig. 1.—Hydrolysis at Different pH Values, in Acetate Buffer, of 6 inM p-Nitrophenyl a-D-Mannoside by Limpet a-D-Mannosidase.4S [A, no addition in the presence of A, 0.1 mM ZnS04 O, 0.1 M NaCl, and , 0.1 M NaCl + 0.1 mM ZnS04. The a-D-man-nosidase activity is expressed as a percentage of the maximum value obtained in the presence of Zn2+ and Cl-.]...

The firm binding of toxic cations by limpet a-D-mannosidase at pH 5 has already been mentioned (see Section 11,5 p. 412). This fact is not evident on assay at pH 3.5, because, at that pH, immediate exchange occurs with Zn2+ in the assay medium. On assay at pH 5 (used to arrest any cation exchange), an enzyme preparation may exhibit only about one-quarter of its potential activity at this pH. It is possible to accomplish replacement of endogenous, toxic cations by Zn2+, either directly by incubation with this cation, or indirectly, after incubation with EDTA (see Section III,4 p. 431). Subsequent assay at pH 5 then reveals activation. The lower the pH of incubation, the faster the removal of toxic cations.48... 

Fig. 2.—Effect of Concentration of Substrate on the Rate of Hydrolysis4 of p-Nitro-phenyl a-D-Mannoside in Acetate Buffer, pH 3.5, at 37° by a Crude Preparation of Limpet a-D-Mannosidase in the Presence of , 0.1 mM ZnS04 and 0.1 M NaCl A, 0.1 M NaCl only and O, 0.1 mM ZnS04 only.

As will be seen later (see Section III,3 p. 424), a-D-mannosidase freed from Zn2+ by the action of EDTA cannot combine with the substrate. The results for Zn2+, shown in Fig. 3, fit Frieden s formula for this limiting case, in which reaction 1 and, hence, reaction 3 do not occur. When Cl- is regarded as a modifier of limpet a-D-mannosidase, (67) C. FriedenJ. Biol. Chem., 239, 3522 (1964). 

D-Mannono-1,5-lactone68 gave a value of 0.07 mM for Kj with rat-epididymal a-D-mannosidase, compared with 12 mM for Km for p-nitrophenyl a-D-mannopyranoside,88 and 57 mM for phenyl a-D-mannopyranoside.65 With jack-bean a-D-mannosidase, the values... 

As discussed under Purification and pH and Stability [see Sections 11,4 (p. 409) and 11,6 (p. 413)], a-D-mannosidase is unstable at low pH values unless Zn2+ is added. In the following experiments employing a partially purified, jack-bean meal preparation (see Table V, stage 3 p. 410) to which Zn2+ had not been added, the enzyme was pre-incubated at 37° and pH 5 before assay at the same pH with p-nitrophenyl a-D-mannopyranoside as the sub-... 

In its behavior with Zn2+, other metal ions, and EDTA, rat-epi-didymal a-D-mannosidase closely resembles the jack-bean enzyme. The greater ease of dissociation of the epididymal protein-zinc complex has been discussed under pH and Stability (see Section 11,6 p. 413). 

Apart from the effects of the strongly bound, toxic cations in the native preparation (see Section 11,5 p. 412), limpet a-D-mannosidase also resembles the jack-bean enzyme in its zinc-dependence. Fortunately, at the relatively low pH optimum (3.5) of the enzyme, the metals are freely dissociable, and toxic cations can be displaced by the addition of Zn2+. Full activity is only shown by the limpet enzyme at the pH optimum when an excess of Zn2+ is present in the assay medium (see Sections 11,3 (p. 408) and 11,5 (p. 412)]. 

Many of the effects of metals on a-D-mannosidase from different sources, reported by other authors, are merely examples of activation or inhibition by the metal in the assay they do not illustrate metal-dependence of the type just discussed. However, a-D-mannosidase from pig kidney,18 soy bean,62 and P. vulgaris64 has also been shown to be completely stabilized by the addition of Zn2+. Activity lost by the action of EDTA on rat-liver a-D-mannosidase was reversed by the addition of Zn2+ to the assay mixture.76 We have made a similar observation with the enzyme from rat liver.27... 

This Section presents direct evidence for the conclusions arrived at indirectly from kinetic data (see Section 11,8 p. 416), namely, that Cl- accelerates the hydrolysis of the substrate by limpet a-D-mannosidase, whereas Zn2+ is an essential component of the enzyme in its catalytically active form. 

To establish that a-D-mannosidase is a metalloenzyme, the proportion of bound zinc in the active molecule must first be determined. This experiment has been performed for the enzyme from jack-bean meal.27 Two types of preparation were employed. The first was purified, throughout, at pH 8 without any addition of Zn2+ (see Section 11,4 p. 409). The second enzyme preparation was purified in the presence of added Zn2+ at pH 5 up to stage 5 of the original procedure (see Table V p. 410). It was then freed from unbound Zn2+ by dialysis against glycine buffer of pH 8, and passed through a column of Sephadex G-100 in the same buffer. As may be seen from Table IX, the final specific activity was, in each instance, slightly less than that shown in Table V. 

Although the a-D-mannosidase activity of several different organs in mammals has been found to alter under a variety of conditions (see Section 11,1 p. 402),11,12-14,28,28 the most striking changes are those produced by sex hormones in the epididymis and uterus.26,28 In rats and mice, a-D-mannosidase activity in the epididymis increases with the age of the animal, up to maturity orchidectomy causes a dramatic fall in the enzyme activity of the adult tissue (up to 100-fold), but activity can be partially restored by the administration of testosterone. Ovariectomy results in a 10-fold fall in the activity of a-D-mannosidase in mouse uterus, and this is completely reversed by estrone. 

In zinc-deficient rats, the zinc content of the epididymis was only about half the normal value,28,87 and the level of a-D-mannosidase activity was also little over half the value usually observed.26 However, the zinc concentration of the tissue was still in vast excess over that required for stoichiometric combination with the enzyme protein, calculated on any probable estimate of its specific activity and molecular weight (see Section III,5 p. 433). 

P-4, and gave two products, in approximately equimolar amounts, having D-mannose hexosamine ratios of 5 4 and 1 2, respectively. The first was considered to have resulted from the action of a-D-man-nosidase on Montgomery s fractions A, B, and C (see Table X), but, theoretically, it should only be produced from fraction C. The second product was attributed to the action of a-D-mannosidase on fractions D and E. 

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