Mannosidase incubation

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. 

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... 

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-... 

Fig. 4.—Effect of Zn2+ and EDTA on a-D-Mannosidase Activity85 in a Partially Purified, Jack-bean Meal Preparation Incubated for Various Periods of Time at 37° and pH5. [ , Enzyme alone , enzyme + 1 mAf ZnS04 andO, enzyme-1- 1 mMEDTA. The results are expressed as a percentage of the activity in the unincubated, enzyme preparation.39]...

One of the most striking indications of the importance of Zn2+ for a-D-mannosidase activity was obtained with preparations that had been inactivated by incubation with EDTA. On addition of an excess of Zn2+ to the assay mixture, complete activity was regained instantaneously, regardless of the extent of prior inactivation. (When the EDTA-inactivated enzyme described in Fig. 4 was assayed in the presence of Zn2+, the points followed the line for the Zn2+-stabilized enzyme.) Again, no other cation that we have examined can replace Zn2+, leaving little doubt as to the identity of the activating cation in the original material. It also follows that EDTA must withdraw Zn2+ from the protein-metal complex. Had EDTA merely formed a... 

When the ovalbumin was incubated with purified, jack-bean a-D-mannosidase, at least two molecules of D-mannose per molecule were released, suggesting that these residues are a-D-linked in a terminal position. Two glycopeptides having different contents of hexosamine were subjected to the action of purified 2-acetamido-2-deoxy-/ -D-glucosidase, and, in each case, two hexosamine residues per molecule were either inaccessible, or resistant to attack by the enzyme. 

For degradation of the carbohydrate moiety of 50 mg ARS2 sample, 10 or 25 U exo-P-galactosidase were added and the mixture was incubated at 37°C for 18 hr 10 ml of 100 mM acetate buffer (pH 4.2) containing 25 U endo-P-1,4-galactanase or 1 mg crude endo-P-1,6-galactanase [43] were added and the mixture was incubated at 45°C for 48 hr. Fifty unit exo-a-mannosidase or 0.5 U endo-P-galactosidase in 50 mM potassium phosphate buffer (pH 6.0) at 37 °C for 18 h was also used. 

The differences in the rates of hydrolysis of various linkage types by a particular glycosidase can be used to provide information about this aspect of structure. Jack-bean a-D-mannosidase cleaves a-(l- 2) and a-(l- 6) linkages much faster than a-(l -> 3). Oligosaccharides, obtained by endo-N-acetyl-/J-D-glucosaminidase hydrolysis of ovalbumin, were subjected to acetolysis, which selectively cleaved the a-(l - 6) bonds. A tetrasaccharide isolated after this treatment was then incubated with jack-bean o-d-... 

For thermostability of cysteine mutants, wild-type (WT), C334A, C363A, and C443A were incubated at 55°C, and the half-lives of residual activity (/ /2) at 30°C were 119, 5.2, 11.3, and 24.4 min respectively (Table 20). Mutation at Cys334, Cys363, and Cys443 drastically decreased the thermostability of 1,2-a-D-mannosidase. It is evident that the thermostability of A saitoi 1,2-a-D-mannosidase is significantly influenced by mutations. 

An a-D-mannosidase (acid pH optimum) that is Zn " -dependent and three other a-D-mannosidase activities (neutral pH optima) have been found in the tissues of rats. The enzymes most active at a neutral pH value were inhibited by heavy-metal ions and were stabilized, to some extent, by thiols. The reversible effects of Fe , Co and Mn ions and H edta on the activity of these enzymes were investigated. Tissue preparations varied widely in the levels of the enzymes most active at pH 6.5, although latent enzymic activity was released on incubation with activating metal ions. 

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