Mannosidase assay

Cycloaddition reaction of nitrone (—)-(394) with dimethyl maleate D14 has been used for the synthesis of two new polyhydroxyl pyrrolizidines (687) and (688) (Schemes 2.293, 2.294). These compounds are analogs of alkaloids ros-marinecine and crotanecine, which were assayed for their inhibitory activities toward 22 commercially available glycosidase enzymes. One of them ((-)- a-epi-crotanecine) (—)-(688) is a potent and selective inhibitor of a-mannosidases (310). The reaction of (—)-(394) with dimethyl maleate gave a 9.6 6 1 mixture of cycloadducts (—)-(680), (+ )-(680), and (—)-(681), which arise from anti-exo,... 

Fig. 23. Methyl a-septanoside mono-, di-, and tri-saccharides 243-245 assayed against jack-bean a-mannosidase.

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. 

Although a-D-mannosidase from mammalian, plant, and molluscan sources is dependent upon zinc for its catalytic activity, the addition of this ion has a marked effect in the enzyme assay only at those pH values where the active, protein-metal complex dissociates appreciably despite the presence of substrate. (Dissociation, which is greater at lower values of pH, is lessened in the presence of substrate.) The presence of zinc ion in the assay (0.1 mM) is thus of particular importance in the case of the limpet enzyme, where the pH of optimal activity is 3.5. Jack-bean and rat-epididymal a-D-mannosidase are both assayed at pH 5, and up to 10% activation may be observed with zinc. 

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. 

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-... 

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... 

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... 

EDTA inhibited the Ca2+-free wild type 1,2-a-D-mannosidase. The inhibition was investigated by fixed concentrations of EDTA in the assay buffer and by varying the concentrations of substrate. A Lineweaver-Burk plot showed a pattern consistent with competitive inhibition (Figure 30) and gave a K, of 0.91 mM for EDTA. 

A simple assay based on potent and specific inhibition of jack bean a-mannosidase has been devised for determining low concentrations of 162 (up to 0.5 cm ) in M anisopliae cultures (110). The new assay was used to demonstrate that the addition of L-lysine (163) to the culture medium stimulated production of the alkaloid by approximately fourfold. Other early metabolic precursors of 162 in this fungus, including a-aminoadipic acid, saccharopine (164), L-pipecolic acid (165), and L-lysine itself, were quantified by reverse-phase HPLC analysis of mycelial extracts derivatised with 9-fluorenylmethyl chloroformate (FMOC) (111). 

A review of enzyme analysers has described the automation of fixed-time and continuous-monitoring assays and the uses of partly or completely automated analysers and multi-channel systems. Automated analyses of polysaccharide hydrolases, in soluble or insoluble form, have been based on determination of the liberated reducing sugars with 3,5-dinitrosalicylic acid. A sensitive and specific method for locating glycoside hydrolases (e.g. oc-D-mannosidases) in polyacryl-... 

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