Glucosidase assay

Daniels LB, Glew RH, Diven WF, Lee RE, Radin NS (1981) An improved fluorometric leukocyte beta-glucosidase assay for Gaucher s disease. Clin Chim Acta 115 369-375... 

Although the (3-glucosidase assay can be performed on any tissue, the tissues of choice are peripheral blood leukocytes or cultured fibroblasts grown from a punch biopsy of the skin. Antenatal diagnosis can be made using extracts of cultured amniotic cells obtained from amniocentesis. There are several different (3-glucoside substrates that can be used in the assay the natural substrate glucocerebroside or structurally similar, artificial substrates. 

Since radiolabeled molecules are hazardous and difficult to dispose, most researchers and clinical laboratories prefer to use nonisotopic methods of labeling molecules. For this reason, investigators have established (3-glucosidase assay conditions that allow the estimation of glucocerebrosidase content of human tissues when nonphysiological (3-glucosides serve as the substrate. The artificial substrate of choice,... 

Daniels LB, Glew RH 3-Glucosidase assays in the diagnosis of Gaucher s disease. Clin Chem 28 569-577,1982. 

An example of a direct spectrophotometrical assay is the use of synthetic peptide -nitroanilide substrates to determine protease activity. The /)-nitroani1ine group Hberated from the substrates by the protease can be determined spectrophotometricaHy at 410 nm. An example of an indirect (coupled) spectrophotometric assay is the determination of a-amylase using -nitrophenyLmaltoheptaoside. Initially, the substrate is cleaved by the a-amylase and subsequentiy one of the reaction products, -nitrophenyLmaltotrioside, is cleaved by a-glucosidase, hberating -nitrophenyl, a chromophore... 

The / -glucosidase activity was determined by measuring the release of p-nitrophenol from p-nitrophenyl-/i-D-glucopyranoside one unit of / -glucosidase activity (U) is defined as the amount of enzyme that releases 1 [mu] mol p-nitrophenol per minute. AU samples were assayed in potassium phosphate buffer (50 mM, pH 7.0) at 50 °C under conditions that activity was proportional to enzyme concentration. 

Figure 1. pH-dependence of the half-life for loss of activity in native and glutaraldehyde-modified j9-D-glucosidase upon preincubation at the indicated pH values and at a range of temperatures from 55o to 70 oC, with subsequent assay at pH 5.0, 45 oC. A,B Linear-ordinate and logarithmic-ordinate plots, respectively, for the glutaraldehyde-modified enzyme. 

The ability of an esterase or a -glucosidase to hydrolyze the in vitro generated metabolite was tested. An assay mixture that had been incubated for 22 h (ca. 50% conversion of salicylic acid) was incubated with either 10 units of hog-liver esterase (E.C. 3.1.1.1, Sigma Chemical Co.) at pH 8.0 or 20 units of -glucosidase (E.C. 3.2.1.21, Sigma) at pH 5.0 for 1 h at 37 °C. Salicylic acid and the metabolite were separated by thin layer chromatography with BAW and quantified by liquid scintillation chromatography. 

Leukocytes contain other a-glucosidases in the neutral pH range, interfering with the assay they can be suppressed by acarbose (see section 4.6.15). 

Incorporation of [14C]-glucose into glycogen by the reverse reaction of the hydrolase amylo-l,6-glucosidase. Measurement of the incorporated radioactivity in the precipitated glycogen [20, 39]. This assay can only test the enzymes function of the hydrolysis of the 1-6 bond (see section 4.6.16.1). 

Amylo-l,6-Glucosidase (Debranching Enzyme, EC 3.2.1.33), Assay with [14C]-Glucose... 

Table 4.6.11 Mixtures for liquid scintillation counting of amylo-1,6-glucosidase (debranching enzyme assay with [14C]-glucose) for liver and muscle, and for RBC...

All assays (21) contained C 180 / g protein, 1000 units of Cx (CM-cellulose) activity (26), and 100 units of /3-glucosidase where necessary, /3-glucosidase isolated from the cellulase of F. solani (21) was added. The a-celluloses were the residues left after delignification and extraction with 18% (w/v) NaOH of oat straw, birch wood, and ryegrass. 

The categories of substrates which are used for assays of cellulase enzymes are shown in Table I. The use of crystalline, insoluble forms of cellulose as substrates makes assays difficult and has led to such trivial names as Avicelase activity. These assays are useful as indications of the capacity of an enzyme system to degrade native cellulose and indicate the presence of CBH enzyme which cannot be assayed in the presence of endoglucanases or / -glucosidase. The susceptibility to enzymatic attack generally increases with the hydration of the polymer chains that accom-... 

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