Abstract
A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in the brush border, can be semi-quantified using nano LC-MS/MS.
Highlights
It is a common goal in physiology to assess the function of enzymes as well as to identify effector proteins in subcellular compartments
We describe a method with the following two advantages: (1) It can perform both steps on the same sample, and (2) the enzyme identification is fast and accurate
We used the method to reveal the hydrolytic activity of α-glucosidases in brush border membrane of intestinal epithelial cells, and their identification, by combining a gel electrophoresis for sample protein separation, a colored reaction to reveal the enzyme activity on the substrate, i.e., zymography [1], and a proteomic method, for enzyme identification-nano-liquid chromatography coupled to mass spectrometry of the solubilized stained bands of the gels
Summary
It is a common goal in physiology to assess the function of enzymes as well as to identify effector proteins in subcellular compartments. Numerous methods have been proposed based on split assays, one to measure the function of the enzyme and another to identify the protein that catalyzes the reaction The latter step often requires an additional sample (not the one in which function was measured), and relies on the use of antibodies. According to the classification system of the carbohydrate-active enzymes (CAZy, [6]), all of the subunits belong to the glycoside hydrolase 31 family (GH31) subgroup 1, characterized by the WIDMNE sequence in their catalytic centers [7] All these similarities make it time consuming and costly to distinguish the different complexes. Because some of the subunits share substrate hydrolytic specificity [8,9], and in the reaction assay the activity gives similar colored products, distinguishing between the enzymes poses an even greater challenge. According to the specificities of these enzymes, the proteomic analyses of the gel slices should reveal the presence of both proteins
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