Physicochemical characterization of human intestinal lactase.
Human lactase was isolated from solubilized small-intestinal brush-border membranes by a combination of chromatography on concanavalin A-Sepharose, Bio-Gel 1.5m and chromatofocusing, with a yield of approx. 1% and a 750-fold purification. The enzyme appeared to be homogeneous on SDS/polyacrylamide-gel electrophoresis under both reduced and non-reduced conditions, with an apparent Mr of approx. 170,000. On gel filtration, however, it displayed an apparent Mr of approx. 380,000. The protein had a pI of 4.8, as judged by the chromatofocusing experiment, and had a lactase activity whose optimum is at pH 6.0. In addition to the beta-galactosidase activity, the protein also hydrolysed to various extents cellobiose, phlorizin, p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-glucoside, o-nitrophenyl beta-D-galactoside and o-nitrophenyl beta-D-fucoside. Antisera had been raised against the purified enzyme in two rabbits. One of the antibody populations could inhibit the enzyme in a concentration-dependent manner. This antibody population was used to set up an antibody-bound Sepharose column for the use in an immunoaffinity purification of lactase from crude intestinal homogenate. A partially purified preparation of lactase could thus be obtained. The antibody population was also used to set up a radioimmunoassay for quantifying the enzyme. The competition assay could detect about 0.5 micrograms of lactase protein/ml.
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005696:Galactosidases / Q000302:isolation & purification*; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D008871:Microvilli / Q000201:enzymology; D011863:Radioimmunoassay; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D005696:Galactosidases; D001616:beta-Galactosidase