Physicochemical characterization of human intestinal lactase.

摘要: 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 yield approx. 1% 750-fold purification. The enzyme appeared to be homogeneous SDS/polyacrylamide-gel electrophoresis under both reduced non-reduced conditions, an apparent Mr 170,000. On gel filtration, however, it displayed 380,000. protein had pI 4.8, as judged the chromatofocusing experiment, activity whose optimum is at pH 6.0. In addition beta-galactosidase activity, also hydrolysed various extents cellobiose, phlorizin, p-nitrophenyl beta-D-galactoside, beta-D-glucoside, o-nitrophenyl beta-D-galactoside beta-D-fucoside. Antisera been raised against purified in two rabbits. One antibody populations could inhibit concentration-dependent manner. This population used set up antibody-bound Sepharose column for use immunoaffinity purification crude intestinal homogenate. A partially preparation thus obtained. radioimmunoassay quantifying enzyme. competition assay detect about 0.5 micrograms protein/ml.