Histochemical detection of alpha-D-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-alpha-D-glucoside.

Abstract

5-Br-4-Cl-3-Indoxyl-alpha-D-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of alpha-D-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fuchsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid alpha-D-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane alpha-D-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane alpha-D-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid alpha-D-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal alpha-D-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.