Indigogenic methods for glycosidases. 3. An improved method with 4-Cl-5-Br-3-indolyl-beta-D-fucoside and its application in studies of enzymes in the intestine, kidney and other tissues.

Abstract

8ummary. 4-C1-5-Br-3-indolyl-fl-D-fucoside (IbF) was tested in histochemical and biochemical experiments. Sections from differently treated parts of the small intestine of suckling and adult rats, of jejunum of adult hamsters, guinea pigs, cocks, monkeys, of human jejunal biopsies, of kidney of suckling and adult rats, adult monkeys, guinea pigs and hamsters, and of some other rat organs were used in histochemical experiments. Neutral and acid {LDgalactosidases prepared from homogenates of the small intestine of suckling rats by chromatography on Sephadex G 200 were used in biochemical experiments. The recommended medium for histoehemical studies consists of 0.1 M citrate phosphate buffer pH6 (brush border enzyme) and pH4 (lysosomal enzyme), 3.6.10-4M IbF and 3.1-10 -a M potassium ferri- and ferrecyanide. IbF is split quite efficiently by the brush border fl-D-galactosidase (~ lactase) and the recommended histochemical method with IbF at pH 6 is the method of choice in studies concerned with the localization of intestinal lactase. In unfixed cold microtome sections the brush border a~tivity can be easily detected within 15-240 minutes, depending on the lactase activity of the studied sample. In this study this activity was shown to be present in the brush border of enterocytes in the upper part of crypts reaching its maximum in differentiated enterocytes covering the sides and tops of villi in the small intestine of suckling (the highest activity) and adult rats (3-4  lower activity according to the time of appearance of the staining), and of hnm~.n, monkey and hamster jejunum. In the cock jejunum traces of brush border staining were seen only after 24 hours of incubation. In enterocytes of patients with celiac sprue the brush border ~ivity was very much reduced in dependence on the stage of the disease. Brush border staining along with a diffuse cytoplasmic reaction was found in the proximal convoluted tubules of the monkey kidney. The question of localization of enzyme activity splitting IbF in the monkey kidney deserves further investigation. IbF is also split by isolated intestinal acid ~-D-galactosidase and histochemically a positive reaction was found in lysosomes of many cells displaying a high activity of fl-Dgalactosidase when IbF was used in the recommended medium of pH 4. The use of aldehyde fixation (glutaraldehyde is to be preferred) is a prerequisite for the assessment of this lysosomal localization. The lysosomal activity splitting IbF is not firmly bound structurally and escapes from cold microtome sections prepared from unfixed tissue samples into the incubation solution. The recommended method is also very suitable for processing zymograms and immunoprecipitation lines of lactase with antisera obtained by Ouehterlony's technic and by immunoelectrophoresis.