[Histochemical and biochemical investigation of alpha-glucosidases by means of 2-naphthyl-alpha-D-glucoside (author's transl)].
Histochemical and biochemical studies yield the following method of choice for the in situ detection of neutral (microvillous) and acid (lysosomal) alpha-glucosidases: 12 mg 2-naphthyl-alpha-D-glucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.6--0.8 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid phosphate buffer for aqueous or 5 ml buffer mixed with equal parts of 2% agar for incubation with semipermeable membranes, pH 5 or 6.5. With this method neutral alpha-glucosidases can be exactly demonstrated in the brush border of the small intestine (glycoamylase, sucrase-isomaltase) and kidney of mammals, birds,fishes, amphibia and reptiles; localization of acid alpha-glucosidases is achieved at the cellular level in many organs and tissues. Fluorometric and photometric measurements prove that 2-naphthyl-alpha-D-glucoside is superior to 6-brom-2-naphthyl-alpha-D-glucoside for the demonstration of alpha-glucosidases in situ due to the lower Michaelis constant and higher maximal reaction velocity of the naphthol derivative.--Among the coupling reagents tested neutral alpha-glucosidases can be localized correctly with hexazotized p-rosaniline (with and without semipermeable membranes) for simultaneous coupling. Fast Blue B delivers false positive results in the suczedaneous and simultaneous coupling procedure using aqueous incubation media; in combination with the membrane technique azo dye can not be observed in the sections. Hexazonium-p-rosaniline inhibits neutral and acid alpha-glucosidases to nearly the same extent as Fast Blue B. Fixation of blocks of tissue in formaldehyde and glutaraldehyde suppresses alpha-glucosidases in the intestine and epididymis. The inhibition rates amount to 50 and 70% respectively. Washing in sugar solution rises enzyme activity to 65 and 50%. Species and organ dependent activity differences of neutral and acid alpha-glucosidases and changes of enzyme activity in the intestine and kidney after castration as well as in the course of pregnancy can be detected by means of biochemistry but not with the histochemical assay including minimal incubation. In comparison with p-nitrophenyl-alpha-D-glucoside the 2-naphthyl derivative is also the substrate of choice for the biochemical determination of alpha-glucosidases.--Agar gel electrophoresis reveals one band in the neutral and acid pH range.
D000818:Animals; D001391:Azo Compounds; D005260:Female; D005404:Fixatives; D005959:Glucosidases / Q000032:analysis* / Q000037:antagonists & inhibitors; D005960:Glucosides; D006168:Guinea Pigs; D006651:Histocytochemistry; D007700:Kinetics; D008297:Male; D051379:Mice; D009284:Naphthols; D051381:Rats; D012394:Rosaniline Dyes
D001391:Azo Compounds; D005404:Fixatives; D005960:Glucosides; D009284:Naphthols; D012394:Rosaniline Dyes; D005959:Glucosidases
Histochemische und biochemische Untersuchung der alpha-Glucosidasen mit 2-Naphthyl-alpha-D-glucosid