Über den histochemischen Nachweis der β-Glueosidase mit 1-Naphthyl-β-glucopyranosid

Summary

A simple simultaneous azo coupling method for the demonstration of β-glucosidase in the small intestine of various mammals is described and compared with other histochemical techniques for this enzyme. Strong evidence occurs that a correct intracellular localization of β-glucosidase can only be obtained by means of the indigogenic and the assay presented here: the azo dye methods published so far with 6-Br-2-naphthyl-β-glucopyranoside as substrate and p-rosaniline for simultaneous or Fast Blue B for postcoupling are not able to reflect the true binding sites of intestinal-β-glucosidase.

The recommended incubation medium consists of 4.5–9.0 mg 1-naphthyl-β-glucopyranoside (dissolved in 0.4 ml NN-dimethyl formamide) and 0.6–0.8 ml 2% hexazonium-p-rosaniline in 9.0 ml 0.1 M citric acid-phosphate buffer, pH 5.5.— Microchemical measurements using the same substrate show that p-rosaniline inhibits β-glucosidase to a similar extent as ferricyanide in the indigogenic medium.

Because of the presumed relationship between β-glucosidase and neutral β-galactosidase the latter enzyme has been demonstrated with the above mentioned assay replacing the 1-naphthyl-β-glucoside by the corresponding β-galactopyranoside.

The strongest β-glucosidase and β-galactosidase activity can regularly be observed in the jejunum of rats, mice and guinea-pigs where both enzymes are localized in the brush border region of the enterocytes. In comparison with β-galactosidase the β-glucosidase reaction is always more intensive and the azo dye production in the microvillous zone of suckling rats and guinea-pigs is far higher than in the intestine of adult animals. Furthermore both enzymes react in a similar way to inhibitors, experiments (thirst, hunger) and pregnancy and do not split naphthol AS BI β-glucopyranoside respectively β-galactopyranoside.

Bloc fixation in formol-calcium and especially in glutaraldehyde improves the localization of the azo dye considerably; but microchemistry reveals that aldehyde fixation supresses the β-glucosidase to ca. 50%. The basis activity of the enzyme following pretreatment with formol is reached within the first minute of fixation.

Zusammenfassung

Es wird eine einfache simultane Azokupplungsmethode zur Darstellung der β-Glucosidase im Dünndarm verschiedener Säuger beschrieben und mit anderen histochemischen Verfahren zum Nachweis dieses Enzyms verglichen. Eine intrazelluläre Lokalisation der β-Glucosidase ermöglichen nur die Indigogen-und die hier angegebene Technik, nicht dagegen die bisherigen Azofarbstoffmethoden mit 6-Br-2-Naphthyl-β-glucopyranosid als Substrat und p-Rosanilin zur Simultanoder Fast Blue B zur Postkupplung.

Das Inkubationsmedium des neuen Verfahrens enthält 4,5–9,0 mg 1-Naphthyl-β-glucopyranosid (gelöst in 0,4 ml Dimethylformamid) und 0,4–0.8 ml 2% hexazotiertes p-Rosanilin in 9,0 ml 0,1 M Citronensäure-Phosphat-Puffer, pH 5,5. —Mikrochemische Messungen mit dem gleichen Substrat zeigen, daß die β-Glucosidase durch p-Rosanilin in ähnlichem Ausmaß wie durch Ferricyanid im Indigogen-Medium gehemmt wird.

Wegen der fraglichen Verwandtschaft von β-Glucosidase und neutraler β-Galactosidase wurde dieses Enzym mit obigem Ansatz und 1-Naphthyl-β-galactopyranosid als Substrat untersucht.