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Histochemische und biochemische Untersuchung der α-glucosidasen mit 2-naphthyl-α-D-glucosid

Histochemical and biochemical investigation of α-glucosidases by means of 2-naphthyl-α-D-glucoside

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Summary

Histochemical and biochemical studies yield the following method of choice for the in situ detection of neutral (microvillous) and acid (lysosomal) α-glucosidases: 12 mg 2-naphthyl-α-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 α-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 α-glucosidases is achieved at the cellular level in many organs and tissues.

Fluorometric and photometric measurements prove that 2-naphthyl-α-D-glucoside is superior to 6-brom-2-naphthyl-α-D-glucoside for the demonstration of α-glucosidases in situ due to the lower Michaelis constant and higher maximal reaction velocity of the naphthol derivative. — Among the coupling reagents tested neutral α-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 α-glucosidases to nearly the same extent as Fast Blue B.

Fixation of blocks of tissue in formaldehyde and glutaraldehyde suppresses α-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 α-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-α-D-glucoside the 2-naphthyl derivative is also the substrate of choice for the biochemical determination of α-glucosidases. — Agar gel electrophoresis reveals one band in the neutral and acid pH range.

Zusammenfassung

Vergleichend histochemisch-biochemische Untersuchungen ergeben folgende Methode der Wahl zum in situ-Nachweis der neutralen (mikrovillären) und sauren (lysosomalen) α-Glucosidasen: 12 mg 2-Naphthyl-α-D-glucosid (gelöst in 0,5 ml N,N-Dimethylformamid) und 0,6–0,8 ml Hexazonium-p-rosanilin in 10 ml 0,1 M Citronensäure-Phosphat-Puffer zur wäßrigen oder in 5 ml Pufferlösung 1:1 gemischt zur Inkubation mit semipermeablen Membranen, pH 5 und 6,5.

Mit diesem Verfahren können die neutralen α-Glucosidasen in Bürstensaum von Dünndarm (Glucoamylase, Saccharase-Isomaltase) und Niere bei Säugern, Fischen, Vögeln, Reptilien und Amphibien exakt dargestellt werden; die Lokalisation der sauren α-Glucosidase gelingt auf Zellebene in zahlreichen Organen und Geweben.

Fluorometrische und photometrische Messungen zeigen, daß 2-Naphthyl-α-D-glucosid dem Alternativsubstrat 6-Br-2-Naphthyl-α-glucosid zur histochemischen Untersuchung wegen seiner kleineren Michaelis-Konstante und höheren maximalen Reaktionsgeschwindigkeit überlegen ist. — Unter den geprüften Kupplungssubstanzen kann nur Hexaonium-p-rosanilin als Simultankuppler mit und ohne Membrantechnik vor allem die neutralen α-Glucosidasen korrekt erfassen; Fast Blue B in wäßrigen Medien lokalisiert bei Post- und Simultankupplung falsch-positiv und liefert in Verbindung mit semipermeablen Membranen negative Resultate. Die Hemmung der α-Glucosidasen durch hexazotiertes p-Rosanilin entspricht etwa der durch Fast Blue B.

Stückfixation in Form- und Glutaraldehyd inhibiert die neutralen und sauren α-Glucosidasen in Dünndarm und Nebenhoden zu ca. 50 bzw. 70%; nach Auswaschen liegen die Aktivitäten bei 65 bzw. 50%.

Art- und organspezifische Aktivitätsdifferenzen der sauren und neutralen α-Glucosidasen und Änderungen der Enzymaktivität nach Kastration sowie während der Gravidität in Dünndarm und Niere deckt zuverlässig nur die biochemische Untersuchung auf; der histochemische α-Glucosidasen-Nachweis versagt hier selbst bei Minimalinkubation weitgehend. Verglichen mit p-Nitrophenyl-α-glucosid ist das 2-Naphthylderivat auch das biochemische Substrat der Wahl. — Mittels Agargelelektrophorese kann im alkalischen und sauren pH-Bereich 1 Bande nachgewiesen werden.

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  1. Anatomisches Institut der Universität Würzburg, Koellikerstraße 6, D-8700, Würzburg

    R. Gossrau

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Gossrau, R. Histochemische und biochemische Untersuchung der α-glucosidasen mit 2-naphthyl-α-D-glucosid. Histochemistry 49, 193–211 (1976). https://doi.org/10.1007/BF00492375

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