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Untersuchung der N-Acetyl-β-glucosaminidase mit 1-Naphthyl-N-Acetyl-β-glucosaminid

Investigation of N-acetyl-β-glucosaminidase by means of 1-naphthyl-N-acetyl-β-glucosaminide

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Summary

Using 1-naphthyl-N-acetyl-β-gIucosaminide as substrate N-acetyl-β-glucosaminidase (N-A-Gase) has been investigated histochemically (simultaneous coupling of 1-naphthol and hexazonium p-rosaniline) and microchemically (fluorometric measurement of 1-naphthol) in various organs of rats, mice, and guinea-pigs.

The histochemical incubation medium consists of 5–12 mg 1-naphthyl-N-acetyl-β-glucosaminide (dissolved in NN-dimethyl formamide) and 0.8–1 ml hexazotized p-rosaniline in 9 ml 0.1 M citrate buffer, pH 5; for microchemical purposes 2 mg/ml of this substrate (6 mM) in the same buffer, pH 4.5 were used.

For the in situ demonstration of N-A-Gase formol fixation is recommended because of its lower inhibition rate (32%); in highly active tissues glutaraldehyde is also suitable (54% inhibition in the proximal convoluted tubules of the rat kidney). The total activity of N-A-Gase can better be detected in fresh frozen sections in connection with semipermeable membranes.

Following fixation in glutaraldehyde the enzyme occurs in the lysosomes of many organs, e.g. kidney, epididymis, bronchi, adrenal gland, intestine, uterus, and vesicular gland, especially in rats. Furthermore spezies-dependent differences exist: the suprarenal gland, epididymis, and spleen of guinea-pigs display the highest amount of N-A-Gase. In rats and mice the enzyme activity of these organs is lower; the adrenal cortex is nearly free of N-A-Gase. — The kidney reacts intensely in rats, the sex differences of which can only be detected by means of microchemistry. In the mouse kidney they are more pronounced. Therefore the histochemical N-A-Gase assay reveals them, too. — Organs containing considerable quantities of mucopolysaccharides, e.g. the submandibular gland, urinary bladder, and colon are also rich in N-A-Gase.

The activity and distribution pattern of N-A-Gase obtained with the naphthol AS and 1-naphthyl technique are completely in correspondance with one another. In some cases the quality of the intracellular enzyme localization using naphthol AS-BI N-acetyl-β-glucosaminide as substrate surpasses the possibilities of the 1-naphthyl derivate, in others the latter one enables identical results.

Zusammenfassung

Mit 1-Naphthyl-N-acetyl-β-glucosaminid als Substrat wird die N-Acetyl-β-glucosaminidase (N-A-Gase) histochemisch (Simultankupplung von 1-Naphthol an Hexazonium-p-rosanilin) und mikrochemisch (fluorometrische Messung von 1-Naphthol) in Ratten-, Mäuseund Meerschweinchenorganen untersucht.

Das histochemische Inkubationsmedium enthält 5–12 mg 1-Naphthyl-N-acetyl-β-glucosaminid (gelöst in NN-Dimethylformamid) und 0,8–1 ml 2% Hexazonium-p-rosanilin in 9 ml 0,1 M Citrat-Puffer, pH 5; das mikrochemische 2 mg/ml dieses Substrats (6 mM) im gleichen Puffer, pH 4,5.

Zum Nachweis in situ empfiehlt sich wegen der geringeren Hemmrate (32%) Formol-Fixation, in hochaktiven Geweben auch Glutaraldehyd (54% Inhibition im proximalen Konvolut der Rattenniere). Die histochemische Gesamtaktivität der N-A-Gase ist besser an frischen Schnitten mit semipermeablen Membranen zu erfassen.

Nach Fixation in Glutaraldehyd kann das Enzym vor allem bei Ratten in den Lysosomen zahlreicher Organe nachgewiesen werden (u.a. Niere, Nebenhoden, Bronchien, Darm, Uterus und Samenblase), wobei artspezifische Unterschiede bestehen: Über die höchste N-A-Gase-Aktivität verfügen Nebenniere, Nebenhoden und Milz von Meerschweinchen. Weniger Enzym enthalten diese Organe bei Ratten und Mäusen; speziell die Nebenniere ist weitgehend N-A-Gase-frei. Am kräftigsten reagiert die Niere bei Ratten. Geschlechtsspezifische Differenzen sind hier nur mikrochemisch faßbar, in der Niere der Maus aber schon histochemisch. — Reich an N-A-Gase sind Speicheldrüsen, Harnblase und Colon sowie andere Organe mit mucopolysaccharid-haltigen Strukturen.

Die mit der Naphthol-AS und 1-Naphthyl-Methode für die N-A-Gase erhaltenen Aktivitätsund Verteilungsmuster entsprechen sich. Die Qualität der intrazellulären Enzymlokalisation mit 1-Naphthyl-N-acetyl-β-glucosaminid als Substrat kommt den Möglichkeiten des Naphthol-AS-Verfahrens in vielen Organen nahe oder ist ihm gleichwertig.

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  1. Anatomisches Institut der Universität Würzburg, Bundesrepublik Deutschland

    R. Gossrau

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Gossrau, R. Untersuchung der N-Acetyl-β-glucosaminidase mit 1-Naphthyl-N-Acetyl-β-glucosaminid. Histochemie 37, 169–185 (1973). https://doi.org/10.1007/BF00305588

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