HISTOCHEMICAL DEMONSTRATION OF N-ACETYL-β-GLUCOSAMINIDASE EMPLOYING NAPHTHOL AS-BI N-ACETYL-β-GLUCOSAMINIDE AS SUBSTRATE

The N-acetyl-β-glucosaminide of naphthol AS-BI (7-bromo-3-hydroxy-2-naphth- o-anisidide) was obtained by reacting the anisidide with acetochloroglucosamine in aqueous alkaline acetone. After removal of O-acetyl groups with methanolic ammonia and recrystallization from aqueous methanol, the compound may be used as a substrate for N-acetyl-β-glucosaminidase. Formol-calcium fixed frozen sections were incubated with 0.5 mM substrate in the presence of hexazonium pararosanilin or fast garnet GBC at pH 5.2. Staining appeared in the cytoplasm mostly as discrete granules at the presumed site of enzyme activity and was inhibited in the presence of N-acetylglucosaminolactone. In the rat kidney a number of spherical granules which stained for the enzyme were observed in the proximal portion of the proximal convoluted tubules. In the liver staining was recognized as small granules localized at the pericanalicular cytoplasm of parenchymal hepatic cells. The general cytological localization in these tissues was quite similar to that of other lysosomal enzymes. The limitations of the technique were briefly discussed.