A POSTCOUPLING TECHNIQUE FOR β-GLUCURONIDASE EMPLOYING THE SUBSTRATE, NAPHTHOL AS-BI-β-d-GLUCOSIDURONIC ACID

Abstract

A technique is described for visualizing β-glucuronidase sites in tissue employing the substrate, naphthol AS-BI-β-d-glucosiduronic acid. Incubations at 37°C for 30 to 60 minutes of fresh frozen unfixed rat liver and kidney tissues released sufficient naphthol AS-BI at β-glucuronidase sites so that they could be demonstrated by subsequent coupling with a suitable diazo dye at pH 7.4. The experimental data showed that the enzyme in fixed sections was immobilized and that diffusion of enzyme or of product did not present a problem. The staining reaction was inhibited by the presence in the digest of saccharolactone, of a second substrate and of the product, glucuronic acid in a manner expected from biochemical data. The intensity of the stainig reaction was observed to be a function of substrate concentration and pH. The relative preponderance of active sites was pH-dependent, e.g., in the liver, fine cytoplasmic particles at pH 3.5, Kupffer cell granules at pH 4.5, and dense pericanalicular bodies at pH 5.5. Glomerular mesangial cells were most prominent at pH 4.5. Intense β-glucuronidase activity was visible in bodies present in the renal papilla.