Abstract
Biochemical evidence has been collected which supports the proposition that the appearance of the staining reaction in tissue sections incubated in Fishman-Baker substrate solution is dependent on the activity of β-glucuronidase. It was first shown by quantitative analysis that the glucuronic acid is liberated into the medium simultaneously with the deposition of ferric-hydroxyquinoline as the hydrolysis of the substrate, 8-hydroxyquinoline glucosiduronic acid, proceeded. This demonstration was made both in a gel-β-glucuronidase model system and in incubation mixtures containing tissue sections. Next, it was established that the staining reaction was dependent on pH in a manner indistinguishable from the pH dependence of purified rat liver β-glucuronidase acting on 8-hydroxyquinoline glucosiduronic acid. Finally, the staining reaction was invariably prevented by adding to the Fishman-Baker substrate solution an appropriate amount of a second glucosiduronic acid beside that of 8-hydroxyquinoline. This inhibition was produced by naphthol AS-BI-β-glucosiduronic acid, a histochemical substrate for β-glucuronidase. Next, it was observed that the addition of p-nitrophenyl β-glucosiduronic acid to the digest prepared for each staining reaction inhibited the staining reactions of both. The use of the mixed substrate test suggests itself for application to the critical study of other enzymes.