Hydrolysis of conjugated steroids by beta-glucuronidase from Ampullaria and application to the determination of urinary 17-hydroxycorticosteroids.
The usefulness of Ampullaria beta-glucuronidase as a reagent for the hydrolysis of steroid glucuronides was examined. The purified enzyme showed an optimum pH of 4, and was stable between pH 4.5 and 7.5. The enzyme from Ampullaria and Helix pomatia hydrolyzed various artificial substrates and conjugated steroids. Affinity of the Ampullaria enzyme for 17-hydroxycorticosteroid glucuronide was 7-fold the affinity of the Helix pomatia enzyme. Hydrolysis of urinary steroid conjugates was completed by an incubation with 320 Fishman units of Ampullaria beta-glucuronidase at 60 degrees C for 1 h. A close correlation was obtained between the values of steroid conjugates hydrolyzed by the Ampullaria enzyme and those by the bovine liver enzyme used conventionally. Ampullaria beta-glucuronidase is suitable for hydrolysis of urinary steroid conjugates because of its higher affinity for steroid substrate and the thermal stability.
D015065:17-Hydroxycorticosteroids / Q000652:urine*; D000818:Animals; D005966:Glucuronidase / Q000378:metabolism*; D006372:Helix, Snails / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D012908:Snails / Q000201:enzymology*; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity
D015065:17-Hydroxycorticosteroids; D013256:Steroids; D005966:Glucuronidase