Properties of limpet β-glucuronidase

Abstract

The method is capable of extension to the preparation of other nucleoside polyphosphates labelled with radioactive phosphorus. SUMMARY 1. The reaction of orthophosphate with adenosine 5'-monophosphate, diphosphate and triphosphate in the presence of NN'-dicyclohexylcarbodiimide has been used for the preparation of labelled adenosine polyphosphates.2. The adenosine diphosphate so prepared is labelled exclusively in the terminal phosphate group. The adenosine triphosphate and adenosine tetraphosphate so prepared are labelled in the terminal phosphate group, and to a lesser extent in the penultimate phosphate group.3. A feature of the method is that carrier-free labelled orthophosphate can be used without prior dilution with unlabelled orthophosphate.4. The reaction ofpyrophosphate with adenosine 5'-monophosphate in the presence of NN'-dicyclohexylcarbodiimide has been used for the preparation of labelled adenosine triphosphate. 5. The adenosine triphosphate so prepared is labelled in the f,and y-phosphate groups to an equal extent.6. An improved method for the preparation of NN'-dicyclohexylcarbodiimide is described.I am indebted to Professor H. A. Krebs for the privilege and pleasure of working in his laboratory. I wish to thank Mr R. Hems for discussions and much helpful advice, Mr K. Jones for advice on the use of ultraviolet photography, Dr H. L. Kornberg for advice on autoradiography, Dr W. Bartley for a gift of potato apyrase and Professor J.Baddiley for pointing out to me the necessity of the refluxing procedure in the preparation of N,N'-dicyclohexylthiourea.My thanks go to the Sigma Chemical Co., St Louis, Missouri, for a most generous gift of adenosine tetraphosphate.

Estimation of urinary 5‐hydroxytryptamine‐O‐glucuronide, a metabolite of endogenous 5‐hydroxytryptamine in sheep

“…Passage through Dowex-1 (acetate form) removed an inhibitor of f3-glucuronidase from the urine. Marsh, 1957). The colorimetric method for the estimation of glucaric acid (Ishidate et al, 1965) could not be applied to sheep urine or to the effluent from the columns of Dowex-1 (acetate form) loaded with sheep urine as chromophore formation was inhibited in each instance.…”

“…Sheep urine contained substances which strongly inhibited the breakdown of 5-HT-O-glucuronide by /8-glucuronidase. The liberation of 5-HT from its glucuronide was probably inhibited competitively by other urinary glucuronides and by the 1:4 lactone of glucaric acid, a powerful inhibitor of p-glucuronidase (Levvy, 1952;Levvy et al, 1957) excreted in the urine of sheep and other species (Marsh, 1963). 5-HT-O-Glucuronide was displaced from a column of Dowex-1 (hydroxide form) by acetic acid indicating that it had only weak acidic properties.…”

Metabolism of d-glucuronolactone in mammalian systems. Identification of d-glucaric acid as a normal constituent of urine

“…in body fluids. Recourse may thus be necessary to the much more sensitive, if less simple, fluorimetric method with 4-methylumbelliferone f-D-glucosiduronic acid as substrate (Mead et al 1955;Levvy, Hay & Marsh, 1957); this method requires short incubation periods and a much lower proportion of enzyme preparation in the assay mixture, thus reducing the inhibitor concentration. Values published for the normal output of P-glucuronidase in urine are probably therefore low.…”

A CHEMICAL METHOD FOR THE QUANTITATIVE DETERMINATION OF ESTRIOL, 16-epiESTRIOL, ESTRADIOL-17β AND ESTRONE IN HUMAN MALE AND NON-PREGNANCY URINE

“…Acid hydrolysis and alkali treatment of the phenolic fraction were adopted as the method of choice. When the hydrolysis with limpet(3-glucuronidase (Levvy et al, 1957) was performed, fluorescence due to non-estrogen impurities increased in the eluted estriol fraction and when refluxing with alkali was omitted, determination of 1 to 3 pg of estrogens in some samples became impossible due to high fluorescence background and coloration after sulfuric acid treatment. Estriol could be determined accurately in all cases examined(male and nonpregnancy urine).…”