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PubMed1983

Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-14C]phosphoribosylpyrophosphate.

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-alpha-D-ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction. 5-beta-phosphoribosyl-1-amine, using [1-14C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent Km values of the human lymphoblast enzyme are 0.46 mM for glutamine and 0.71 mM for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo-L-norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity.

Amidophosphoribosyltransferase↗

PubMed1986

The measurement of xylulose 5-phosphate, ribulose 5-phosphate, and combined sedoheptulose 7-phosphate and ribose 5-phosphate in liver tissue.

A modification of the method of Kauffman et al. (F. C. Kauffman, J. G. Brown, J. V. Passonneau, and O. H. Lowry (1969) J. Biol. Chem. 244, 3647-3653) for the spectrophotometric determination of xylulose 5-phosphate, ribulose 5-phosphate, and combined ribose 5-phosphate and sedoheptulose 7-phosphate in tissue extract is presented. Using commercially available enzymes all three assays come to a clear endpoint with the assays described. Values for these metabolites in liver in three dietary states are reported; 48 h starved, ad libitum feeding of standard NIH rat ration, and meal feeding of a fat-free diet. Xylulose 5-phosphate values were 3.8 +/- 0.3, 8.6 +/- 0.3, and 66.3 +/- 8.3 nmol/g. Ribulose 5-phosphate values were 3.4 +/- 0.3, 5.8 +/- 0.2, and 37.1 +/- 5.3 nmol/g. Combined ribose 5-phosphate and sedoheptulose 7-phosphate were 29.3 +/- 0.3, 38.2 +/- 1.2, and 108.2 +/- 14.5 nmol/g. The ratio of measured tissue content of [xylulose 5-phosphate]/[ribulose 5-phosphate] was found to be 1.12 +/- 0.07 in starved animals, 1.48 +/- 0.04 in ad libitum fed animals and 1.78 +/- 0.03 in low-fat meal fed animals. These data are in good agreement with the range of equilibrium constants reported for this reaction, suggesting that the ribulose 5-phosphate 3-epimerase reaction (EC 5.1.3.1) is a near equilibrium reaction despite a more than 10-fold change in the tissue content of these metabolites.

Animals↗

PubMed1987

A method for the determination of 5-phosphoribosyl 1-pyrophosphate concentrations in erythrocytes using high-performance liquid chromatography.

A method for the measurement of erythrocyte 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) using HPLC is described. Inosinic acid formed from the enzyme-catalyzed reaction of hypoxanthine and PP-ribose-P using partially purified hypoxanthine-guanine phosphoribosyltransferase is measured after chromatography on an ion-exchange column (Partisil 10 SAX). The average recovery of PP-ribose-P added to erythrocytes was 96.6%. Normal values found were 1.3 +/- 0.6 nmol PP-ribose-P/ml packed RBC (20 individuals). Replication experiments gave a coefficient of variation of 4.4%. Elevated levels in the range 4.4-7.9 nmol PP-ribose-P/ml packed RBC were found in four patients with gout and partial deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase.

Chromatography, High Pressure Liquid↗

PubMed1987

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts.

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

Adenine Phosphoribosyltransferase↗

PubMed1987

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate.

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.

Animals↗

PubMed1990

Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.

Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.

Adenine Phosphoribosyltransferase↗

PubMed1955