The catalytic effect of Mg2+ and imidazole on the decomposition of 5-phosphoribosyl-alpha-1-pyrophosphate in aqueous solution

The rate of decomposition of aqueous solutions of 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) is greatly enhanced by the presence of certain divalent ions. High levels of Mg(2+) were shown to increase the rate of phosphate removal from PRPP by factors greater than 100-fold. The combined action of Mg(2+) and a nitrogenous compound (imidazole was employed in this study) increases the degradation rate constants by up to 1000-fold. The degradation of PRPP in neutral solutions follows two main paths, a pyrophosphate hydrolysis reaction yielding ribose 5-phosphate and an internal beta-phosphate cleavage yielding 5-phosphoribosyl-1,2 cyclic phosphate. The catalytic effect of Mg(2+) on PRPP degradation appears to suggest a connection between the observed rate enhancements and the dimagnesium form of PRPP. Rate enhancing effects of Mg(2+) are greatest at pH values (i.e. pH>7) which promote strong phosphate-Mg(2+) interaction.     

Biological activities of phosphodiester linkage isomers of 2-5A

To determine the relative importance of the 2',5'-phosphodiester bond of 2-5A in its binding to and activation of the 2-5A-dependent ribonuclease (RNase L, RNase F), a number of phosphodiester linkage isomers of 2-5A were prepared. These isomers were obtained either by lead ion-catalyzed polymerization of adenosine 5'-phosphorimidazolidate or by T4 polynucleotide kinase-catalyzed 5'-phosphorylation of adenylyl(3' leads to 5')adenylyl(3' leads to 5')adenosine followed by reaction of the corresponding phosphorimidazolidates with tri(n-butylammonium)pyrophosphate. The following 2-5A isomers thus were prepared: ppp5'A2'p5'A3'p5'A, ppp5'A3'p5'A2'p5'A, ppp5'A3'p5'A3'p5'A("3-5A"), ppp5'A2'p5'A3'p5'A2'p5'A,and ppp5'A3'p5'A2'p5'-A2'p5'A. The ability of these isomeric 2-5As to interact with the 2-5A-dependent endonuclease was ascertained by three different criteria: (i) ability to prevent the protein synthesis inhibitory effects of 2-5A, (ii) activity as an inhibitor of translation in encephalomyocarditis RNA-programmed L cell extracts, and (iii) ability to prevent binding of the radiolabeled probe, ppp5'A2'p5'A2'p5'A2'p5'A3'[32P]p5'Cp, to the endonuclease of L cell extracts. In certain experiments, degradation of oligonucleotide was minimized or eliminated by altering assay conditions, providing alternate phosphodiesterase substrates, or by using purified endoribonuclease of Ehrlich ascites cells. By all criteria, replacement of 2',5'-bond by a 3',5'-bond led to a substantial decrease in biological activity. Generally, replacement of just one 2',5'-phosphodiester bond with a 3',5'-linkage led to at least a one order of magnitude loss of activity. In accord with this trend, ppp5'A3'p5'A3'p5'A(3-5A) was greater than 10,000 less active than 2-5A in binding to the endonuclease or as an inhibitor of protein synthesis.     

Determination of 5-phosphoribosyl 1-pyrophosphate in mouse liver

Optimum conditions for the determination of 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) in mouse liver are described. PP-Ribose-P is extracted from frozen liver powder with a solution of 182 μm [¹⁴C]adenine (10 μCi/μmole), 3.36 mm 2,3-diphosphoglycerate and 21.3 mm Tris-Cl buffer (pH 7.4) for 20 sec at 100°C. The amount of PP-ribose-P in the extract is calculated from the radioactivity incorporated into AMP, ADP, and ATP during a 60 min incubation at 37°C with adenine phosphoribosyltransferase and 2.5 mm CaCl₂.     

Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP): determination of cellular PRPP pools and assay of PRPP synthetase activity.

Conditions are described where 5-phosphoribosyl-α-1-pyrophosphate (PRPP) can be determined by thin-layer chromatographic methods commonly used for the determination of nucleoside triphosphate pools in ³²P-labeled bacteria. A two-dimensional chromatographic system is described where very small pools of PRPP (about 0.03 μmol per gram dry weight bacteria) can be determined. In a uni-dimensional chromatographic system the lower limit for detection of PRPP pools is about 0.3 μmol per gram dry weight bacteria. This uni-dimensional system offers an assay also for PRPP synthetase activity even in crude extracts using [γ-³²P]ATP as a substrate. The assay is highly specific due to the chromatographic isolation of PRPP and is very sensitive due to the use of ³²P labeling.The chromatographic methods for determination of PRPP pools and of activities of PRPP synthetase have been applied to the analysis of some mutants of Salmonella typhimurium and have provided results that agree well with the results obtained by conventional methods of PRPP analysis.     

Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine.

Purification and partial characterization of the poliovirus RNA-linked protein (VPg) are described. VPg has been freed from the RNA by ribonuclease digestion and phenol extraction. Gel filtration chromatography of VPg-pUp (labeled with 32P) in 0.5% sodium dodecyl sulfate or 6 M guanidine HCl indicates that it has a molecular weight of about 12,000. VPg is bound to the 5' end of poliovirion RNA by a phosphodiester bond between a tyrosine residue in the VPg molecule and the 5'-terminal uridine. After acid hydrolysis of [3H]tyrosine-labeled VPg-pU, free tyrosine can be released by venom phosphodiesterase. Acid hydrolysis of VPg-p labeled with either 32P or [3H] tyrosine yields tyrosine-phosphate. There appears to be only 1 tyrosine residue per VPg molecule.     

Determination of the intracellular concentration of 5-phosphoribosyl-1-pyrophosphate in cultured mammalian fibroblasts

The properties of an assay for the 5-phosphoribosyl-1-pyrophosphate (PRPP) content of cultured mammalian fibroblasts are described. The assay is based upon the PRPP-dependent release of ¹⁴CO₂ from [carboxyl-¹⁴C]orotic acid by a commercially available preparation of yeast orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. The advantages of the assay include the fact that it is based on the enzymatic recognition of PRPP, employs an irreversible reaction, and does not involve either the chromatographic separation of substrate and product or the purification of a phosphoribosyltransferase. The disadvantage of the assay is that the efficiency of PRPP measurement varies somewhat, in part because the yeast enzyme preparation contains 5′-nucleotidase activity. A calibration procedure is described which corrects for variation in efficiency both between and within experiments. This procedure seems to yield highly reliable estimates of PRPP content. The assay will readily detect 0.6 nmol, and the cell strain studied contained $\text{7.76 ± 1.14 nmol of PRPP}\text{10}{}^7\text{cells}$.     

A sensitive method for estimating 5-phosphoribosyl 1-pyrophosphate in Escherichia coli

A sensitive method, which uses a PRT'ase-catalysed reaction to couple PRPP with a labeled base, has been described for estimating the PRPP content of E. coli. Although the method is basically that of Henderson and Khoo (2), a new chromatographic system, which allows the complete separation of [¹⁴C]nucleoside 5′-phosphate from any contaminating [¹⁴C]-labeled base, has been devised. Further, the extraction process and the conditions for the PRT'ase reaction have both been modified for application to bacterial cultures. Finally, a choice between methods using either [¹⁴C]adenine or [¹⁴C]guanine, with their respective PRT'ase enzymes, allows for the estimation of PRPP in extracts which contain unlabeled purine or pyrimidine bases.     

The concentration of 5-phosphoribosyl 1-pyrophosphate in monolayer tumor cells and the effect of various pyrimidine antimetabolites

5-Phosphoribosyl 1-pyrophosphate (PRPP) was determined in several murine and human cancer cell lines grown in monolayer, and harvested by trypsinization. For all cell lines a large variation in the PRPP concentration (5-1300 pmol/ 10(6) cells was found. A 1-hr incubation in Dullbecco's medium reduced the variation in PRPP concentration. After this incubation the highest concentration was found in the murine B16 melanoma cell line (about 200 pmol/10(6) cells). The human melanoma cell lines IGR3 and M5 and the human colon carcinoma cell line WiDr contained about 100 pmol/10(6) cells. After this preincubation of 1-hr these cell suspensions were used to study the effect of several antimetabolites on PRPP concentration. A 2-hr incubation with 1mM N-(phosphonacetyl)-L-aspartate (PALA) increased the PRPP concentration only in M5 cells, whereas methotrexate caused an increase in all cell lines. When 5-fluorouracil (5FU) was added, no significant decrease was found in any cell line. Addition of 5FU after a 2-hr preincubation with PALA resulted in a lower concentration in B16, M5 and WiDr cells. The prodrug, 5-fluoro-5' deoxyuridine altered the PRPP concentration only in in WiDr cells when it was added after PALA. The activity of the 5FU metabolizing enzyme orotate phosphoribosyl transferase was comparable in B16, M5 and WiDr cells, but much lower in IGR3 cells.     

Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.     

Regulation of 5-phosphoribosyl-1-pyrophosphate synthesis in human fibroblasts by the concentration of inorganic phosphate

During the growth cycle of normal fibroblasts and of fibroblasts deficient in glucose-6-phosphate dehydrogenase activity, the concentration of 5-phosphoribosyl-1-pyrophosphate and of P_i, as well as the activity of 5-phosphoribosyl-1-pyrophosphate synthetase, decreased to stable values in confluent cultures. A high degree of correlation (0.89 and 0.91 for two normal and 0.69 for one glucose-6-phosphate dehydrogenase-deficient cell strain, respectively) was shown between intracellular P_i and 5-phosphoribosyl-1-pyrophosphate concentrations under varying culture and incubation conditions. 5-phosphoribosyl-1-pyrophosphate concentrations were elevated in normal fibroblasts incubated with methylene blue only if intracellular P_i levels were high. Neither methylene blue nor 6-aminonicotinamide, singly, affected intracellular P_i concentrations. However, when normal cells were pretreated with 6-aminonicotinamide and then with methylene blue, intracellular P_i decreased, 5-phosphoribosyl-1-pyrophosphate was depleted, and its rate of generation decreased. Under similar conditions, glucose-6-phosphate dehydrogenase-deficient fibroblasts maintained unaltered P_i levels, and 5-phosphoribosyl-1-pyrophosphate concentration and generation were slightly increased. The decrease in intracellular P_i in normal cells after the combined treatment was commensurate with an accumulation of 6-phosphogluconate, which did not take place in mutant cells. The changes in 5-phosphoribosyl-1-pyrophosphate synthesis, whether due to the stage of growth or various experimental manipulations, were always concordant with changes in intracellular P_i level. The regulatory role of P_i is consistent with the known enzymic properties of 5-phosphoribosyl-1-pyrophosphate synthetase.     

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.     

Electrophoretic heterogeneity of 5-phosphoribosyl-1-pyrophosphate synthetase within and among humans

The product of the enzyme 5-phosphoribosyl-1-pyrophosphate (PPriboseP) synthetase is a substrate for purine, pyrimidine, and pyridine biosynthesis and may be rate limiting for purine biosynthesis. A system developed to electrophoretically separate and histochemically detect PPriboseP synthetase in crude cell extracts has facilitated the identification of electrophoretically variant enzyme forms in the erythrocytes of five patients from a patient population of 200. Additional studies of human organs obtained at autopsy revealed a unique electrophoretic mobility for nearly each organ within the same individual.     

Configuration-dependent properties of randomly coiling polynucleotide chains. II. The role of the phosphodiester linkage

The dependence of the unperturbed dimensions of randomly coiling polynucleotides on the rotations about the phosphodiester linkages of the chain has been examined in order to understand the conformational discrepancies, set forth in paper I, regarding these angles (ω′ and ω). Large values of the characteristic ratio 〈r²〉₀/nl² , which agree with the experimental behavior of the chain, are obtained only if a sizeable proportion of the polymer residues have trans ω′ values. The asymmetric torsional potential that is believed to arise from gauche effects associated with the P-O bonds has been approximated using a hard core model. The calculated characteristic ratio exhibits a strong dependence upon the magnitude of this torsional barrier (separating trans and gauche conformations) and shows agreement with experimental values for polyribonucleotides only if this energy difference is 1 kcal/mol or less.