A rapid assay technique for RNA ribose methylases.

A rapid technique for quantitative separation of ribose-methylated nucleosides from base-methylated and non-methylated nucleosides by chromatography on DEAE-cellulose paper in the presence of borate is described. The method has been used as an assay for tRNA ribose methylases from yeast, using under methylated Escherichia coli tRNA as substrate. The main product formed with a partly purified yeast enzyme was characterized as 2'-O-methylcytidine.     

Synthesis of a new ribose modified analogue of cyclic inosine diphosphate ribose

A new analogue of cyclic inosine diphosphate ribose (cIDPR), in which the N-1 and N-9 ribosyl moieties were substituted by an alkyl moiety and an hydroxy-alkyl chain, has been synthesized and characterized.     

Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.     

Intact chloroplast electron flow. Effects of ribose 5-phosphate

Additions of ribose 5-phosphate to intact spinach chloroplasts were used to probe the effects of ADP regeneration on pH-gradient formation and electron-transfer reactions. In weakly illuminated chloroplasts, the ATP/ADP ratio dropped by 64% and the transthylakoid pH gradient decreased by a minimum of 0.2 units in response to ribose 5-phosphate. Nitrite reduction increased 2-fold while, under conditions of cyclic electron flow, the half-time for cytochrome f reduction decreased by a factor of two from 4.1 to 1.9 ms. The results suggest that metabolic ATP consumption, during the conversion of ribulose 5-phosphate to ribulose 1,5-bisphosphate, enhances electron transfer between plastohydroquinone and cytochrome f through decreases in the transthylakoid pH gradient caused by phosphorylation of ADP.     

Influence of ribose on adenine salvage after intense muscle contractions.

The influence of ribose supplementation on skeletal muscle adenine salvage rates during recovery from intense contractions and subsequent muscle performance was evaluated using an adult rat perfused hindquarter preparation. Three minutes of tetanic contractions (60 tetani/min) decreased ATP content in the calf muscles by approximately 50% and produced an equimolar increase in IMP. Effective recovery of muscle ATP 1 h after contractions was due to reamination of IMP via the purine nucleotide cycle and was complete in the red gastrocnemius but incomplete in the white gastrocnemius muscle section. Adenine salvage rates in recovering muscle averaged 45 +/- 4, 49 +/- 5, and 30 +/- 3 nmol. h(-1). g(-1) for plantaris, red gastrocnemius, and white gastrocnemius muscle, respectively, which were not different from values in corresponding nonstimulated muscle sections. Adenine salvage rates increased five- to sevenfold by perfusion with approximately 4 mM ribose (212 +/- 17, 192 +/- 9, and 215 +/- 14 nmol. h(-1). g(-1) in resting muscle sections, respectively). These high rates were sustained in recovering muscle, except for a small (approximately 20%) but significant (P < 0.001) decrease in the white gastrocnemius muscle. Ribose supplementation did not affect subsequent muscle force production after 60 min of recovery. These data indicate that adenine salvage rates were essentially unaltered during recovery from intense contractions.     

Adaptive alterations in Lactobacillus casei. Gluconeogenesis in cells grown on ribose.

Adaptive alterations of the enzymes involved in gluconeogenesis have been studied in homofermentative Lactobacillus casei after growth on ribose. Among the enzymes induced were phosphoenolpyruvate carboxykinase, fructose 1,6-diphosphatase and glucose 6-phosphatase. The activities of phosphoglucomutase and fructose 1,6-diphosphate aldolase, measured in the direction of condensation of triose phosphates, were also observed to be enhanced. Oxalacetate, the substrate of phosphoenolpyruvate carboxykinase, appears to be formed through aspartate aminotransferase activity developed in ribose-grown cells. The gluconeogenic enzymes were repressed when glucose was added to the pentose-containing medium during the growth of the organism. The relative participation of precursors, assessed from the extent of incorporation of radioactivity into cellular polysaccharides, suggested that the products of ribose fermentation did not contribute to new glucose synthesis.     

Poly(adenosine diphosphate ribose) glycohydrolase in Physarum polycephalum.

Poly(adenosine diphosphate ribose) glycohydrolase, which has thus far only been found in mammalian tissues, was found for the first time in the primitive eukaryotic slime mold Physarum polycephalum. The hydrolytic product of poly(adenosine diphosphate ribose) with this enzyme was identified as adenosine diphosphate ribose by paper and thin-layer chromatography. It is likely that the enzyme caused exoglycosidic hydrolysis. The optimal pH of this enzyme was 6.0, and the K_m value was 4.3 μm, as adenosine diphosphate ribose residues of polymer. Adenosine diphosphate ribose, ADP and ATP at a concentration of 0.1mm strongly inhibited the enzyme activity. 3′,5′-Cyclic AMP was inhibitory at a concentration of 1mm. The molecular weight of this enzyme was estimated to be 57,000.     

Poly(adenosine diphosphate ribose) polymerase in Physarum polycephalum.

1. The isolated nuclei of the slime mould Physarum polycephalum contain an enzyme that will incorporated [adenine-3H] NAD+ into an acid-insoluble product, which is shown to be poly(ADP-ribose). 2. This incorporation has an optimum pH of 8.2 and a temperature optimum below 10degreesC. 3. Optimum stimulation is given by 15 mM-Mg2+. 4. 2-Mercaptoethanol or dithiothreitol also stimulates the incorporation, the latter at an optimum concentration of about 1 mM. 5. Under optimum conditions the Km value for the reaction is 0.28 mM at 15degreesC. Nicotinamide inhibits the incorporation with a Ki of 5.7 muM. 6. Exogenous DNA stimulates the incorporation by about 100%. 7. Preincubation of the nuclei with deoxyribonuclease, but not with ribonuclease, almost completely inactivates the incorporation of NAD+. 8. The enzyme is unstable at both 0degrees and 15degreesC in the absence of dithiothreitol. The presence of dithiothreitol at a concentration of 1 mM stabilizes the enzyme at both these temperatures. 9. The activity of this enzyme per nucleus was shown in three separate experiments to fall by about one-half in early S phase and then to rise to its pre-mitotic value after about 3 h, that is in late S phase. 10. The possible physiological function of this enzyme system is discussed.     

Presence and turnover of adenosine diphosphate ribose in human erythrocytes.

ADP-ribose was detected in human red blood cells (RBC) at 0.45 +/- 0.1 microM concentrations. These levels could be estimated after purification of ADP-ribose by means of three sequential HPLC fractionations of RBC extracts. Extraction was performed by sonication of RBC either in trichloroacetic acid, followed by centrifugation, or in carbonate-bicarbonate buffer, pH 10.0, followed by rapid ultrafiltration. Neither procedure of extraction caused artefactual formation of ADP-ribose. Prolonged incubation of intact RBC in isotonic buffer containing labeled orthophosphate resulted in the slow incorporation of radioactivity into ADP-ribose. Identification of the labeled ADP-ribose was confirmed upon incubation of the purified metabolite with nucleotide pyrophosphatase, yielding radioactive 5'-AMP and ribose 5-phosphate, while its exposure to a nonspecific deaminase resulted in the quantitative formation of labeled inosine diphosphate ribose.     

Analogues of ribose 5-phosphate and 5-phosphoribosyl pyrophosphate. The preparation and properties of ribose 5-phosphorothioate and 5-phosphoribosyl 1-methylenediphosphonate

1. 5-Phosphoribosyl 1-methylenediphosphonate was isolated after reaction of ribose 5-phosphate and O-adenylyl methylenediphosphonate with 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. 2. The analogue reacted with adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase [K(m) (analogue)/K(m) (5-phosphoribosyl pyrophosphate) 0.17, 0.19 and 6.3 respectively; V(max.) (analogue)/V(max.) (5-phosphoribosyl pyrophosphate) 0.011, 0.26 and 1.1 respectively]. 3. The analogue was not a substrate for 5-phosphoribosyl pyrophosphate amidotransferase or orotate phosphoribosyltransferase. 4. Ribose 5-phosphorothioate was synthesized by allowing ribose to react with thiophosphoryl chloride in triethyl phosphate. The analogue was a substrate for 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. When this reaction was coupled to either adenine phosphoribosyltransferase or hypoxanthine phosphoribosyltransferase, adenosine 5'-phosphorothioate or inosine 5'-phosphorothioate was formed respectively.     

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.