Abstract: To delineate the normal function of purine nucleoside phosphorylase and to understand the pathogenesis of the immune dysfunction associated with deficiency of this enzyme, we studied purine metabolism in a patient deficient in purine nucleoside phosphorylase, her erythrocytes and cultured fibroblasts. She exhibited severe hypouricemia and hypouricosuria but excreted excessive amounts of purines in her urine, the major components of which were inosine and guanosine. Her urine also contained deoxyinosine, deoxyguanosine and uric acid 9-N riboside. The patient's erythrocytes but not her cultured fibroblasts contained increased concentrations of phosphoribosylpyrophosphate and inosine. The metabolic abnormalities resembled those in the erythrocytes of patients with the Lesch-Nyhan syndrome. Purine nucleoside phosphorylase is a necessary component of the major, if not the sole, pathway for the conversion of purine nucleosides and nucleotides to uric acid. The increased intracellular concentrations of inosine may, by inhibiting adenosine deaminase, be related to the immunologic dysfunction.

Abstract: A simple, convenient, and sensitive spectrophotometric procedure is described for quantitative measurement of nucleoside phosphate binding sites constructed by the dinucleotide fold. The procedure involves difference spectral titration of such enzymes with the dye Cibacron blue F3GA in a spectral region remote from the intrinsic absorbance of proteins or natural ligands. The titration curves can be analyzed to determine the affinity of nucleoside phosphate binding sites for both the dye and the natural ligand over a potentially wide range of experimental conditions. The interaction of the dye with two proteins which contain the dinucleotide fold, lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), is illustrated.

Abstract: A possible prebiotic phosphorylation method has been investigated in which formamide served as the reaction medium. Nucleotides and nucleotide derivatives were formed when nucleosides were allowed to react with different orthophosphate, hydrogen phosphate or dihydrogen phosphate salts or with different condensed phosphate salts. The reaction products obtained from the phosphorylation of adenosine were 2′, 3′ and 5′-AMPs, 2′, 5′ and 3′, 5′-ADPs, and 2′, 3′-cyclic AMP. The extent of phosphorylation in formamide exceeded 50% under favorable conditions after 15 days at 70°. The acidic dihydrogen phosphates and condensed hydrogen phosphates proved to be the best phosphorylating agents. The presence of water in the medium decreased the yield of nucleotide derivatives, but some phosphorylation of adenosine was detected using dihydrogen phosphate in formamide containing water. The phosphorylation reactions were also observed for deoxynucleosides. Little decomposition of the nucleosides was detected during the reaction time needed to form nucleotide derivatives. The facility with which phosphorylation takes place in formamide under very mild conditions may justify further studies both of prebiotic phosphorylation and synthetic phosphorylation using this solvent.

Abstract: Envelope-specific and sarcoma-specific nucleotide sequences have been located within the 10,000 nucleotides of the RNA of nondefective Schmidt-Ruppin Rous sarcoma virus (nd SR). For this purpose, about 30 RNase-T1-resistant oligonucleotides were ordered relative to the 3'-poly(A) terminus of the RNA, to construct an oligonucleotide map of the nd SR RNA. A cluster of seven envelope-specific oligonucleotides, identified by their absence from an otherwise very similar oligonucleotide map of an envelop-defective deletion mutant (which lacks the major viral glycoprotein), mapped at a distance of 2800-5000 nucleotides from the poly(A) end of nd SR RNA. A cluster of two sarcoma-specific oligonucleotides, identified by their absence from an otherwise nearly identical oligonucleotide map of a transformation-defective deletion mutant, mapped at a distance of 1000-2000 nucleotides from the poly(A) end of nd SR RNA. The oligonucleotide maps of nd SR and of the two deletion mutants were the same from the poly(A) end up to 650 nucleotides and included one terminal oligonucleotid, termed C, which is found in all avian tumor viruses tested so far. A possible gene order consistent with our data suggests that sarcoma-specific nucleotide sequences map between envelope-specific nucleotide sequences and the poly(A) end of the RNA.

Abstract: A novel phosphodiesterase was purified from cultured tobacco cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5'-phosphate, p-nitrophenyl thymidine 3'-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides, and poly(adenosine diphosphate ribose), which is a polymer synthesized from NAD+. However, it did not hydrolyze highly polymerized polynucleotides. The molecular weight of the native enzyme was estimated as 270 000 to 280 000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with molecular weights calculated to be 75 000. The enzyme did not require divalent cations for activity being fully active in the presence of ethylenediaminetetraacetic acid. The pH optimum for the enzyme was approximately 6 with p-ni-trophenyl thymidine 5'-phosphate or adenosine cyclic 3',5'monophosphate, and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5'-phosphate gave two apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least two active sites.

Abstract: A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.

Abstract: Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of alpha-amanitin (1 mug/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45-S ribosomal precursor was completely resistant even to high concentrations of alpha-amanitin (150 mug/ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45-S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4-8 S) was synthesized by HeLa cell nuclei in the presence of 1 mug/ml alpha-amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 mug/ml) but clearly sensitive to high (150 mug/ml) concentrations of alpha-amanitin. Consequently the synthesis of all these small-molecular-weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5' end of the RNA molecule with the gamma-phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small-molecular-weight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45-S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.

Abstract: The interactions among Escherichia coli elongation factor G (EF-G), guanine nucleotides, ribosomes, and fusidic acid were investigated by a number of physical techniques. Equilibrium dialysis studies demonstrated the existence of a binary EF-G-GDP complex. This complex forms with a stoichiometry of ca 1:1 and an apparent Ka of 2.5 X 10(5) M-1. While no evidence was obtained for the formation of a ribosome-GDP complex, in the presence of ribosomes, the apparent Ka for guanosine diphosphate (GDP) increased 40-fold over that for binding to EF-G alone. Although the apparent Ka increased, the stoichiometry remained ca. 1 mol of GDP/mol of EF-G. An upper limit of 1.3 X 10(7) M-1 was calculated for the Ka for binding of ribosomes to the EF-G-GDP complex. Fusidic acid had no effect on the apparent Ka's for either the EF-G-GDP or EF-G-beta, gamma-methyleneguanosine triphosphate (GMP-P(CH2)P)=ribosome complexes, but markedly increased the Ka for GDP in the EF-G-GDP-ribosome complex without altering the stoichiometry. The apparent Ka for GDP was shown to be dependent upon the fusidic acid concentration. In addition, the rate of GDP exchange into the quaternary EF-G-GDP-ribosome-fusidic acid complex was inversely related to the fusidic acid concentration. All of the data obtained in these studies suggest that the formation and dissociation of complexes involving EF-G and guanine nucleotides is ordered. GDP is the first component to bind to EF-G, followed by the ribosome, and, finally, fusidic acid. This conclusion is consistent with the kinetic mechanism for the hydrolysis of GTP by EF-G and the ribosome proposed in the preceding paper of this issue (Rohrbach and Bodley (1976b). In addition to these binding studies, guanine nucleotides have also been shown to protect EF-G against both limited trypsinolysis and chemical modification by N-ethylmaleimide. These observations offer additional evidence for the existence of a guanine nucleotide binding site on EF-G.

Abstract: Publisher Summary The term nicotinamide nucleotide transhydrogenase has been used in this chapter to denote those enzymes that catalyze the reversible transfer of hydrogen between the two naturally occurring nicotinamide nucleotides, NAD (H) and NADP (H). The term does not include those enzymes that catalyze transhydrogenation between only one naturally occurring nicotinamide nucleotide and an artificial nicotinamide nucleotide analog. Nicotinamide nucleotide transhydrogenases may be divided into two classes. One class is present in certain bacteria, and possibly in some plants, is an easily extractable, water-soluble enzyme. The other class is present in both certain bacteria and mitochondria, and is a firmly membrane-bound water-insoluble enzyme, functionally linked to the energy-transfer system of the bacterial or mitochondria1 membrane.