2'',3''=Carbonates in the synthesis of uridine 5''-deoxy and 2'',5''-dideoxy derivatives.

Detritylation of 2',3'-O-carbonyl-5'-O-trityluridine (Ia) with ethereal hydrogen chloride affords 2',3'-O-carbonyluridine (Ib; 83%) which is converted by mesylation to the 5'-mesylcarbonate Ic (75%). Reaction of compound, Ic with tetrabutylammonium bromide in DMF affords the 5'-bromo carbonate Id (77%) which is reduced with tributyltin hydride to the 5'-deoxyuridine 2',3'-cyclic carbonate Ie (70%). When heated with imidazole, compound Ie affords the 2,2'-anhydro derivative IIa (76%) which is converted to the 2'-chloro derivative IIIa (88%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIa gives 2',5'-dideoxyuridine (IIIb; 68%). When heated with NaHCO3 in DMF, the 5'-bromo carbonate Id affords the anhydro bromo derivative IIb (50%) which is converted to the 2',5'-dichloro derivative IIIc (86%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIc affords the 2',5'-dideoxy derivative IIIb (59%). Alkaline hydrolysis of the 2,2'-anhydro derivative IIa affords the arabinosyl derivative IVa which is converted to the diacetyl derivative IVb (34%) by acetylation. When refluxed in water, the 2',3'-cyclic carbonates Ib, Id, and Ie are hydrolysed to the parent nucleosides, namely, uridine (Va; 81%), 5'-bromo-5'-deoxyuridine (Vb; 78%), and 5'-deoxyuridine (Vc; 83%). Hydrolysis of carbonates Ib and Ie is accompanied by the formation of the 2,2'-anhydro derivatives IIc (10%) and IIa (5%) as by-products.     

Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485.

Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp. strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth. Both enzymes released acetic acid from chemically acetylated larch xylan. Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II). The isoelectric points were 4.2 and 4.3, respectively. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively. Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively. At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.     

Acyloxy neighboring-group participation in the acid-catalyzed cleavage of methyl 2,3-anhydro-beta-D-ribofuranoside.

The reaction of methyl 2,3-anhydro-beta-D-ribofuranoside with hydrogen bromide in an acetic acid-acetic anhydride solution leads to the formation of methyl 2,3-di-O-acetyl-5-bromo-5-deoxy-alpha,beta-D-xylofuranoside. Similar treatment of methyl 2,3-anhydro-5-O-benzoyl-beta-D-robofuranoside provided methyl 2-O-acetyl-3-O-benzoyl-5-bromo-5-deoxy-alpha,beta-D-xylofuranosides. The position of halogen substitution was ascertained by hydrogenolysis to the resultant 5-deoxy sugars, which were characterized by their n.m.r. spectra. Confirmation of the structural assignment for methyl 2-O-acetyl-3-O-benzoyl-5-deoxy-alpha,beta-D-xylofuranoside was obtained by synthesis from 1,2-O-isopropylidene-alpha-D-xylofuranose. The formation of the 5-bromo derivatives under the reported conditions probably occurred through the intermediacy of the 3,5-acyloxonium ions. Similar conversions were observed when the starting compound was treated with hydrogen chloride, acetyl bromide, or acetyl chloride in acetic acid-acetic anhydride solutions.     

Chicken liver purine nucleoside phosphorylase activity dependency of the redox state of sulfhydryl groups

1. Double reciprocal plots (1/v vs 1/S) for nucleoside substrates of chicken liver purine nucleoside phosphorylase were non linear at high inosine or deoxyinosine concentrations (greater than 0.1 mM). The appearance of downward curvatures may be correlated with the oxidation of sulfhydryl groups of the enzyme. 2. 5,5'-Dithiobis-(2-nitrobenzoic acid) reacts with four sulfhydryl groups in the native enzyme, but upon denaturation with sodium dodecylsulfate six sulfhydryl groups react with this reagent. 3. Inosine, ribose-1-phosphate, hypoxanthine and orthophosphate partially protect sulfhydryl groups from the reaction with Ellman's reagent. 4. Inhibition of purine nucleoside phosphorylase by p-chloromercuribenzoate and 5,5'-dithiobis-(2-nitrobenzoic acid) follows a second order reaction kinetics.     

Synthesis and biological evaluation of 9-(5',5'-difluoro-5'-phosphonopentyl)guanine derivatives for PNP-inhibitors

9-(5',5'-Difluoro-5'-phosphonopentyl)guanine (DFPP-G) and its hypoxanthine analogue (DFPP-H) were modified by introducing a methyl group to all possible positions of the linker connecting a purine and difluoromethylenephosphonic acid moiety to evaluate the effects of the methyl group on inhibition against purine nucleoside phosphorylase. The methyl group on the linker affected the inhibition in a positional-dependent manner. Inhibitory potency of alpha-methyl and beta-methyl-substituted analogues of DFPP-H increased by about 600- to 1000-fold upon converting to cyclopropane nucleotide analogue (+/-)-4.     

5' terminal nucleotide sequences of the messenger RNA's of Dictyostelium discoideum.

As in the mRNA from all other eucaryotic cells examined, the 5' nucleotide in messenger RNA from Dictyostelium discoideum is linked by a 5'-5' triphosphate bridge to the unusual nucleoside 7-methyl guanosine. In mammalian cellular mRNA, the 5' terminal sequences have the general formula m7GpppXmpYp(m), where X and Y can be either purine or pyrimidine nucleotides and Y, as well as X, may contain a 2'0-methylated ribose. Although at least 32 5' terminal sequences are possible in cellular mRNA, only four are present in Dictyostelum mRNA. They are (I) m7GppppAp (65%); (II) m7GpppGp (10%); (III) m7GpppAmpAp (10%); (IV m7GpppAp (65%); (II) m7gpppGp (10%); (III) m7GpppAmpAp (10%); (IV) m7GpppAmpUp (10%). Sequences I and II are simpler than those previously reported for mammalian cells because they lack 2'0-methylated nucleosides. Another difference is that in all Dictyostelium mRNAs. the nucleoside X is a purine. The nucleoside 6-methyl adenosine which is found internal to the 5' end of the mRNA of mammalian cells is not detectable in Dictyostelium mRNA. Thus neither 2'0-methylated nucleotides nor 6-methyl adenosine can represent sites for processing of a primary nuclear transript to yield mRNA.     

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12.

A xanthosine-inducible enzyme, inosine-guanosine phosphorylase, has been partially purified from a strain of Escherichia coli K-12 lacking the deo-encoded purine nucleoside phosphorylase. Inosine-guanosine phosphorylase had a particle weight of 180 kilodaltons and was rapidly inactivated by p-chloromercuriphenylsulfonic acid (p-CMB). The enzyme was not protected from inactivation by inosine (Ino), 2'-deoxyinosine (dIno), hypoxanthine (Hyp), Pi, or alpha-D-ribose-1-phosphate (Rib-1-P). Incubating the inactive enzyme with dithiothreitol restored the catalytic activity. Reaction with p-CMB did not affect the particle weight. Inosine-guanosine phosphorylase was more sensitive to thermal inactivation than purine nucleoside phosphorylase. The half-life determined at 45 degrees C between pH 5 and 8 was 5 to 9 min. Phosphate (20 mM) stabilized the enzyme to thermal inactivation, while Ino (1 mM), dIno (1 mM), xanthosine (Xao) (1 mM), Rib-1-P (2 mM), or Hyp (0.05 mM) had no effect. However, Hyp at 1 mM did stabilize the enzyme. In addition, the combination of Pi (20 mM) and Hyp (0.05 mM) stabilized this enzyme to a greater extent than did Pi alone. Apparent activation energies of 11.5 kcal/mol and 7.9 kcal/mol were determined in the phosphorolytic and synthetic direction, respectively. The pH dependence of Ino cleavage or synthesis did not vary between 6 and 8. The substrate specificity, listed in decreasing order of efficiency (V/Km), was: 2'-deoxyguanosine, dIno, guanosine, Xao, Ino, 5'-dIno, and 2',3'-dideoxyinosine. Inosine-guanosine phosphorylase differed from the deo operon-encoded purine nucleoside phosphorylase in that neither adenosine, 2'-deoxyadenosine, nor hypoxanthine arabinoside were substrates or potent inhibitors. Moreover, the E. coli inosine-guanosine phosphorylase was antigenically distinct from the purine nucleoside phosphorylase since it did not react with any of 14 monoclonal antisera or a polyvalent antiserum raised against deo-encoded purine nucleoside phosphorylase.     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

NADH binding to cytochrome b5 reductase blocks the acetylation of lysine 110

Lysine residues outside of the NADH-binding site in the soluble catalytic fragment of cytochrome b5 reductase were modified with ethyl acetimidate and acetic anhydride while the binding site was protected by formation of the stable oxidized nucleotide-reduced flavoprotein complex. This treatment had a minimal effect on enzyme activity; the turnover number with potassium ferricyanide was 45,300 in the native reductase and 39,200 in the derivative. Subsequent reaction with [3H]acetic anhydride after the removal of NADH resulted in the loss of 91% of the enzyme activity and the incorporation of 1.9 eq of acetyl groups into the protein. Treatment with 1 M hydroxylamine at pH 13 indicated that only lysine residues were acetylated, and fragmentation of the derivative with cyanogen bromide and subfragmentation with trypsin and chymotrypsin demonstrated that only Lys110 was labeled at high specific activity, with a stoichiometry of 0.83 acetyl groups/mol, in good agreement with the loss of enzyme activity observed. The remaining label was distributed at low levels among four or more additional lysine residues. These results demonstrate that only Lys110 is specifically protected by NADH and is therefore the residue which provides the epsilon-amino group implicated in NADH binding in cytochrome b5 reductase.     

Enzymatic Synthesis of 5-Methyluridine from Adenosine and Thymine with High Efficiency

5-Methyluridine (5MU) was synthesized efficiently from adenosine, thymine, and phosphate by a combination of adenosine deaminase (ADA), purine nucleoside phosphorylase (PUNP), pyrimidine nucleoside phosphorylase (PYNP), and xanthine oxidase (XOD). Adenosine was converted into inosine first by ADA. 5MU and hypoxanthine were synthesized from inosine and thymine by PUNP and PYNP. The hypoxanthine formed was converted into urate via xanthine by XOD. After inosine was completely consumed, an equilibrium state, in which 5MU, thymine, ribose-1-phosphate, and phosphate were involved, was achieved. At the equilibrium state, the maximum yield of 5MU was obtained. The yield of 5MU was 74%, when the initial concentrations of adenosine, thymine, and phosphate were 5 mM each. On the other hand, in the absence of ADA or XOD the yield of 5MU was 1.8%. Several kinds of nucleosides were also synthesized with high yield by the same method.     

Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity.

Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 x 10(-4) s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 x 10(-3) s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E.hypoxanthine complex dissociates at a rate of 1.9 x 10(-6) s-1 at 4 degrees C and 1.3 x 10(-4) s-1 at 30 degrees C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 x 10(-12) M at 30 degrees C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E.hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 microM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 microM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.     

Toxoplasma gondii purine nucleoside phosphorylase biochemical characterization, inhibitor profiles, and comparison with the Plasmodium falciparum ortholog

Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development. The intracellular pathogen Toxoplasma gondii was therefore tested for sensitivity to immucillins, transition state analogs that exhibit high potency against PNP in the malaria parasite Plasmodium falciparum. Growth of wild-type T. gondii is unaffected by up to 10 microm immucillin-H (ImmH), but mutants lacking the (redundant) purine salvage pathway enzyme adenosine kinase are susceptible to the drug, with an IC50 of 23 nm. This effect is rescued by the reaction product hypoxanthine, but not the substrate inosine, indicating that ImmH acts via inhibition of T. gondii PNP. The primary amino acid sequence of TgPNP is >40% identical to PfPNP, and recombinant enzymes exhibit similar kinetic parameters for most substrates. Unlike the Plasmodium enzyme, however, TgPNP cannot utilize 5'-methylthio-inosine (MTI). Moreover, TgPNP is insensitive to methylthio-immucillin-H (MT-ImmH), which inhibits PfPNP with a Ki* of 2.7 nm. MTI arises through the deamination of methylthio-adenosine, a product of the polyamine biosynthetic pathway, and its further metabolism to hypoxanthine involves PfPNP in purine recycling (in addition to salvage). Remarkably, analysis of the recently completed T. gondii genome indicates that polyamine biosynthetic machinery is completely lacking in this species, obviating the need for TgPNP to metabolize MTI. Differences in purine and polyamine metabolic pathways among members of the phylum Apicomplexa and these parasites and their human hosts are likely to influence drug target selection strategies. Targeting T. gondii PNP alone is unlikely to be efficacious for treatment of toxoplasmosis.     

Hexameric purine nucleoside phosphorylase II from Escherichia coli K-12. Physico-chemical and catalytic properties and stabilization with substrates

Some properties of hexameric purine nucleoside phosphorylase II (EC 2.4.2.1) from Escherichia coli K-12 were studied. The enzyme obeys the Michaelis-Menten kinetics with respect to purine substrates (Km for inosine, deoxyinosine and hypoxanthine are equal to 492, 106 and 26.6 microM, respectively) and exhibits negative kinetic cooperativity towards phosphate and ribose-1-phosphate. The Hill coefficient is equal to approximately 0.5 for both substrates. Hexameric purine nucleoside phosphorylase II is not a metal-dependent enzyme; its activity is inhibited by Cu2+, Zn2+, Ni2+ and SO4(2-). The enzyme is the most stable at pH 6.0; it contains essential thiol groups. All substrates partly protect the enzyme against inactivation by 5.5'-dithiobis(2-nitrobenzoic acid) and heat-inactivation and, with the exception of phosphate-against inactivation by p-chloromercuribenzoate. Hypoxanthine, especially in combination with phosphate, afford the best protection against inactivation.     

Alpha-5-phosphoribosyl-1-pyrophosphate-independent salvage of purines in cultured Chinese hamster lung fibroblasts

A variant clone of cultured chinese hamster lung fibroblasts (V79), selected for resistance to 8-azaguanine (V79 azagrst), although lacking hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), is able to convert hypoxanthine into IMP via purine-nucleoside phosphorylase (EC 2.4.2.1) and nucleoside kinase. In addition to the phosphoribosylation pathway, we also present evidence for the occurrence of a kinase-mediated pathway of recovery of hypoxanthine in the wild-type cells. The lower rate of formation of IMP in the V79 azagrst cells, apparently correlated with the phosphorylation of the nucleoside, suggests possible differences in the catalytic and/or regulatory properties of nucleoside kinase in the two cell lines. This fact might be of particular relevance in evaluating the mechanisms of resistance to purine analogs displayed by several cell types.     

Hematopoiesis and the inosine modification in transfer RNA

Human promyelocytic leukemia (HL-60) cells were used to begin to evaluate the role in hematopoiesis of inosine biosynthesis in the tRNA anticodon wobble position; a reaction involving the enzymatic insertion of performed hypoxanthine. Dimethyl sulfoxide (DMSO) and hypoxanthine were found to induce the differentiation of HL-60 cells in a synergistic manner, and the induced differentiation was independent of changes in the purine catabolic enzymes adenosine deaminase and purine nucleoside phosphorylase. The short-term exposure of HL-60 cells to DMSO plus hypoxanthine resulted in enhanced leucine incorporation, and a model is presented showing how the inosine modification reaction in tRNA may be involved. A means by which hypoxanthine insertion into tRNA may modulate the synthesis of regulatory proteins (e.g., lymphokines and cell surface receptors) is also outlined.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.     

Characterizing Substrate Properties of Purine-Related Compounds with Purine Metabolism Enzymes for Enzymatic Peak-Shift HPLC Method

We have extended peak-shift method for measuring purine bases to make it suitable for other purine-related compounds. We optimized the reactions of the purine metabolism enzymes 5′-nucleotidase (EC 3.1.3.5), purine nucleoside phosphorylase (PNP) (EC 2.4.2.1), xanthine oxidase (XO) (EC 1.17.3.2), urate hydroxylase (EC 1.7.3.3), adenosine deaminase (ADA) (EC 3.5.4.4), and guanine deaminase (EC 3.5.4.3) by determining their substrate specificity and reaction kinetics. These enzymes eliminate the five purine base peaks (adenine, guanine, hypoxanthine, xanthine, and uric acid) and four nucleosides (adenosine, guanosine, inosine, and xanthosine). The bases and nucleosides can be identified and accurately quantified by comparing the chromatograms before and after treatment with the enzymes. Elimination of the individual purine compound peaks was complete in a few minutes. However, when there were multiple substrates, such as for XO, and when the metabolites were purine compounds, such as for PNP and ADA, it took longer to eliminate the peaks. The optimum reaction conditions for the peak-shift assay methods were an assay mixture containing the substrate (10 μL, 0.1 mg/mL), the combined enzyme solution (10 μL each, optimum concentration), and 50 mM sodium phosphate (up to 120 μL, pH 7.4). The mixture was incubated for 60 minutes at 37°C. This method should be suitable for determining the purine content of a variety of samples, without interference from impurities.     

The existence of a group translocation transport mechanism in animal cells: Uptake of the ribose moiety of inosine

After exposure to inosine, transport-competent plasma membrane vesicles isolated from SV -40-transformed Balb/c 3T3 cells accumulate intravesicular ribose 1-PO₄ at a concentration 200-fold greater than the extravesicular concentration. An analysis of the purine nucleoside phosphorylase activity distribution in various subcellular fractions, relative to other enzyme activities, indicated the presence of plasma membrane-associated purine nucleoside phosphorylase activity. The plasma membrane vesicles appear relatively impermeable to hypoxanthine. However, hypoxanthine, which is a competitive inhibitor of the transport reaction, is the only compound tested capable of mediating efflux of already accumulated ribose 1-PO₄. In addition, hypoxanthine does not result in the efflux of transported uridine which is accumulated in these membrane vesicles as uridine. Exogenous ribose 1-PO₄ neither results in counterflow nor does it inhibit the original uptake reaction. The following transport reaction is proposed: uptake occurs by group translocation, mediated by membrane-localized purine nuceloside phosphorylase. The data are consistent with sites for inosine and hypoxanthine being on the outer membrane surface whereas the ribose 1-PO₄ site is only on the inner surface.