Pentose phosphates in nucleoside interconversion and catabolism

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.     

Purification and Properties of a Bacterial Enzyme Catalyzing Purine Nucleoside Transribosylation

An enzyme catalyzing the ribosyl group transfer from inosine to adenine was purified from Aerobacter cloacae No. 172–1 by means of ammonium sulfate fractionation and DEAE-cellulose and hydroxylapatite column chromatographies. A. cloacae was found to have much activities of this enzyme in its cell free extract. The enzyme activity was increased about 90-fold. By using the purified enzyme, some properties of the reaction were investigated. The enzyme was considerably stable and essentially required inorganic orthophosphate for the reaction. It was suggested that the enzyme might be a purine nucleoside phosphorylase and that the reaction might be a coupled reaction with ribose-1-phosphate as an intermediate. The enzyme could not transfer the ribosyl group between purine and pyrimidine bases.     

Phosphate inhibition of the conversion of ribose-1-phosphate--a product of purine nucleoside phosphorylase splitting in the phosphoribomutase reaction

Different phosphate concentrations were studied for their effect on the degradation of adenosine by rat liver homogenates. It is shown that phosphate considerably inhibits the phosphoribomutase reaction without a significant effect on adenosine deaminase and purine nucleoside phosphorylase activities, that leads to the ribose-1-phosphate accumulation and to the disturbance of nucleoside utilization in reactions of the pentose phosphate pathway. It is supposed that the inhibition of the phosphoribomutase reaction by phosphate is important for regulation of nucleoside and nucleotide formation in a cell.     

Metabolisms of Nucleosides in Bacteria

The mechanism of trans-N-ribosylation in Corynebacterium sepedonicum was investigated. Using the DEAE-cellulose colum chromatography, this enzyme activity was divided into two fractions. One cleaved uridine to uracil and ribose phosphate, and the other decomposed inosine into hypoxanthine and ribose phosphate, in the presence of inorganic phosphate. The ribose phosphate was isolated and crystallized.

Several analytical data indicated that the ribose phosphate was ribose-1-phosphate. These two enzyme fractions catalyzed the formation of nucleosides from ribose-1-phosphate and bases.

Most of bacteria, which had the activity to transfer N-ribosyl group between purine and pyrimidine, could synthesize the nucleoside from base and ribose-1-phosphate.     

Microsequecing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts

Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.Abbreviations RPI ribose-5-phosphate isomerase - OPPP oxidative pentose phosphate pathway - CNBr cyanogen bromide - R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate     

Kinetic properties of Cellulomonas sp. purine nucleoside phosphorylase with typical and non-typical substrates: implications for the reaction mechanism

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m7Guo), with either nucleoside or phosphate (Pd) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of Pi, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

Ribose-5-phosphate biosynthesis in Methanocaldococcus jannaschii occurs in the absence of a pentose-phosphate pathway

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-2H2]- and [U-13C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of D-arabino-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the D-ara-bino-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from D-arabino-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.     

PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii.

A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.     

Purine nucleoside phosphorylase is associated with centrioles and basal bodies

We have localized a fraction of the enzyme, purine nucleoside phosphorylase (PNP), to the centrioles and basal bodies of mammalian, avian, and protozoan cells. Two completely independent methods were used, one based on the ultrastructural cytochemistry of the enzyme activity and one based on immunofluorescence microscopy using an antibody raised in rabbit against purified human PNP. PNP catalyzes the reversible conversion of purine nucleosides and inorganic phosphate to the corresponding purine bases and ribose-1-phosphate. Its partial localization to centrioles and basal bodies raises the possibility that purine compounds are involved in centriole replication and/or in the regulation of microtubule assembly in vivo. No centriolar PNP could be detected in primary skin fibroblast from two infants with severe immunodeficiency disease associated with the absence of soluble PNP. This raises the possibility that defects in centriole function may contribute to the impaired division and maturation of T lymphoid precursor in this inherited disorder. Initially, the immunofluorescence analyses were complicated by a residual centriole-binding antibody that persisted in immunoglobulins from immune animals after complete removal of anti-PNP by affinity chromatography. Binding was abolished by exposure of cells to sodium periodate, indicating that this (and possibly other) "spontaneous" anticentriole antibodies in rabbit serum may be directed against carbohydrates.     

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.     

KINETIC PROPERTIES OF CELLULOMONAS SP. PURINE NUCLEOSIDE PHOSPHORYLASE WITH TYPICAL AND NON-TYPICAL SUBSTRATES: IMPLICATIONS FOR THE REACTION MECHANISM

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m⁷Guo), with either nucleoside or phosphate (P_i) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of P_i, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

A New Synthesis of Hepialone [(R)-2-Ethyl-6-methyl-2,3-dihydro-4H-pyran-4-one], the Principal Component from Male Sex Scales of the Ghost Moth,Hepialus californicus B

Microorganisms that produce 5-methyluridine (ribothymidine) directly from purine nucleosides and thymine were screened from our stock cultures. Of the 400 strains tested, Erwinia carotovora AJ- 2992 was found to possess the most potent ability as to production of 5-methyluridine from guanosine and thymine. In the presence of intact cells of Er. carotovora AJ-2992 as the enzyme source, 222 mm 5-methyluridine was produced from 300 mm guanosine and 300 mm thymine at 60°C on 48 hr incubation. The enzymatic production of 5-methyluridine by Er. carotovora AJ-2992 was found to involve the following two successive reactions via ribose-1-phosphate as an intermediate: phosphorolysis of purine nucleosides to ribose-1-phosphate and purine bases by purine nucleoside phosphorylase, followed by condensation of ribose-1-phosphate and thymine into 5-methyluridine by pyrimidine nucleoside phosphorylase.     

Synthesis of deoxyribomononucleotides in Mollicutes: dependence on deoxyribose-1-phosphate and PPi.

Cell extracts of Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, and Mycoplasma gallisepticum S6 were examined for 37 cytoplasmic enzyme activities involved in the salvage and biosynthesis of purines. All of these organisms had adenine phosphoribosyltransferase activity (EC 2.4.2.7) and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8). All of these organisms had purine-nucleoside phosphorylase activity (EC 2.4.2.1) in the synthetic direction using ribose-1-phosphate (R-1-P) or deoxyribose-1-phosphate (dR-1-P); this activity generated ribonucleosides or deoxyribonucleosides, respectively. The pyrimidine nucleobase uracil could also be ribosylated by using either R-1-P or dR-1-P as a donor. The synthesis of deoxyribonucleosides from nucleobases and dR-1-P has been reported from only one other procaryote, Escherichia coli (L. A. Mason and J. O. Lampen, J. Biol. Chem. 193:539-547, 1951). The reverse of this phosphorylase reaction is more widely known, and we found such activity in all mollicutes studied. Some Acholeplasma species but not the Mycoplasma species can phosphorylate deoxyribonucleosides to deoxyribomononucleotides by a PPi-dependent deoxyribonucleoside kinase activity, which was first reported in this group for the ribose analogs (V. V. Tryon and J. D. Pollack, Int. J. Syst. Bacteriol. 35:497-501, 1985). This is the first report of PPi-dependent purine deoxyribonucleoside kinase activity. An ATP-dependent purine deoxyribonucleoside kinase activity is known only in salmon milt extracts (H. L. A. Tarr, Can. J. Biochem. 42:1535-1545, 1964). Deoxyribomononucleotidase activity was also found in cytoplasmic extracts of these mollicutes. This is the first report of deoxyribomononucleotidase activity.     

Determination of plasma activities of purine nucleoside phosphorylase by high-performance liquid chromatography: estimates of nonparenchymal cell injury after porcine liver transplantation

An assay is described for measurement of purine nucleoside phosphorylase (PNP) in plasma by high-performance liquid chromatography (HPLC). A plasma sample was incubated with hypoxanthine and ribose-1-phosphate in phosphate-free medium at pH 7.4 to catalyse the production of inosine by plasmatic PNP. The reaction was stopped by addition of perchloric acid to inactivate the enzyme and to precipitate plasma proteins. After centrifugation and neutralization of the supernatant with NaOH the increase in the substrate inosine was determined by HPLC.

Plasma activities of PNP averaged 5.0 mU/ml before and 12.3 mU/ml (p < 0.001), 5 min after porcine liver transplantation. At the same time points, the plasma activities of the frequently used liver enzymes lactate dehydrogenase or alanine aminotransferase remained virtually unchanged. Thus, plasmatic activities of PNP may be a suitable and early indicator of ischemic alterations to the graft in vivo.     

Purine and pyrimidine salvage in whole rat brain. Utilization of ATP-derived ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate generated in experiments with dialyzed cell-free extracts.

The object of this work stems from our previous studies on the mechanisms responsible of ribose-1-phosphate- and 5-phosphoribosyl-1-pyrophosphate-mediated nucleobase salvage and 5-fluorouracil activation in rat brain (Mascia, L., Cappiello M., Cherri, S., and Ipata, P. L. (2000) Biochim. Biophys. Acta 1474, 70-74; Mascia, L., Cotrufo, T., Cappiello, M., and Ipata, P. L. (1999) Biochim. Biophys. Acta 1472, 93-98). Here we show that when ATP at "physiological concentration" is added to dialyzed extracts of rat brain in the presence of natural nucleobases or 5-fluorouracil, adenine-, hypoxanthine-, guanine-, uracil-, and 5-fluorouracil-ribonucleotides are synthesized. The molecular mechanism of this peculiar nucleotide synthesis relies on the capacity of rat brain to salvage purine and pyrimidine bases by deriving ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate from ATP even in the absence of added pentose or pentose phosphates. The levels of the two sugar phosphates formed are compatible with those of synthesized nucleotides. We propose that the ATP-mediated 5-phosphoribosyl-1-pyrophosphate synthesis occurs through the action of purine nucleoside phosphorylase, phosphopentomutase, and 5-phosphoribosyl-1-pyrophosphate synthetase. Furthering our previous observations on the effect of ATP in the 5-phosphoribosyl-1-pyrophosphate-mediated 5-fluorouracil activation in rat liver (Mascia, L., and Ipata, P. L. (2001) Biochem. Pharmacol. 62, 213-218), we now show that the ratio [5-phosphoribosyl-1-pyrophosphate]/[ATP] plays a major role in modulating adenine salvage in rat brain. On the basis of our in vitro results, we suggest that massive ATP degradation, as it occurs in brain during ischemia, might lead to an increase of the intracellular 5-phosphoribosyl-1-pyrophosphate and ribose-1-phosphate pools, to be utilized for nucleotide resynthesis during reperfusion.     

The purine nucleoside phosphorylase from Trichomonas vaginalis is a homologue of the bacterial enzyme

Trichomonas vaginalis is a parasitic protozoan and the causative agent of trichomoniasis. Its primary purine salvage system, consisting of a purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase, presents potential targets for designing selective inhibitors as antitrichomonial drugs because of lack of de novo synthesis of purine nucleotides in this organism. cDNA encoding T. vaginalis PNP was isolated by complementation of an Escherichia coli strain deficient in PNP and expressed, and the recombinant enzyme was purified to apparent homogeneity. It bears only 28% sequence identity with that of human PNP but 57% identity with the E. coli enzyme. Gel filtration showed the enzyme in a hexameric form, similar to the bacterial PNPs. Steady-state kinetic analysis of T. vaginalis PNP-catalyzed reactions gave K(m)'s of 31.5, 59.7, and 6.1 microM for inosine, guanosine, and adenosine in the nucleosidase reaction and 45.6, 35.9, and 12.3 microM for hypoxanthine, guanine, and adenine in the direction of nucleoside synthesis. This substrate specificity appears to be similar to that of bacterial PNPs. The catalytic efficiency of this enzyme with adenine as substrate is 58-fold higher than that with either hypoxanthine or guanine, representing a distinct disparity with the mammalian PNPs, which have negligible activity with either adenine or adenosine. The kinetic mechanism of T. vaginalis PNP-catalyzed reactions, determined by product inhibition and equilibrium isotope exchange, was by random binding of substrates (purine base and ribose 1-phosphate) with ordered release of the purine nucleoside first, followed by inorganic phosphate. Formycin A, an analogue of adenosine known as an inhibitor of E. coli PNP without any effect on mammalian PNPs, was shown to inhibit T. vaginalis PNP with a K(is) of 2.3 microM by competing with adenosine. T. vaginalis PNP thus belongs to the family of bacterial PNPs and constitutes a target for antitrichomonial chemotherapy.     

The effect of ribose 5-phosphate and 5-phosphoribosyl-1-pyrophosphate availability on de novo synthesis of purine nucleotides in rat liver slices.

The effect of increasing cellular ribose 5-phosphate (ribose-5-P) availability by methylene blue-induced acceleration of the oxidative pentose phosphate pathway on the rate of 5-phosphoribosyl-1-pyrophosphate (P-ribose-PP) generation, was studied in slices of rat liver at varying Pi concentration. It was found that at Pi concentration prevailing in the tissue of extracellular physiological Pi concentration, ribose-5-P availability is saturating for P-ribose-PP generation, as gauged by the rate of adenine incorporation into tissue nucleotides. The effect of altering P-ribose-PP availability on the rate of de novo purine production gauged by the rate of formate incorporation into purines, was also studied. It was found that the physiological P-ribose-PP concentration in rat liver tissue is limiting for purine synthesis de novo. Depletion of cellular P-ribose-PP, achieved by increase of P-ribose-PP consumption, decelerated purine synthesis, while increase of P-ribose-PP availability, achieved by activation of P-ribose-PP synthetase occurring at elevated Pi concentration, resulted in acceleration of purine synthesis.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.     

Some inhibitors of purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes reversible phosphorolysis of purine deoxy- and ribonucleosides with formation (d)Rib-1-P and corresponding bases. PNP plays a leading role in the cell metabolism of nucleosides and nucleotides, as well as in maintaining the immune status of an organism. The major aim of the majority of studies on the PNP is the detection of highly effective inhibitors of this enzyme, derivatives of purine nucleosides used in medicine as immunosuppressors, which are essential for creating selective T-cell immunodeficiency in a human body for organ and tissue transplantation. The present work is devoted to the study of the effects of some synthetic derivatives of purine nucleosides on activity of highly purified PNP from rabbit spleen and also from human healthy and tumor tissues of lung and kidneys. Purine nucleoside analogues modified at various positions of both the heterocyclic base and carbohydrate residues have been investigated. Several compounds, including 8-mercapto-acyclovir, 8-bromo-9-(3,4-hydroxybutyl)guanine, which demonstrated potent PNP inhibition, could be offered for subsequent study as immunosuppressors during organ and tissue transplantation.     

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.