Biotransformation, by Enterobacter agglomerans, of pyrimidine acyclonucleosides to acyclonucleotides.

The ability of Enterobacter agglomerans to transform naturally occurring nucleosides into nucleotides has been utilized to transform newly synthesized pyrimidine acyclonucleosides into the corresponding acyclonucleotides. Unselected bacteria were used as the source of nucleoside phosphotransferase, the phosphate group donor being 4-nitrophenyl phosphate in the presence of Zn2+ ions. Optimal reaction conditions are outlined.     

Biotransformation,byEnterobacter agglomerans,of pyrimidine acyclonucleosides to acyclonucleotides

The ability ofEnterobacter agglomerans to transform naturally occurring nucleosides into nucleotides has been utilized to transform newly synthesized pyrimidine acyclonucleosides into the corresponding acyclonucleotides. Unselected bacteria were used as the source of nucleoside phosphotransferase, the phosphate group donor being 4-nitrophenyl phosphate in the presence of Zn²⁺ ions. Optimal reaction conditions are outlined.     

Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5′-nucleotidase

Human erythrocyte pyrimidine 5′-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.Received 25 March 2005; received after revision 3 May 2005; accepted 13 May 2005     

Detection of pyrimidine 5'-nucleotidase deficiency using 1H- or 31P-nuclear magnetic resonance

We describe here a further Japanese family with pyrimidine 5'-nucleotidase (P5'N) deficiency diagnosed using a nuclear magnetic resonance (NMR) spectrum, in Kumamoto prefecture where two families having the disease have been reported before. The specific spectra in 1H-NMR of P5'N deficient erythrocytes were due to three methyl protons of CDP-choline at 3.22 ppm and to H-2, H-8 and ribose-1' of pyrimidine nucleotide phosphate(s) in the lower fields (at 5.82 and 8.00 ppm). The other specificities in 31P-NMR spectra were due to CDP-choline, CDP-ethanolamine and UDP-glucose. Those spectra were not detected in other types of hemolytic anemia.     

Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product

Carbamoyl phosphate synthetase (CPS) catalyzes one of the most remarkable reactions ever described in biological chemistry, in which carbamoyl phosphate is produced from one molecule of bicarbonate, two molecules of Mg2+ ATP, and one molecule of either glutamine or ammonia. The carbamoyl phosphate so produced is utilized in the synthesis of arginine and pyrimidine nucleotides. It is also employed in the urea cycle in most terrestrial vertebrates. Due to its large size, its important metabolic role, and the fact that it is highly regulated, CPS has been the focus of intensive investigation for nearly 40 years. Numerous enzymological, biochemical, and biophysical studies by a variety of investigators have led to a quite detailed understanding of CPS. Perhaps one of the most significant advances on this topic within the last 2 years has been the successful X-ray crystallographic analysis of CPS from Escherichia coli. Quite unexpectedly, this structural investigation revealed that the three active sites on the protein are widely separated from one another. Furthermore, these active sites are connected by a molecular tunnel with a total length of approximately 100 A, suggesting that CPS utilizes this channel to facilitate the translocation of reaction intermediates from one site to another. In this review, we highlight the recent biochemical and X-ray crystallographic results that have led to a more complete understanding of this finely tuned instrument of catalysis.     

Antiproliferative effects of 4',5'-unsaturated adenine nucleosides on leukemia L1210 cells in vitro

Several 4',5'-unsaturated adenine nucleosides were shown to have antiproliferative activity against L1210 leukemia cells in vitro. The active nucleosides were cytotoxic to the L1210 cells as demonstrated by Trypan Blue uptake. The cytotoxicity was not induced by alterations in the ribonucleoside and deoxyribonucleoside triphosphate levels of the L1210 cells.     

Detection of pyrimidine 5′-nucleotidase deficiency using1H-or31P-nuclear magnetic resonance

Summary We describe here a further Japanese family with pyrimidine 5-nucleotidase (P5N) deficiency diagnosed using a nuclear magnetic resonance (NMR) spectrum, in Kumamoto prefecture where two families having the disease have been reported before. The specific spectra in¹H-NMR of P5N deficient erythrocytes were due to three methyl protons of CDP-choline at 3.22 ppm and to H-2, H-8 and ribose-1 of pyrimidine nucleotide phosphate(s) in the lower fields (at 5.82 and 8.00 ppm). The other specificities in³¹P-NMR spectra were due to CDP-choline, CDP-ethanolamine and UDP-glucose. Those spectra were not detected in other types of hemolytic anemia.     

Antiproliferative effects of 4′, 5′-unsaturated adenine nucleosides on leukemia L 1210 cells in vitro

Summary Several 4, 5-unsaturated adenine nucleosides were shown to have antiproliferative activity against L 1210 leukemia cells in vitro. The active nucleosides were cytotoxic to the L 1210 cells as demonstrated by Trypan Blue uptake. The cytotoxicity was not induced by alterations in the ribonucleoside and deoxyribonucleoside triphosphate levels of the L 1210 cells.     

Determination of erythrocyte pyrimidine 5′-nucleotidase activity by31P nuclear magnetic resonance: Comparison of normal controls and multiple sclerosis patients

Summary A technique to assay erythrocyte pyrimidine 5-nucleotidase activity in situ using³¹P nuclear magnetic resonance spectroscopy is presented. The assay is chemically specific, simple and applicable to untreated lysates. A comparison of enzyme levels in normal controls and in multiple sclerosis patients employing the assay yielded no significant differences between both groups. Difficulties encountered in the quantitative analysis of the assay using¹H-NMR spectroscopy are briefly discussed.     

Inhibition of the 3' to 5' exonuclease activity of the DNA polymerase I of Escherichia coli by deoxyribonucleotides

The 3' à 5' exonuclease activity of E. coli DNA-polymerase I is inhibited by nucleotides and deoxynucleotides at concentrations (< 1 mM) where polymerase activity is not affected. This inhibitory effect depends on the nature of the excised deoxynucleotide, excision of purines being much less inhibited than that of pyrimidines. It does not depend on the purine or pyrimidine nature of the inhibitor.     

Ein für Massenspektrometrie und Gaschromatographie geeignetes Argininderivat

Summary Arginine has been converted into a suitable pyrimidine derivative to permit its qualitative analysis in the presence of other amino acids by means of mass spectrometric and gas-chromatographic techniques.     

2-Methoxybenzoyl phosphate: a new substrate for continuous fluorimetric and spectrophotometric acyl phosphatase assays

A new aromatic acyl phosphate, 2-methoxybenzoyl phosphate, has been synthesized. The compound shows an intrinsic fluorescence; it displays an intense emission band at 390 nm upon excitation in the near UV region. This band practically disappears after hydrolysis of the product. On the other hand, the product displays differences in the near UV absorption spectra measured before and after hydrolysis. The at 301 nm is 2720 M^–1 cm^–1, a value that is 4.3-fold higher than that of benzoyl phosphate (the usual substrate for acylphosphatase assay) at 283 nm. The main kinetic parameters of three different acylphosphatase molecular forms (the muscular isoenzyme and two subtypes of the organ common isoenzyme) were determined using both benzoyl phosphate and 2-methoxybenzoyl phosphate as substrates, and then compared. These kinetic data and the UV absorption and fluorescence properties of 2-methoxybenzoyl phosphate sugest that this compound has better substrate features than benzoyl phosphate, and can be used for both high sensitivity continuous fluorimetric and UV absorption spectrophotometric assays of acylphosphatase.     

Biochemistry of frog ribonucleases

Frogs have unique pyrimidine base-specific RNases, with structures similar to those of turtle, iguana and chicken RNases. Among the four frog RNases discussed here, three from Rana pipiens, R. catesbeiana and R. japonica oocyte cells show anti-tumour activity, and the latter two show lectin activity towards sialic acid-rich glycoproteins. In this review, (i) we compare their unique primary structures with respect to the locations of their disulphide bridges, three-dimensional structure, base specificity and heat stability as compared with RNase A, and (ii) we summarize current knowledge about the mode of action of lectin and the antitumour activities of the three frog RNases.     

Early effects of growth hormone on blood and urinary inorganic phosphorus in the rat (author's transl)]

From the kinetic study of the effects of one single growth hormone (GH) injection on the phosphate metabolism, it appears that the influence of GH on the serum phosphate level is biphasic: a decrease is followed by an increase. Conversely GH leads to an early decrease of the urinary phosphate excretion.     

Apparent inhibition of erythrocyte glutamic oxaloacetic transaminase in subjects on large dosages of B-6

Summary An apparent inhibition of the erythrocyte glutamic oxaloacetic transaminase E-GOT system of Kishi and Folkers has been observed with erythrocytes from individuals on large amounts of B-6. Evidence is presented that this inhibition is an artefact due to excess pyridoxal phosphate reacting with 2,4,dinitrophenylhydrazine. Excess pyridoxal phosphate competes with pyruvic acid for the available 2,4,dinitrophenylhydrazine which results in a product that is not extractable into toluene, therefore, less chromophore is present as more pyridoxal phosphate is available.We would like to acknowledge the Tom and Marion Peters' Foundation for their support of this research.     

Adenosine uptake in pyrimidine 5'-nucleotidase deficient human erythrocytes via a high affinity transport system

Using a pulse-labeling technique, 14C-adenosine uptake into pyrimidine 5'-nucleotidase (P5N) deficient erythrocytes (RBC) was found to be impaired. The Lineweaver-Burk plot showed Km values of 2.0 X 10(-3) mM and 0.2 X 10(-3) mM for normal RBC and P5N deficient RBC, respectively. These results indicate that P5N is one of regulators of the adenosine transport system and/or is associated with adenosine carrier protein.     

The purine nucleoside cycle in cell-free extracts of rat brain: evidence for the occurrence of an inosine and a guanosine cycle with distinct metabolic roles

The purine nucleoside cycle is a cyclic pathway composed of three cytosolic enzymes, hypoxanthine-guanine phosphoribosyltransferase, IMP-GMP specific 5'-nucleotidase, and purine-nucleoside phosphorylase. It may be considered a 'futile cycle', whose net reaction is the hydrolysis of 5-phosphoribosyl-1-pyrophosphate to inorganic pyrophosphate and ribose 1-phosphate. The availability of a highly purified preparation of cytosolic 5'-nucleotidase prompted us to reconstitute the purine nucleoside cycle. Its kinetics were strikingly similar to those observed when dialyzed extracts of rat brain were used. Thus, when the cycle is started by addition of inorganic phospate (Pi) and hypoxanthine or inosine (the 'inosine cycle'), steady-state levels of the intermediates are observed and the cycle 'turns over' as far as 5-phosphoribosyl-1-pyrophosphate is being consumed. In the presence of ATP, which acts both as an activator of IMP-GMP-specific 5'-nucleotidase and as substrate of nucleoside mono- and di-phosphokinases, no IDP and ITP are formed. The inosine cycle is further favored by the extremely low xanthine oxidase activity. Evidence is presented that ribose 1-phosphate needed to salvage pyrimidine bases in rat brain may arise, at least in part, from the 5-phosphoribosyl-1-pyrophosphate hydrolysis as catalyzed by the inosine cycle, showing that it may function as a link between purine and pyrimidine salvage. When the cycle is started by addition of Pi and guanine (the 'guanosine cycle'), xanthine and xanthosine are formed, in addition to GMP and guanosine, showing that the guanosine cycle 'turns over' in conjunction with the recycling of ribose 1-phosphate for nucleoside interconversion. In the presence of ATP, GDP and GTP are also formed, and the velocity of the cycle is drastically reduced, suggesting that it might metabolically modulate the salvage synthesis of guanyl nucleotides.