Ribonucleotide Reductase reduces all four ribonucleoside diphosphates t o deoxynucleoside diphosphates and functions a s the major source of deoxyribonucleotides for DNA synthesis i n vivo. Ribonucleotide reductase i s cell cycle regulated and most of t h e increased activity during S phase results from a 6 t o 1 0 fold increase in M2 subunit activity. Hydroxyurea resistant cell lines have increased M 2 activity and M2 gene amplification but normal cell cycle regulation of ribonucleotide reductase activity. We investigated M2 specific mRNA content of cell cycle specific populations using a 1487 b p long mouse M2 cDNA t o probe northern dot blots o f total cellular RNA from wild type, cyclic AMP dependent protein kinase deficient, and two hydroxyurea resistant cell lines one with and one without CAMP dependent protein kinase activity. Five sequential cell cycle fractions were obtained by centrifugal elutriation. Hydroxyurea resistant cell lines had greater M2 specific RNA per mg than wild type though their cell cycle regulation appeared t o be largely t h e same. All cell types have low concentrations o f M2 specific RNA in very early G1, a dramatic increase i n late Gl/early S , a rapid decline in later S and finally a gradual rise in G 2 phase. These data suggest transcriptional regulation o f M 2 during the cell cycle i s a t least in part responsible for cell cycle variation i n ribonucleotide reductase activity. 3Anna Di Stefano, Maria Pizzichini, Germano Resconi, Enrico Marinello. University of Siena. Department of Biological Chemistry, Italy.Allantoin -the key compound in purine catabolism -is determined in biological systems using the Rimini's reaction, which involves treatment with 0.1 N alkali (10O0C) and formation of allantoic acid. 0.025 N HC1 (100°C). and production of urea and glyoxylic .~..rd; final determination of the latter compound as a dinitrophenylhydrazine derivative. The reaction is subject to several interferences. The purification of allantoin is also difficult and thus it cannot be analyzed after labelled precursor administration. We evaluated the allantoin content in rat tissue extracts using two procedures: (1) allantoinase treatment (instead of alkali), subsequently proceeding as described above, (2) NaOH and HC1 treatment, but a final determination of urea with urease and glutamate dehydrogenase. Results were lower compared with the conventional dinitrophenylhydrazine reaction. Precipitation of allantoin with Hg-acetate (followed by ion -exchange chromatography) guarantees excellent recovery of pure allantoin from tissue extracts. This has proved to be a simple procedure for determination of true allantoin content and of its labelling with isotopes for kinetic studies in different tissues. Chronic urate nephropathy in the past decades was a frequent cause of renal failure in gouty patients but this entity has become very rare in the recent years and in many centers of hemodialysis the registries of chronic renal failure populations do not list gouty nephropathy as a cause.Some author...
The C-8 substituted guanine nucleosides are a new class of immunologically active purine analogues. Naturally occurring guanosine is immunosuppressive to B-cells. 8-Mercaptoguanosine (8-SGuo) and 8-bromoguanosine (8-BrGuo) have polyclonal B-cell activating properties in murine lymphocytes. 8-Aminoguanosine (8-AH Guo) has no immunologic effects on cells in culture but is a potent inhibitor of the purine catabolic enzyme, purine nucleosids phosphoylase. This study examined the metabolic fate of guanosine and the C-8 substituted analogues in dialyzed human splenic cytosol. Required cofactors for the involved enzyme systems were added to the cytosol. The metabolism of the ribonucleoside substrate to other purine forms was monitored by reverse-phase and anion exchange HPLC at 15 minute intervals for 2 hours. Guanosine declines linearly over 90 minutes with linear accumulations in guanine, xanthine, GTP and GDP when incubated at 37'~ in the presence of PRPP. 8-SGuo and 8-BrCuo remain essentially non-metabolized to other forms after 120 minutes. 8-NH Guo is rapidly metaholized to 8-NH guanine and a minor peak of 8-NH GDP appearing after 60 minutes. These preliminary studies in human spleen demonstrate that 2 C-8 substituted purine analogues (8-SCuo and 8-BrGuo) that stimulate B-cell proliferation and differentiation are not substantially metabolized by cytoplasmic purine enzymes. Conversely guanosine is extensively metabolized to both higher (GTP and GDP) and lower (guanine and xanthine) forms. These findings may help define the molecular basis for both immune-suppressive and immune-potentiating effects of guanosine and its C-8 substituted analogues. A EEOEL OF GOUT NEPHWIPPIREI. B. T. m , R.
Trichomonas uaginalis, a pathogenic protozoa, i s ~ncapable of de novo purine synthesis and i s thus dependent on preformed purlnes Unltke other protozoa. T . uaginalis i s devotd of purine phosphoribosyltransfer act~vity but has both purine nucleoside phosphorylase (PNP) and purine nucleoside kinase act~vity. These enzymes comprise a potential route for the salvage of purlnes In this organism. Both enzymes have been purlfied and characterized.The purified PNP (mol. wt. 95.000) catalyzed the synthesis and cleavage of guanoslne, adenosine and inosine at maximal velocities (pmollminlmg) of 590:360:240 and 81:16:390 respectively. K, values ranged from 17-54 p M for these purine nucleosides and 21-25 p M for the purine bases. Initial velocity studies in both the synthetic and cleavage direction indicate a sequential mechanism for this enzyme.As a result of the extreme lability of the nucleoside kinase, only a limited purification was possible. The purified enzyme (mol. wt. 16.000) had a specific activity of 34 nmol of G M P forrnedlminlmg. I t was free of interfering activities and catalyzed the phosphorylation (K, pMIRel. V , , , ) of guanosine (11100).adenosine (20011 11) and inosine (20167). This enzyme appears to be the only purine ribonucleoside kinase activity in extracts of this organism.The finding that both of these enzymes catalyze reactions involving the common purine nucleosides, guanosine and adenosine, suggests that they may act as a coordinated set of enzyme activities to salvage purines and purine ribonucleosides. Uric acid binding to human plasma proteins has been studied in various physiological and paraphysiological conditions,including the following groups: I-a group of children aged 4-10 years; 2-a group of healthy males; 3-a group of subjects treated with estrogen preparations for prostatic cancer; 4-a group of regularly menstruating females with normal cycle; 5-a group of post-menopausal females; 6-a group of pregnant females; 7-a group of females treated with oral contraceptives; 8-a group of females treated with estrogen f reparations for post menopausal symptoms. The results obtained demonstrate a significant correlation between the increase of plasma estrogen and the increase of the uric acid binding to human plasma proteins. Procedures for the HPLC analysis of nucleosides and bases have generally been designed to fulfil specific requirements. No single method exists capable of separating all the purines and pyrimidines found in the biological fluids of patients in the different inherited disorders in a single run and within a reasonable time. Because of a need for such a system an appropriate method has been devised. EFFECTSThe system has been in continuous use for over two years and three to four thousand samples have been analysed. It has proved reliable and reproducible throughout the life of many columns. Problems encountered during this period have resulted from artifacts and errors inherent to different isolation/extraction procedures. Pitfalls frequently noted have in the m...
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