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The ribonucleotides in the chorioallantoic membrane of 12-day-old chick embryos have been isolated by ion-exchange chromatography and characterized by their spectrophotometric and paper chromatographic properties. The following nucleotides were identified: adenosine-5' phosphate (AMP), uridine-5' phosphate (UMP), cytidine-5' phosphate (CMP), uridine-5' diphosphate galactose (UDPGal), uridine-5' diphosphate N-acetyl hexosamine (UDPNAHexosamine), guanosine-5' phosphate (GMP), cytidine-5' diphosphate (CDP), uridine-5' diphosphate (UDP), adenosine-51 diphosphate (ADP), guanosine-5' diphosphate (GDP), cytidine-5' triphosphate (CTP), uridine-5' triphosphate (DTP), adenosine-5' triphosphate (ATP), and guanosine-5' triphosphate (GTP). Quantitative determinations of these nucleotides were made on the basis of their ultraviolet absorption at 260 mμ. Similarly, concentrations of these nucleotides were estimated in 12-day-old chorioallantoic membranes after infection with vaccinia virus. Larger amounts of ribonucleoside-5' phosphates were present in the infected tissue at 4 and 12 hours after infection. The amounts of ribonucleoside-5' triphosphates were decreased. In tissues where, it is believed, synchronous infection occurred, the amounts of ribonucleoside-5' diphosphates and triphosphates were markedly lower than in controls after 12 hours of infection. Infection in the presence of tritium₌labelled thymidine showed that the amount of labelled thymidine-5' mono-, di-, and triphosphates had increased after 4 hours and that the amounts of these nucleotides subsequently decreased. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Cyclic nucleotides are important inhibitory regulators of platelet function. These second messengers are hydrolysed by cyclic 3' ,5'-nucleotide phosphodiesterases (PDEs). Three PDEs have been detected in human platelets: cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMP-binding, cGMP-selective phosphodiesterase (PDE5). This research investigates the contribution of PDE2 to the regulation of platelet cyclic nucleotide concentrations, and the effects that PDE2 activity has on the inhibition by cAMP and cGMP of platelet aggregation in response to thrombin or collagen. Increases in platelet cAMP were initiated by stimulation of adenylyl cyclase with prostacyclin (PGI₂), whereas the accumulation of cGMP was induced by nitroprusside (NP). The contributions of PDE2 to the hydrolysis of these cyclic nucleotides were evaluated using a novel inhibitor of the enzyme, 𝘦𝘳𝘺𝘵𝘩𝘳𝘰-9-(2-hydroxy-3-nonyl)adenine (EHNA). Before EHNA was used in experiments on platelet function, its effects on partially purified preparations of the three platelet PDEs were studied. These investigations demonstrated that EHNA is a selective and potent inhibitor of platelet PDE2, and indicated that this compound is a more effective inhibitor of cAMP hydrolysis in the presence than in the absence of cGMP. To measure changes in cyclic nucleotide concentrations, platelets were preincubated with [³H]adenine and ³H]guanine to label the metabolic nucleotide pools. NP caused large concentration-dependent increases in platelet [³H] cGMP levels, and this was associated with highly significant but much smaller increases in [³H] cAMP accumulation, which were optimal with 10 μM NP. Higher concentrations of NP had much less effect on platelet [³H] cAMP. A previous study had shown that the increases in platelet cAMP caused by NP were attributable to the inhibition of PDE3 by cGMP (Maurice and Haslam, 1990a), but the inhibitory component observed with high concentrations of NP had not been explained. The present research showed that the accumulation of cAMP and cGMP induced by high NP concentrations is enhanced by EHNA, and so provides the first demonstration that PDE2 activity restricts NP-induced cyclic nucleotide accumulation. To assess whether these changes in platelet cyclic nucleotide levels were important, platelet aggregation in response to thrombin and collagen was monitored. In these studies, EHNA markedly increased the inhibitory action of NP on platelet aggregation. All the effects of NP on cyclic nucleotide accumulation and on platelet aggregation were blocked by a guanylyl cyclase inhibitor, 1𝘏-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one, confirming that NP acts solely through activation of this enzyme and that the increases in cAMP are secondary to cGMP formation. However, experiments with the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine, which diminished the accumulation of cAMP but not that of cGMP, indicated that the inhibition of platelet aggregation is more closely correlated with the increases in cAMP than with those in cGMP. In experiments in which platelet PDE3 was selectively blocked by lixazinone, the accumulation of [³H]cAMP was greatly increased and a corresponding inhibition of thrombin-induced platelet aggregation was observed. Both of these effects were greatly diminished when PDE2 was stimulated by NP (or cGMP). This research demonstrates for the first time that activation of PDE2 by cGMP has marked effects on platelet function, restricting the inhibition of platelet aggregation by agents that increase platelet cAMP. To investigate the importance of PDE2 in regulating different platelet cAMP levels, the effects of EHNA were studied in the presence of 1 or 20 nM PGI₂. Whereas no significant increase in cAMP accumulation was caused by EHNA in the presence of 1 nM PGI₂, at the higher PGI₂ concentration a marked increase was detected when PDE2 was inhibited. NP potentiated the increase in cAMP seen with low PGI₂ but inhibited that seen with a high PGI₂ concentration, indicating a shift in the relative importance of PDE3 and PDE2 as platelet cAMP was increased. These studies show that in the presence of a high concentration of cAMP alone, or of regulatory cGMP, PDE2 makes a major contribution to the hydrolysis of platelet cAMP. Moreover, the results suggest that PDE2 inhibitors could be of value in the therapeutic modification of platelet responses. / Thesis / Master of Science (MSc)

Nucleotide pools were extracted and quantified from Pyr^+ and Pyr^- strains of P. aerucjinosa. Strains were grown in succinate minimal medium with and without pyrimidines, and nucleotides were extracted using trichloracetic acid (TCA; 6% w/v). The pyrimidine requirement was satisfied by uracil, uridine, cytosine or cytidine. Pyr^- mutants were starved for pyrimidines for two hours before nucleotide levels were measured. This starvation depleted the nucleotide pools which were restored to wild type levels by the addition of pyrimidines to the medium. When the pyrimidine analogue, 6-azauracil, known to inhibit OMP decarboxylase, was added to cultures of the wild type strain, the uridine and cytidine nucleotides were depleted to near zero. Thus, the nucleotide pool levels of Pseudomonas strains can be manipulated.

Asparate transcarbamoylase catalyzes the first committed step in the de novo synthesis of pyrmidine nucleotides UMP, UDP, UTP, and CTP. The archetype enzyme found in Escherichia coli (310 kDa) exhibits sigmodial substrate binding kinetics with positive control by ATP and negative control with CTP and UTP. The ATCase characterized in this study is from the extreme thermophilic Archaebacterium, Methanococcus jannaschii. The enzyme was very stable at elevated temperatures and possessed activity from 20 degrees Celsius to 90 degrees Celsius. M. Jannaschii ATCase retained 75% of its activity after incubation at 100 degrees Celsius for a period of 90 minutes. No sigmodial allosteric response to substrate for the enzyme was observed. Velocity substrate plots gave Michaelis-Menten (hyperbolic) kinetics. The Km for aspartate was 7 mM at 30 degrees Celsius and the KM for carbamoylphosphate was .125 mM. The enzyme from M. jannaschii had a broad pH response with an optimum above pH 9. Kinetic measurements were significantly affected by changes in pH and temperature. The enzyme catalyzed reaction had an energy of activation of 10,300 calories per mole. ATCase from M. jannaschii was partially purified. The enzyme was shown to have a molecular weight of 110,000 Da., with a subunit molecular weight of 37,000 Da. The enzyme was thus a trimer composed of three identical subunits. The enzyme did not possess any regulatory response and no evidence for a regulatory polypeptide was found, DNA from M. jannaschii did hybridize to probes corresponding to genes for both the catalytic and regulatory subunits from E. coli. Analysis of DNA sequences for the M. jannaschii ATCase genes showed that the gene for the catalytic subunits shares significant homology with the pyrB genes from E. coli, and maximum homology amongst known ATCase genes to pyrB from Bacillus. An unlinked gene homologous to E. coli pyrl encoding the regulatory subunit was identified, though its expression and true function remain uncharacterized.