Call number: LD2668 .T4 1958 M88

The objective of this research was to study the pyrimidine salvage pathways of Rhizobium. Three approaches were used to define the pyrimidine salvage pathways operative in two species of Rhizobium, R. meliloti and R. leguminosarum . The first approach was to ascertain the pyrimidine bases and nucleosides that could satisfy the pyrimidine requirement of pyrimidine auxotrophs. Uracil, cytosine, uridine or cytidine all satisfied the absolute pyrimidine requirement. The second approach was to select for mutants resistant to 5-fluoropyrimidine analogues which block known steps in the interconversion of the pyrimidine bases and nucleosides. Mutants resistant to 5-fluorouracil lacked the enzyme uracil phosphoribosyltransferase (upp ) and could no longer use uracil to satisfy their pyrimidine requirement. Mutants resistant to 5-fluorocytosine, while remaining sensitive to 5- fluorouracil, lacked cytosine deaminase (cod) and thus could no longer use cytosine to satisfy their pyrimidine auxotrophy. The third approach used a reversed phase HPLC column to identify the products that accumulated when cytidine, uridine or cytosine was incubated with cell extracts of wild type and analogue resistant mutants of Rhizobium. When cytidine was incubated with cell extracts of Rhizobium wild type, uridine, uracil and cytosine were produced. This Indicated that Rhizobium had an active cytidine deaminase (cdd) and either uridine phosphorylase or uridine hydrolase. By dialyzing the extract and reincubating it with cytidine, uridine and uracil still appeared. This proved that it was a hydrolase ( nuh ) rather than a phosphorylase that degraded the nucleoside. Thus, Rhizobium was found to contain an active cytidine deaminase and cytosine deaminase with no uridine phosphorylase present. The nucleoside hydrolase was active with cytidine, uridine and to a far lesser extent with purines, adenosine and inosine. When high concentrations of cytidine were added to mutants devoid of hydrolase, cytosine was produced from cytidine - 5-monophosphate by the sequential action of uridine ( cytidine ) kinase and nucleoside monophosphate glycosylase. Both ft meliloti and ft leguminosarum had identical salvage pathways.

The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression control (Crc) protein was indirectly involved in pyr gene regulation, and shown to negatively regulate expression of PyrR at the posttranscriptional level.

Salvage of pyrimidine nucleosides and bases by S. griseus and the regulation of aspartate transcarbamoylase (ATCase) were studied. The velocity-substrate curve for S. griseus ATCase was hyperbolic for both aspartate and carbamoylphosphate. The enzyme activity was diminished in the presence of ATP, CTP, or UTP. The synthesis of ATCase was repressed in cells grown in the presence of exogenous uracil. The specific activity of cells grown with uracil was 43 percent of that for cells grown in minimal medium only. Maximal ATCase and dihydroorotase activities were found in the same column fraction after size-exclusion chromatography, suggesting that both activities could reside in the same polypeptide. The pyrimidine salvage enzymes cytosine deaminase and uridine phosphorylase were identified in S. griseus using HPLC reversed-phase chromatography.

In Chapter 1 a review of the literature concerning aspects of erythrocyte membrane transport and metabolism, and purine and pyrimidine metabolism is presented. The effects of pH, pO₂ and inorganic phosphate (Pi) on the uptake and metabolism of hypoxanthine by erythrocytes has been studied in Chapter 2. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low pO₂. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favouring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate (PRPP). Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3- bisphosphoglycerate (2,3-DPG). These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely PRPP synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue. In Chapter 3 the kinetics and mechanism of transport of orotate across the human erythrocyte membrane and the effect of pH and inorganic phosphate on its metabolism (in the erythrocyte) have been studied. It has been shown that orotate enters erythrocytes with non-saturable kinetics and with a capacity of 190 μmoles/1 packed cells/min at a concentration of 4-6 mmolar. The presence of competition for transport by a number of anions and the lack of competition by uridine is indicative of transport by a general anion transporter, with the ability for concentrative uptake in the absence of other external anions being compatible with transport via a ping-pong mechanism. Inhibition of transport by the specific band 3 inhibitors DIDS and CHCA confirm that transport is via the band 3 anion transporter. This explains the lack of significant uptake of orotate by most differentiated tissues which lack the intact band 3 protein. However, the demonstration of band 3 in rat hepatocytes (Cheng and Levy, 1980) provides a mechanism for the orotate transport which has been observed in liver (Handschumacher and Coleridge, 1979). Changes in pH and inorganic phosphate (Pi) concentrations have been shown to have marked effects on the relative quantities of metabolic products produced by the erythrocyte from orotate. There was an increase in orotate metabolised with increasing Pi, an effect augmented by lowering the pH, and most easily explained by the allosteric activation of PRPP synthetase by Pi. The increase in UTP levels with decreasing pH may be the consequence of both increased PRPP availability for the formation of uridine nucleotide from orotate, and decreased conversion of UMP to uridine by pyrimidine 5'-nucleotidase, which is known to be inhibited by phosphate. The accumulation of UDP sugars is optimal at a phosphate concentration of 10 mmolar, which is unexplained but would be compatible with an inhibitory effect of Pi on CTP synthetase. A PRPP wasting cycle at alkaline pH values is proposed to explain the apparent paradox where no PRPP was observed to accumulate in erythrocytes (Chapter 2) at pH values of 7.6 and above in the presence of 10 mmolar phosphate and no added hypoxanthine, yet the metabolism of orotate, which is a PRPP utilising reaction, at alkaline pH values was readily demonstrable here. This (apparent paradox) can be resolved if one assumes that even in the absence of added hypoxanthine and demonstrable intracellular IMP there are sufficient quantities of hypoxanthine and/or IMP to maintain a PRPP wasting cycle at alkaline pH values. The cycle is interrupted at acidic pH values as phosphate levels rise and inhibit 5'-nucleotidase, an effect augmented by the decreasing levels of 2,3-DPG which accompany decreasing pH. This wasting cycle has recently been confirmed by P. Berman (unpublished). The kinetics of orotate uptake by erythrocytes and its eventual release as uridine provides a role for the erythrocyte in the transport and distribution of pyrimidines to peripheral tissues. A model is proposed and involves the de novo production of orotate in the liver. In the next step erythrocytes take up the orotate secreted by the liver into the circulation, convert it into an intermediate buffer store of uridine nucleotides, whose distribution is a function of pH and phosphate concentration, and eventually release it as uridine, which is a readily available form of pyrimidine for utilisation by peripheral nucleated cells. The enhancement of uptake of labelled orotate into nucleic acids of cultured cells is demonstrated here. The degradative half of the cycle proposes that uracil and palanine are the predominant degradative forms of pyrimidines produced by peripheral cells, and their ultimate metabolic fate is complete catabolism in the liver to CO₂ and water. In the final chapter the possible role of the human erythrocyte in the prevention of reperfusion injury has been investigated. The development of a model of renal ischaemia in the rat is described. The ability of human erythrocytes, "primed" by preincubating in acid medium of high Pi concentration and low pO₂, to take up hypoxanthine in a concentrative manner when perfused through ischaemic rat kidney is demonstrated. Attempts to demonstrate improved survival and renal function in rats with "primed" human erythrocytes prior to reperfusion were, however, unsuccessful. It is further demonstrated that "unprimed" human erythrocytes, resident in ischaemic rat kidney for 3 hours, take up hypoxanthine and convert it to IMP. that erythrocytes could play a physiological prevention of reperfusion injury.

In this study, high-performance liquid chromatography (HPLC) was employed to detect and quantify pyrimidine salvage enzymes by monitoring the disappearance of substrates or formation of products.