Aldo S. Bagnara

Adenosine metabolism in human erythrocytes: A study of some factors which affect the metabolic fate of adenosine in intact red cells in vitro☆

Abstract Adenosine metabolism was studied in intact human erythrocytes over a range of adenosine and intracellular phosphate concentrations. The concentration of adenosine greatly influenced its partition between deamination via adenosine deaminase (EC 3.5.4.4.) and phosphorylation via adenosine kinase (EC 2.7.1.20). For example, for cells incubated in a medium containing 1 m m phosphate, a single addition of 40 μ m adenosine was largely deaminated (91% deaminated, 9% phosphorylated). Adenosine added to the incubation at lower concentration (0.5 μ m ) was largely phosphorylated (81% phosphorylated, 19% deaminated). This partition could be predicted from the known K m and V parameters of the kinase and deaminase reactions. Intracellular phosphate concentrations also affected the metabolism of adenosine, apparently by stimulating adenosine kinase activity in situ . The ratio of adenosine phosphorylated to adenosine deaminated was increased significantly by increases in the intracellular phosphate concentration. Thus, at 4 μ m adenosine, approximately 30% of the adenosine was phosphorylated when the intracellular phosphate concentration was 1 m m . However, when the intracellular phosphate concentration was increased to 7.5 m m , the proportion of 4 μ m adenosine phosphorylated increased to 70%. Our studies also revealed that the kinase was inhibited by high adenosine concentrations, especially when 50–100 μ m adenosine was added to the incubation.

Factors affecting the rate of purine ribonucleotide dephosphorylation in human erythrocytes

Purine ribonucleotide dephosphorylation was measured in intact human erythrocytes in vitro to evaluate those factors which might regulate this process in vivo. It was found that purine nucleotides which exist predominantly in the triphosphate form (e.g. ATP and GTP) are protected from dephosphorylation while those nucleotides normally present as the monophosphate (e.g. IMP) are susceptible to dephosphorylation. This point was emphasised by studying an individual whose erythrocytes accumulated ITP rather than IMP; erythrocytes from this individual has a more stable pool of inosine phosphates than did erythrocytes from normal individuals. The concentration of intracellular phosphate was also shown to affect the rate of dephosphorylation. The dephosphorylation of IMP was inhibited at intracellular phosphate concentrations above approx. 3 mM. AMP dephosphorylation (in cells whose AMP concentration was increased by incubating them in the presence of 2-deoxyglucose) was inhibited by phosphate more strongly than was found for IMP. In contrast, the dephosphorylation of GMP did not appear to be affected by phosphate concentration. High oxygen tension was a powerful stimulator of IMP dephosphorylation while low oxygen tension protected IMP from dephosphorylation. This finding shows that human erythrocytes are similar to those of other mammals in this regard and points to a possible physiological determinant of purine turnover in these cells.

Purine salvage and metabolism in Babesia bovis.

Studies of the incorporation of radio-labelled purine precursors into the erythrocytic forms ofBabesia bovis under tissue-culture conditions have confirmed the presence in the parasite of enzymatic activities responsible for the salvage of preformed purines. The results also revealed that the parasite was capable of a variety of nucleotide interconversions, such that exogenous hypoxanthine and adenosine were incorporated into both adenine and guanine nucleotides followed by the incorporation of these nucleotides into the adenine and guanine moieties of RNA and DNA. No evidence was found for salvage of preformed pyrimidines. Evidence was also obtained for the insertion of a parasite-specific nucleoside/nucleobase transporter into the membrane of the bovine (host) red cell. Thus, whereas normal (non-parasitised) bovine red cells are essentially incapable of transporting nucleosides across their membranes, the invasion of these cells byB. bovis introduces a transporter that can be inhibited by classic nucleoside transport inhibitors.

Characterisation and expression of the carbamate kinase gene from Giardia intestinalis.

The arginine dihydrolase pathway in Giardia intestinalis produces energy via the carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) reaction. Characterisation of the CBK gene from the Portland 1 strain indicated that it is located on either chromosome 3 or 4, does not appear to contain introns and is expressed in both the trophozoite and early cyst stages. Heterologous expression of CBK in Escherichia coli, using the pQE-30 expression system (QIAGEN), enabled a one-step purification of the recombinant enzyme via affinity chromatography. The expressed protein was identified by enzyme assay and mass spectrometry. The native and recombinant forms of the enzyme have similar physical properties and the recombinant enzyme appears to be active as the homodimer.

Mitochondrial function in Babesia bovis

A variety of anti-mitochondrial drugs that had previously been found to inhibit the growth of the malarial parasite Plasmodium falciparum were tested on Babesia bovis in vitro. Several of these drugs were found to be non-toxic towards B. bovis. However, those drugs that were found to inhibit babesial growth included compounds (shown in parentheses) that have the following putative mitochondrial targets in the parasite: ATP synthetase complex (rhodamine 123, oligomycin, Janus Green); ATP-ADP translocase (bongkrekic acid); electron transport (rotenone, n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), antimycin A); ubiquinone (CoQ) function (BW58C, menoctone); protein synthesis (tetracycline); and the proton pump (CCCP). We have also investigated the effects of some of these drugs on pyrimidine biosynthesis de novo by following the incorporation of [14C]bicarbonate into pyrimidine nucleotides and into the pyrimidine moieties of nucleic acids. The ubiquinone analogues BW58C and menoctone inhibited this pathway in the nM-microM range of concentrations. Inhibitors of electron transport (antimycin A and oligomycin) and an uncoupler (CCCP) were also effective inhibitors of pyrimidine biosynthesis de novo. We conclude that B. bovis has a functional mitochondrion that contributes significantly to pyrimidine biosynthesis de novo and to the overall energy metabolism of the parasite.

The toxicity of antifolates in Babesia bovis

A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [3H]hypoxanthine into the parasites nucleic acids. Inhibitors of folate synthesis (including 7-methylguanosine and several sulpha drugs) were without effect but several structural analogues of folate were toxic. The most potent folate analogues were the lipophilic compounds piritrexim and trimetrexate, each causing 50% inhibition of [3H]hypoxanthine incorporation (IC50) at a concentration of 2.9 nM; other classical anti-folates such as pyrimethamine, methotrexate and trimethoprim were at least 100-fold less effective with IC50 values of 1.2, 0.29 and 0.50 microM, respectively. From these results we conclude that B. bovis does not synthesize folate de novo under cell culture conditions. However, the toxic effects of piritrexim and trimetrexate suggest that dihydrofolate reductase (DHFR) activity is essential for the parasite, most probably because of the role of this enzyme in the synthesis of thymidine nucleotides via thymidylate synthase.

Evidence against the compartmentation of adenosine kinase and adenosine deaminase activities in human erythrocytes.

In most tissues, the metabolism of exogenous adenosine is initiated by either adenosine kinase (EC 2.7 .1.20) or adenosine deaminase (EC 3 5.4.4) activities. A general finding is that phosphorylation of adenosine is favored at low adenosine concentrations while deamination is favored at higher concentrations [l-4]. Several workers have suggested that this concentration-dependent metabolic fate of adenosine arises from the compartmentation of adenosine kinase activity adjacent to the membrane transport system for adenosine [5-71. In contrast, results obtained with human red cells incubated in the presence and absence of the nucleoside transport inhibitor p-nitrobenzylthioguanosine (2-amino-6 [(4-nitrobenzyl)thio]-9

Characterisation and Sequence Analysis of a Carbamate Kinase Gene from the Diplomonad Hexamita inflata1

3-D-ribofuranosylpurine) led Agarwal and Parks to conclude that adenosine deaminase activity may be closely associated with the nucleoside transporter on the red cell membrane [8]. The simple experiments reported here do not support these hypotheses of compartmentation of either adenosine kinase or adenosine deaminase in human red cells. Rather, our results indicate that the metabolic fate of adenosine in these cells is influenced mainly by the maximal activity and Km parameters for adenosine kinase and adenosine deaminase.

Sequences upstream and downstream from the glutamine-dependent carbamoyl phosphate synthetase-encoding gene from the protozoan Babesia bovis ☆

Abstract Hexamita inflata can derive energy from the degradation of arginine via the arginine dihydrolase pathway. Carbamate kinase catalyses the third enzymatic step of the pathway synthesising ATP from the catabolism of carbamyl phosphate. This study reports the identification and characterisation of a carbamate kinase gene from this free-living diplomonad, together with measurements of carbamate kinase enzyme activity in cell-free extracts and a preliminary analysis of the carbamate kinase mRNA by reverse-transcription polymerase chain reaction. Analysis of the carbamate kinase gene revealed the use of non-canonical codons for glutamine. Phylogenetic studies showed a consistent close relationship between carbamate kinase sequences of H. inflata and Giardia intestinalis.

Adenosine kinase from human erythrocytes: kinetic studies and characterization of adenosine binding sites

In the protozoan parasite, Babesia bovis, the glutamine-dependent carbamoyl phosphate synthetase-encoding gene (CPSII) contains contiguous amidotransferase- and synthetase-encoding sequences. Unlike the organisation in most eukaryotes, the gene is not fused with other genes encoding enzymes of pyrimidine biosynthesis de novo. The nucleotide sequences immediately upstream and downstream from the gene contain motifs which may be involved in regulating its expression.