Regulation of purine metabolism in lymphocytes.

Three general questions regarding nucleosides and lymphocytes are discussed: (a) Why are so many measurements being made of adenosine deaminase activity, what do the results mean, and why is there still disagreement about some of the conclusions; (b) what do we understand about nucleosides and lymphocyte death; and (c) to what extent do we really understand nucleoside and nucleotide metabolism in lymphocytes? Experimental studies show that treatment of mice with deoxycoformycin, to produce accumulation of deoxyadenosine, leads to rapid thymus involution, elevated dATP concentrations in thymus and liver, and inhibition of adenosylhomocysteine hydrolase in these tissues. Deoxyguanosine inhibits the growth of mouse lymphoma L5178Y cells, and this toxicity is prevented by deoxycytidine plus adenine. In cells treated with deoxyguanosine, concentrations of both GTP and dGTP are elevated, and this is not affected by deoxycytidine. Adenine, however, reduces GTP concentrations to normal, and prevents most of the elevation in dGTP concentrations. Contrary to previous belief, it has been demonstrated that lymphocytes and nucleated bone marrow cells will synthesize purine nucleotides de novo if incubated in an appropriate medium; carbon dioxide is particularly important for this process.

Variation in erythrocyte purine metabolism among mouse strains.

Erythrocytes of five strains of mice had ATP concentrations of ca 2.7 mumol/ml packed cells, while those of CBA mice were 23% lower, and those of BALB/C mice were 40% lower. The ratio of the concentrations of ATP and GTP were ca 3.3 in four strains but greater than 27 in three other strains. When erythrocytes from different mouse strains were incubated with radioactive precursors, appreciable strain differences were found in the apparent activities of adenine and hypoxanthine-guanine phosphoribosyltransferase, adenosine kinase, adenosine deaminase, guanine deaminase and xanthine oxidase. The activities of adenosine deaminase and guanine deaminase in sera of mice of different strains also varied.

Relationships among purine nucleoside metabolism, adenosine triphosphate catabolism, and glycolysis in human erythrocytes.

In human erythrocytes incubated with both naturally occurring purine nucleosides and with a variety of purine nucleoside analogs, ATP catabolism was accelerated and lactate accumulation was increased. Tubercidin was a particularly potent inducer of ATP catabolism. In cells incubated with tubercidin, the major route of adenylate metabolism was deamination, whereas in cells incubated with deoxyglucose, the major pathway was dephosphorylation.

Guanosine triphosphate catabolism in human and rabbit erythrocytes: role of reductive deamination of guanylate to inosinate.

The reductive deamination of guanylate to inosinate was demonstrable but occurred at low rates in human and rabbit erythrocytes incubated in vitro with or without glucose. However, the process was considerably accelerated in erythrocytes incubated with deoxyglucose. In human erythrocytes incubated with deoxyglucose, deamination was the major pathway of catabolism of guanylate; little or no guanylate was dephosphorylated. In rabbit erythrocytes, guanylate was both deaminated and dephosphorylated. Inosinate formed from guanylate was metabolized only by dephosphorylation in human erythrocytes, but in rabbit erythrocytes, it was also converted to xanthylate.

Effects of ethidium and isometamidium on adenosine triphosphate catabolism and purine nucleotide synthesis in Ehrlich ascites tumor cells in vitro.

Ethidium and isometamidium induce the breakdown of intracellular adenosine triphosphate in Ehrlich ascites tumor cells incubated in vitro. Ethidium induces appreciable adenosine triphosphate breakdown only when cells are incubated without glucose, whereas isometamidium produces this effect both in the presence and absence of glucose. In cells treated with isometamidium, purine nucleoside monophosphates accumulate, whereas these are mostly dephosphorylated when ethidium is used. Both ethidium and isometamidium inhibit purine nucleotide synthesis and incorporation of precursors into nucleic acids, although the magnitudes of these effects varied with the precursor used. Isometamidium inhibited the conversion of inosinate to adenine and guanine nucleotides, and both compounds partially inhibited the accumulation of phosphoribosyl pyrophosphate.

Factors affecting inosinate synthesis and inosine triphosphate accumulation in human erythrocytes.

Measurements of rates of inosinate synthesis from radioactive hypoxanthine by human erythrocytes show a large degree of individual variation. Rates of inosinate synthesis also vary with the pH and phosphate concentration of the incubation medium. This may be due to changes in the rate of phosphoribosyl pyrophosphate synthesis, and the stimulatory effect of phosphate on this process seems to be more important than the inhibitory effect of 2,3-diphodphoglycerate. The rate of inosinate synthesis, and especially the extent of accumulation of inosine triphosphate, increase disproportionately with time of incubation up to at least 24 h. Storage of erythrocytes also tends to increase inosinate synthesis and inosine triphosphate accumulation.

Inhibitors of hypoxanthine metabolism in Ehrlich ascites tumor cells in vitro.

One hundred and sixty-one purine analogs and derivatives were tested for their ability to inhibit ten parameters of purine metabolism in Ehrlich ascites tumor cells incubated in vitro with radioactive hypoxanthine. Sixty-seven compounds were inhibitory against at least one parameter and 30 were inhibitory against two or more.

Relationships between nucleoside triphosphate pyrophosphohydrolase activity and inosine triphosphate accumulation in human erythrocytes.

The relationship between nucleoside triphosphate pyrophosphohydrolast (NTPH) (EC 3.6.1.19) activity in erythrocyte lysates and accumulation of radioactive inosine triphosphate (ITP) in human erythrocytes incubated in vitro with [14C]hypoxanthine, was studied in 93 humans. When ITP accumulation, expressed as percentage of total radioactive nucleotides, was plotted against NTPH specific activity, an inverse relationship was found to exist. A continous spectrum of NTPH specific activities and ITP accumulation values exists in the human population and the relationship between these two parameters follows the relationship of substrate concentration to enzyme activity predicted by Michaelis-Menten enzyme kinetics. One interpretation of these data is that the ITP concentration in human red blood cells is controlled by the degradation of ITP to IMP and pyrophosphate catalyzed by NTPH.