Purine metabolism in human lymphocytes.

In peripheral human blood lymphocytes the uptake and metabolism of adenine, guanine, and hypoxanthine was investigated. This was achieved by incubation of purified lymphocytes with 14C-purine bases, separation of cells from the incubation medium by a rapid filtration technique, and subsequent separation of the acid soluble material by thin-layer chromatography. No perferential uptake for one of the purine bases was observed. In all cases only traces of 14C-purine bases not added originally and labeled nucleosides could be demonstrated. Approximately 2/3 of adenine and 1/2 of guanine or hypoxanthine were converted to nucleotides. Separation of formed nucleotides showed that adenine and guanine were metabolized mainly to their corresponding nucleotides; hypoxanthine was converted to a considerable amount to adenine nucleotides and only to a small proportion into its own nucleotides. These results demonstrate the predomonance of adenine nucleotide formation in normal human lymphocytes.     

Purine nucleoside phosphorylase in lymphocytes of various mammalian species

Synopsis The distribution of purine nucleoside phosphorylase has been assessed by light and electron microscopy in peripheral lymphocytes of man, the rabbit, rat, mouse, guinea-pig, pig and dog. The enzyme activity was detected in the cytosol of the majority of lymphocytes in all species. The amount of reaction product was high in the rabbit, man, guinea-pig and dog, moderate in the rat and very low in the pig and mouse. Other blood cell types are reactive as well, although there is a variation between species. A possible relationship of purine nucleoside phosphorylase with particular subpopulations of lymphocytes is suggested.     

Purine nucleoside kinases in human T- and B-lymphoblasts

Purine nucleoside kinases in human B- and T-lymphoblasts were fractionated by DEAE-cellulose chromatography. Human B-lymphoblast cell extracts showed three peaks of nucleoside kinase activities, adenosine kinase (EC 2.7.1.20), deoxyguanosine kinase and deoxycytidine kinase (EC 2.7.1.74). However, T-lymphoblast cell extracts showed a nucleoside kinase activity which phosphorylates deoxycytidine, deoxyadenosine and deoxyguanosine, similar to deoxycytidine kinase, in addition to the three nucleoside kinases. The Km values of T-lymphoblast-specific nucleoside kinase for deoxyadenosine and deoxyguanosine, 15 and 26 microM, respectively, were smaller than those of deoxycytidine kinase, 150 and 330 microM, respectively. Deoxyadenosine phosphorylation by deoxycytidine kinase was strongly inhibited by dCTP, but the phosphorylation by T-lymphoblast-specific nucleoside kinase was only weakly inhibited by dCTP. Deoxyadenosine phosphorylating activity in B-lymphoblast extracts was more distinctly inhibited by dCTP than that in T-lymphoblast extracts.     

Purine nucleoside phosphorylase from bovine liver.

1. Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, E.C. 2.4.2.1) from liver of cattle, Bos taurus, was purified to homogeneity. Some properties of the enzymes from three different bovine tissues were compared and discussed. 2. The enzyme has a molecular weight of 83,000, a sedimentation coefficient of 5.3 S, a Stokes' radius of 3.71 nm, a frictional ratio of 1.30 and a subunit molecular weight of 30,000. 3. Optimal pH for xanthosine degradation is around 5.5, whereas a broad pH activity profile for inosine degradation was observed between 5.0 and 7.5. Lineweaver-Burk plots curved downward at high concentrations of substrates, inosine, phosphate and arsenate.     

Purine nucleoside diphosphate regulation of yeast hexokinases.

The low activity state of hexokinase P-II, originally produced by Kosow and Rose by lowering the pH from 8 to 7 in certain sulfonated buffers, is not observed in Tris or imidazole buffers at pH 7 unless low concentrations of ADP or GDP are added. At pH values below 7 in imidazole buffer, partial inhibition occurs by protonation alone, and ADP or GDP causes further inhibition. As in the Kosow-Rose experiments, the enzyme in the low activity state can be activated either by excess ATP or by low concentrations of citrate, 3-phosphoglycerate and other metabolites. The inhibition by nucleoside diphosphates is greater at high glucose concentration. Hexokinase P-I is much less susceptible to regulation by nucleoside diphosphates or citrate, suggesting different physiological roles for the two isoenzymes.     

Purine nucleoside phosphorylase synthesis and turnover in human lymphoid cells

We have studied the turnover and synthesis of purine nucleoside phosphorylase by using a polyclonal rabbit antiserum to this protein. The turnover of purine nucleoside phosphorylase was studied in the B lymphoblast cell, WI-L2, by specific immunoprecipitation of [3H]leucine-labeled proteins. The half-lives for total protein and purine nucleoside phosphorylase were 14.5 and 14.1 hr, respectively. For cells cultured in the presence of inosine the half-life of purine nucleoside phosphorylase was reduced to 11.2 hr. The synthesis of purine nucleoside phosphorylase was analyzed during phytohemagglutinin-stimulated T cell transformation by pulse labeling cells with [35S]methionine. Purine nucleoside phosphorylase synthesis increased greater than 10-fold during the first 12 hr of transformation and continued to a maximum of 30-fold. The relative rate of purine nucleoside phosphorylase labeled to total proteins was 0.04% in unstimulated T cells and increased to 0.18% 12 hr after stimulation. These studies identify some preferential synthesis of purine nucleoside phosphorylase during the early stages of T cell transformation.     

Purine nucleoside phosphorylase from human erythrocytes: physiocochemical properties of the crystalline enzyme.

The major physicochemical properties of human erythrocytic purine nucleoside phosphorylase (PNPase) have been described. The molecular weight, estimated by ultracentrifugation, molecular sieving and sucrose density gradient centrifugation, ranged from 87 000 to 92 000. Other physical constants of erythrocytic PNPase were: sedimentation coefficent (s20, w), 5.4 S obtained by sedimentation analysis and 5.5 S by the sucrose density gradient procedure; Stokes radius, 38 A; calculated diffusion coefficient (D20, w), 5.7 X 10(-7) cm2 s-1; frictional ration, 1.29; and partial specific volume calculated from amino acid analysis, 0.73 cm3 g-1. The CD spectra of the human erythrocytic and bovine spleen PNPases were almost identical and indicated a very low alpha-helical content. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the molecular weight of the PNPase subunit is 30 000 +/- 500. These results corroborate earlier reports that the native enzyme is a homologous trimer. Comparative studies with crystalline bovine spleen PNPase confirmed that it is also a trimer but is somewhat smaller than the human erythrocytic enzyme with a molecular weight of about 86 000.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.     

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.