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• W2321692650 abstract "Peripheral mammalian lymphocytes grown in culture have low rates of RNA and protein synthesis and do not synthesize significant amounts of DNA. On addition of the mitogen phytohaemagglutinin, the cells undergo a series of changes including increases in RNA and protein synthesis, and culminating in DNA synthesis and cell division. DNA synthesis commences about 24h after stimulation. The present communication describes changes in the nuclear enzyme, poly(ADP-ribose) polymerase, in phytohaemagglutinin-stimulated pig lymphocytes. This chromatin-bound enzyme converts NAD+ into poly(ADP-ribose) with the elimination of nicotinamide, the product being covalently attached to chromosomal proteins (see, e.g., Nishizuka et af . , 1969). The reaction is absolutely dependent on the presence of DNA. The function of the polymerase and the polymer remain to be elucidated. Poly(ADP-ribose) polymerase activity was measured as the uptake of [3H]NAD+ into acid-insoluble material in isolated nuclei. The activities of the polymerase 24 and 48h after phytohaemagglutinin stimulation were respectively 1.5 and 2.9 times that in unstimulated cells. Thus approximately one-quarter of the increase in activity occurred in the first 24h after stimulation, before the onset of DNA synthesis, the major increase occurring concomitantly with DNA synthesis. The increase in activity of poly(ADP-ribose) polymerase could produce either more poly(ADP-ribose) chains of the same length (i.e. more initiation of new chains) or the same number of longer chains (more elongation). To distinguish between these alternatives, nuclei were incubated with [3H]NAD+and after various digestion procedures the polymer was hydrolysed at the phosphodiester linkages. This gives rise to AMP and 2'-(5-phosphoribosyl)-5'-AMP. The amounts of radioactivity in AMP and phosphoribosyl-AMP were measured after separation on Dowex formate, and the ratio of total radioactivity to radioactivity in AMP gives a measure of the chain length of the polymer (Fujimura & Sugimura, 1971). This length was found to be the same (about eight nucleotides) for unstimulated and 48 h-stimulated cells. Thus the increase in polymerase activity is associated with increased initiation of poly(ADP-ribose) chains, not with longer chains. The effects of culturing the lymphocytes after phytohaemagglutinin stimulation in the presence of various metabolic inhibitors were studied. Although hydroxyurea (an inhibitor of DNA synthesis) partially prevented the increase in poly(ADP-ribose) polymerase activity, fluorodeoxyuridine, at concentrations that completely prevented DNA synthesis, produced only a l0-15% inhibition of the increase in activity seen after 48h. Low doses of actinomycin D (5-25ng/ml), which prevented the increases in DNA and protein synthesis to varying degrees, inhibited the increasein poly(ADP4bose) polymerase activity to similar extents. Thus the stimulation of this activity seems to depend on general macromolecular synthesis, but to be independent of concomitant DNA synthesis." @default.
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• W2321692650 date "1973-05-01" @default.
• W2321692650 modified "2023-09-27" @default.
• W2321692650 title "Changes in Poly(Adenosine Diphosphate Ribose) Polymerase on Stimulation of Pig Lymphocytes with Phytohaemagglutinin" @default.
• W2321692650 doi "https://doi.org/10.1042/bst0010694" @default.
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