THE CYTOLOGICAL AND PHYSIOLOGICAL MODE OF AP4A ACTION

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Diadenosine 5' ,5'''-P1,P4-tetraphosphate (Ap4A), a nucleotide formed in the back reaction of the aminoacid activation step, is ubiquitously present in bacterial and animal cells. The enzyme-catalyzed synthesis of Ap4A from ATP and certain aminoacyladenylates is enhanced by the presence of Zn2+. Several lines of evidence point to possible involvement of Ap4A in aspects of cellular proliferation, DNA replication, heat shock response and cell oxidation. An Ap4A binding protein, which is associated with the high molecular weight form of DNA polymerase alpha of calf thymus and HeLa cell and which binds Ap4A noncovalently with high affinity and great substrate specificity, has been isolated and characterized. Relatively high levels of Ap4A are present in blood platelets and these Ap4A pools are quantitatively released when platelets are induced to aggregate. Our working hypothesis is that Ap4A may be a signal for the induction of the heat shock or stress response in bacterial and animal cells as well as a possible signal for the induction of the host acute phase responses to a variety of infections, injury, and other physical insults in live animals. The physiological response of live animals could be mediated by increases in plasma Ap4A levels produced by platelets' aggregation, which is induced by the above-mentioned trauma. The heat shock or acute phase response is manifested by induction of the synthesis of groups of proteins (different proteins for each of these two phenomena) which presumably enable the individual cells or the whole animal to adapt to external conditions which affect cellular growth or survival. The proposed project is designed to: (1) elucidate the mechanism of cellular Ap4A synthesis in bacterial and animal cells; (2) study the mechanism of Ap4A interaction with the DNA replicative machinery in mammalian cells; (3) establish the physiological response of animals (rats, mice) to increase in serum Ap4A levels which may be produced by thrombocytopenia; and, (4) identify the role of Ap4A in tumor metastasis in light of the recently identified correlations between cellular Ap4A metabolism and the metastatic propensity of cells, and the well-established relationship between platelets' aggregation and tumor metastases in vivo. The long term objectives of these studies are the understanding of Ap4A action at the cellular and physiological levels. The significance of the proposed project is in establishing the mode and mechanism of action of a potential intracellular mediator which signals the cellular response to external conditions affecting growth or survival. (G)