Phosphorylation of 2',3'-dideoxyinosine by cytosolic 5'-nucleotidase of human lymphoid cells.
2',3'-Dideoxyinosine (ddlno) is a potent and selective inhibitor of human immunodeficiency virus in human lymphoid cells and monocytes/macrophages. Earlier studies [J. Biol. Chem. 263:15354 (1988)] showed that anabolism of ddlno in human lymphoid cells is mediated via an initial step of phosphorylation and subsequent amination to dideoxy-AMP via adenylosuccinate synthetase/lyase. Evidence was obtained that neither adenosine kinase nor deoxycytidine kinase is involved in the phosphorylation of this compound in human lymphoid cells. We now find that, in the presence of MgCl2, KCl, and inosine-5'-monophosphate as phosphate donor, purified cytosolic 5'-nucleotidase catalyzed the phosphorylation of ddlno. Although not phosphate donors, ATP, diadenosine tetraphosphate, and glycerate-2,3-bisphosphate stimulate this phosphorylation by the nucleotidase 4-5-fold. In addition to ddlno, the antiviral nucleoside analogs 2',3'-dideoxyguanosine and carbovir were substrates for this enzyme. The relative phosphorylation of these compounds varied with the concentration of the phosphate donor IMP. Approximate Km values of the nucleotidase for inosine, ddlno, dideoxyguanosine, and carbovir were, respectively, 3.4, 0.5, 0.9, and 1.7 mM. Although the substrate activity of dideoxynucleosides is inefficient, it appears likely that this nucleotidase is responsible for the metabolism of these compounds to their active nucleotides, yielding antiviral activity in human lymphoid cells.
Johnson
M A
MA
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Fridland
A
A
D015720:5'-Nucleotidase; D000255:Adenosine Triphosphate / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D016049:Didanosine; D015224:Dideoxynucleosides / Q000378:metabolism*; D054306:Dideoxynucleotides; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D009708:Nucleotidases / Q000378:metabolism*; D010766:Phosphorylation; D013601:T-Lymphocytes / Q000378:metabolism*
D003838:Deoxyadenine Nucleotides; D015224:Dideoxynucleosides; D054306:Dideoxynucleotides; D015226:Dinucleoside Phosphates; D007291:Inosine Monophosphate; C037773:2',3'-dideoxyadenosine triphosphate; C020733:diadenosine tetraphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D015720:5'-Nucleotidase; D016049:Didanosine