Johnson M A, Ahluwalia G, Connelly M C, Cooney D A, Broder S, Johns D G, Fridland A
Division of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
J Biol Chem. 1988 Oct 25;263(30):15354-7.
The pathways of 2',3'-dideoxyadenosine (ddAdo) metabolism, a selective inhibitor of the replication of human immunodeficiency virus, were investigated with use of the human T-lymphoid cell line CCRF-CEM which is deficient in either deoxycytidine kinase or adenosine kinase activity, or both. At an extracellular concentration of 10 microM, which blocks the cytopathic effect of human immunodeficiency virus in vitro, ddAdo was found to be metabolized to its mono-, di-, and triphosphates and to dideoxyinosine monophosphate (ddIMP). The metabolism of ddAdo in the kinase-deficient mutants was found to be unchanged by comparison with that in parental cells; however, the inhibition of ddAdo deamination to 2',3'-dideoxyinosine (ddIno) by the adenosine deaminase inhibitor, 2'-deoxycoformycin, reduced ddAdo nucleotide formation in deoxycytidine kinase-deficient, adenosine kinase-deficient, and doubly kinase-deficient mutants by 42, 54, and 80%, respectively. Incubation of the CCRF-CEM cells with 20 microM L-alanosine, an amino acid antagonist that inhibits purine biosynthesis at the level of adenylosuccinate/lyase synthetase, resulted in 80% inhibition in the accumulation of ddAdo nucleotides in both wild-type and kinase-deficient mutants and also increased ddIMP accumulation 2- to 3-fold. These findings indicate that ddAdo activation in human T-lymphoblasts can occur by three metabolic pathways: directly, by phosphorylation to ddAMP by the action of either deoxycytidine kinase or adenosine kinase and, indirectly, through deamination to ddIno with consequent phosphorylation of ddIno to ddIMP, and reamination to ddAMP in a reaction catalyzed by adenylosuccinate synthetase/lyase. However, in the absence of 2'-deoxycoformycin, the activation of ddAdo to ddATP in T-lymphoid cells is primarily a function of the indirect route.
利用人T淋巴细胞系CCRF - CEM研究了2',3'-二脱氧腺苷（ddAdo）的代谢途径，该细胞系缺乏脱氧胞苷激酶或腺苷激酶活性，或两者均缺乏。在细胞外浓度为10 microM时，可在体外阻断人类免疫缺陷病毒的细胞病变效应，此时发现ddAdo可代谢为其一磷酸、二磷酸和三磷酸形式以及二脱氧肌苷单磷酸（ddIMP）。与亲代细胞相比，激酶缺陷型突变体中ddAdo的代谢未发生变化；然而，腺苷脱氨酶抑制剂2'-脱氧助间型霉素对ddAdo脱氨生成2',3'-二脱氧肌苷（ddIno）的抑制作用，使脱氧胞苷激酶缺陷型、腺苷激酶缺陷型和双激酶缺陷型突变体中ddAdo核苷酸的形成分别减少了42%、54%和80%。用20 microM L - 丙氨酸（一种在腺苷琥珀酸/裂合酶合成酶水平抑制嘌呤生物合成的氨基酸拮抗剂）孵育人CCRF - CEM细胞，导致野生型和激酶缺陷型突变体中ddAdo核苷酸的积累均受到80%的抑制，同时ddIMP的积累增加了2至3倍。这些发现表明，人T淋巴母细胞中ddAdo的活化可通过三种代谢途径发生：直接途径是通过脱氧胞苷激酶或腺苷激酶的作用磷酸化为ddAMP；间接途径是先脱氨生成ddIno，随后ddIno磷酸化为ddIMP，再通过腺苷琥珀酸合成酶/裂合酶催化的反应重新氨化生成ddAMP。然而，在没有2'-脱氧助间型霉素的情况下，T淋巴细胞中ddAdo活化生成ddATP主要是间接途径的作用。
