Barile M, Valenti D, Hobbs G A, Abruzzese M F, Keilbaugh S A, Passarella S, Quagliariello E, Simpson M V
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi, Bari, Italy.
Biochem Pharmacol. 1994 Oct 7;48(7):1405-12. doi: 10.1016/0006-2952(94)90564-9.
Recent experiments from our laboratory have indicated that the inhibitory effect of 3'-azido-3'-deoxythymidine (AZT) on oxidative phosphorylation may occur directly, in addition to being brought about by its inhibition of mtDNA replication. We report here studies on the effect of AZT on adenylate kinase, an enzyme crucial to oxidative phosphorylation. AZT decreased the aromatic residues fluorescence of rabbit muscle adenylate kinase, indicating binding of AZT to the enzyme. Of three other enzymes studied as controls, AZT bound only to those that possessed ATP/ADP binding sites. Up to concentrations of 15 microM, AZT was a more potent effector of fluorescence quenching than were ATP, ADP, AMP, and the AZT control, deoxythymidine. AZT strongly inhibited adenylate kinase in the direction of ATP synthesis (Ki, 8 microM), the inhibition being of the partial competitive type, whereas deoxythymidine inhibition, also partially competitive, was much weaker (Ki, 90 microM). When measured in the direction of ADP synthesis, AZT failed to demonstrate any inhibition at concentrations up to 10 microM. Experiments on isolated intact rat liver mitochondria with the enzyme activity measured in both directions confirmed the isolated enzyme results. Respiratory control by these mitochondria was not affected by AZT. The finding of AZT affinity for ATP/ADP binding sites may open new avenues of approach to the study of AZT toxicity.
我们实验室最近的实验表明，3'-叠氮基-3'-脱氧胸苷（AZT）对氧化磷酸化的抑制作用可能直接发生，此外还可通过其对线粒体DNA复制的抑制作用来实现。我们在此报告关于AZT对腺苷酸激酶（一种对氧化磷酸化至关重要的酶）影响的研究。AZT降低了兔肌肉腺苷酸激酶的芳香族残基荧光，表明AZT与该酶结合。在作为对照研究的其他三种酶中，AZT仅与那些具有ATP/ADP结合位点的酶结合。在浓度高达15 microM时，AZT比ATP、ADP、AMP以及AZT对照脱氧胸苷更有效地淬灭荧光。AZT在ATP合成方向上强烈抑制腺苷酸激酶（Ki，8 microM），这种抑制属于部分竞争性类型，而脱氧胸苷的抑制作用同样为部分竞争性，但要弱得多（Ki，90 microM）。当在ADP合成方向上进行测量时，AZT在浓度高达10 microM时未表现出任何抑制作用。对分离的完整大鼠肝线粒体进行的双向酶活性测量实验证实了分离酶实验的结果。这些线粒体的呼吸控制不受AZT影响。AZT对ATP/ADP结合位点具有亲和力这一发现可能为研究AZT毒性开辟新的途径。
