Gonzalez F A, Wang D J, Huang N N, Heppel L A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
Proc Natl Acad Sci U S A. 1990 Dec;87(24):9717-21. doi: 10.1073/pnas.87.24.9717.
3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, was used as a ligand for a P2Y purinoceptor (adenine nucleotide receptor) present in intact Swiss 3T3 and 3T6 cells and A-431 epidermoid carcinoma cells. Photolysis of serum-starved cells in the presence of 10-50 microM BzATP, followed by extensive washing to remove unincorporated BzATP, induced the release of arachidonic acid. A trace (less than 0.01%) of photoincorporated BzATP was as effective as when 50 microM BzATP or ATP was contained in the incubation medium during the assay. Photoincorporated BzATP also stimulated the production of prostaglandin E2 and the accumulation of cyclic AMP. In previous studies, we demonstrated that these three events are obligatory early steps in a pathway leading to DNA synthesis in the above cell lines. The evidence indicated that the purinoceptor activated by extracellular ATP or BzATP was linked to a pertussis toxin-sensitive GTP-binding protein. Consistent with these observations, we now find that pertussis toxin inhibits the effect of photoincorporated BzATP on arachidonic acid release. These results indicate that BzATP is an effective agonist for the P2Y purinoceptor concerned with stimulation of DNA synthesis in 3T3, 3T6, and A-431 cells. Furthermore, after photolysis it becomes irreversibly associated with intact cells and promotes the activation of early events required for DNA synthesis.
3'-O-(4-苯甲酰基)苯甲酰基-ATP（BzATP）是ATP的一种光亲和类似物，用作完整的瑞士3T3和3T6细胞以及A-431表皮癌细胞中存在的P2Y嘌呤受体（腺嘌呤核苷酸受体）的配体。在10 - 50微摩尔BzATP存在下对血清饥饿细胞进行光解，然后大量洗涤以去除未掺入的BzATP，可诱导花生四烯酸的释放。痕量（小于0.01%）的光掺入BzATP与测定期间孵育培养基中含有50微摩尔BzATP或ATP时一样有效。光掺入的BzATP还刺激前列腺素E2的产生和环磷酸腺苷的积累。在先前的研究中，我们证明这三个事件是上述细胞系中导致DNA合成的途径中必不可少的早期步骤。证据表明，由细胞外ATP或BzATP激活的嘌呤受体与百日咳毒素敏感的GTP结合蛋白相关。与这些观察结果一致，我们现在发现百日咳毒素抑制光掺入的BzATP对花生四烯酸释放的作用。这些结果表明，BzATP是与3T3、3T6和A-431细胞中DNA合成刺激相关的P2Y嘌呤受体的有效激动剂。此外，光解后它与完整细胞不可逆地结合，并促进DNA合成所需早期事件的激活。
