Identification of cyclic ADP-ribose-binding proteins by photoaffinity labeling.
Identification of cyclic ADP-ribose-binding proteins by photoaffinity labeling.
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DOI：
10.1016/s0021-9258(19)74367-1
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发表时间：
1993-12
期刊：
影响因子：
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通讯作者：
T. Walseth;R. Aarhus;J. Kerr;Hon Cheung Lee
中科院分区：
文献类型：
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作者：
T. Walseth;R. Aarhus;J. Kerr;Hon Cheung Lee
我们合成了8-叠氮环ADP-核糖（8 N3-cADPR）和[32 P]8-叠氮环ADP-核糖（[32 P] 8 N3-cADPR），以表征海胆卵匀浆中的环ADP-核糖-（cADPR）结合位点。8 N3-cADPR是cADPR的拮抗剂，因为它不诱导卵微粒体的Ca 2+释放，但确实抑制cADPR这样做的能力。8 N3-cADPR的作用是可逆的，并且可以被高浓度的cADPR克服，这表明两者作用于同一位点。这得到了8 N3-cADPR有效竞争[32 P]cADPR与微粒体结合的事实的支持。相反，[32 P] 8 N3-cADPR的结合也可以被cADPR和8 N3-cADPR选择性地取代，但不能被ADP-核糖取代。这些结果表明，8 N3-cADPR特异性结合到cADPR结合位点，并抑制cADPR释放Ca 2+。用[32 P] 8 N3-cADPR预孵育的微粒体的光解导致140和100 kDa蛋白的特异性标记，这可以被8 N3-cADPR或纳摩尔浓度的cADPR阻止，但不能被微摩尔浓度的ADP-核糖、AMP、ADP、ATP、环AMP或肌醇1，4，5-三磷酸盐阻止。与140-kDa蛋白相比，咖啡因是Ca（2+）诱导的Ca（2+）释放的激动剂，优先抑制100 kDa蛋白的标记。这些结果表明，cADPR可能不直接与兰尼碱受体相互作用，而是通过中间蛋白发挥作用。
We have synthesized 8-azido-cyclic ADP-ribose (8N3-cADPR) and [32P]8-azido-cyclic ADP-ribose ([32P]8N3-cADPR) in order to characterize cyclic ADP-ribose-(cADPR) binding sites in sea urchin egg homogenates. 8N3-cADPR was an antagonist of cADPR since it did not induce Ca2+ release from egg microsomes but did inhibit the ability of cADPR to do so. The effect of 8N3-cADPR was reversible and could be overcome by high concentrations of cADPR, suggesting that both were acting on the same site. This was supported by the fact that 8N3-cADPR effectively competed for [32P]cADPR binding to microsomes. Reciprocally, binding of [32P]8N3-cADPR could also be selectively displaced by cADPR and 8N3-cADPR, but not by ADP-ribose. These results indicate that 8N3-cADPR binds specifically to the cADPR-binding sites and inhibits cADPR from releasing Ca2+. Photolysis of microsomes preincubated with [32P]8N3-cADPR resulted in specific labeling of proteins of 140 and 100 kDa, which could be prevented by 8N3-cADPR or nanomolar concentrations of cADPR, but not by micromolar concentrations of ADP-ribose, AMP, ADP, ATP, cyclic AMP or inositol 1,4,5-trisphosphate. Caffeine, an agonist of Ca(2+)-induced Ca2+ release, preferentially inhibited the labeling of the 100 kDa as compared to the 140-kDa protein. These results suggest that cADPR may not interact directly with the ryanodine receptor, but may instead, exert its effect through intermediate proteins.