Solubilization of receptors for the novel Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate
Solubilization of receptors for the novel Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate
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DOI：
10.1074/jbc.m203224200
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发表时间：
2002-11-15
影响因子：
4.8
通讯作者：
Patel, S
中科院分区：
文献类型：
--
作者：
Berridge, G;Dickinson, G;Patel, S
烟酸腺嘌呤二核苷酸磷酸 (NAADP) 是多种破碎和完整细胞制剂中的有效 Ca2+ 动员剂。在海胆卵匀浆中，NAADP 独立于三磷酸肌醇或兰尼碱受体激活而释放 Ca2+。然而，对于 NAADP 的分子靶点知之甚少。在此，我们首次报道了海胆卵匀浆中 NAADP 受体的溶解。 Triton X-100 处理后制备的上清液级分以与膜制剂相似的亲和力和选择性结合 [P-32]NAADP。此外，NAADP 与其受体结合的不寻常的非解离性质在溶解后得以保留。在用[P-32]NAADP预标记的膜经过去污剂处理后，NAADP受体也可以释放到上清液级分中。以这种方式制备的标记受体很容易通过非变性凝胶电泳解析为单一蛋白质靶标。凝胶过滤和蔗糖密度梯度离心分析表明，NAADP 受体远小于三磷酸肌醇或兰尼碱受体，这为 NAADP 激活新型细胞内 Ca2+ 释放通道提供了进一步的生化证据。
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing agent in a variety of broken and intact cell preparations. In sea urchin egg homogenates, NAADP releases Ca2+ independently of inositol trisphosphate or ryanodine receptor activation. Little, however, is known concerning the molecular target for NAADP. Here we report for the first time solubilization of NAADP receptors from sea urchin egg homogenates. Supernatant fractions, prepared following Triton X-100 treatment, bound [P-32]NAADP with similar affinity and selectivity as membrane preparations. Furthermore, the unusual non-dissociating nature of NAADP binding to its receptor was preserved upon solubilization. NAADP receptors could also be released into supernatant fractions upon detergent treatment of membranes prelabeled with [P-32]NAADP. Tagged receptors prepared in this way, were readily resolved by native gel electrophoresis as a single protein target. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca2+ release channel.