PHOSPHORYLATION OF SOLUBILIZED DARK-ADAPTED RHODOPSIN - INSIGHTS INTO THE ACTIVATION OF RHODOPSIN KINASE
PHOSPHORYLATION OF SOLUBILIZED DARK-ADAPTED RHODOPSIN - INSIGHTS INTO THE ACTIVATION OF RHODOPSIN KINASE
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DOI：
10.1111/j.1432-1033.1993.tb17832.x
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发表时间：
1993-04-15
期刊：
影响因子：
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通讯作者：
AKHTAR, M
中科院分区：
文献类型：
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作者：
DEAN, KR;AKHTAR, M
已经开发了一种从磷酸视蛋白中分离磷酸视紫红质的方案。该方法利用了以下发现：虽然0.5%N，N-二甲基十二烷基胺-N-氧化物完全溶解膜包埋的磷酸视紫红质，但在该浓度的去污剂下，磷酸视蛋白仅微溶。以这种方式溶解的磷酸视紫红质可以通过羟基磷灰石上的色谱法从污染的磷酸视蛋白中释放出来。使用这种方法，视紫红质激酶催化的光激发视紫红质和天然视紫红质的磷酸化研究在视杆外节膜在不同程度的漂白。在分析磷酸化混合物之前，通过用NH 2 OH处理将光激发视紫红质的磷酸化形式转化为磷酸视蛋白。发现，在5%漂白水平下，产生的磷视紫红质的量是磷视蛋白的量的15%，而在60%漂白下，磷视紫红质小于磷视蛋白的1%。在一个完全可溶的系统（使用2%的十二烷基麦芽糖苷）和磷酸盐掺入到视紫红质与视蛋白的模式是相同的，在膜系统中，在不同的漂白条件下的磷酸化反应进行了研究。我们以前提出，视紫红质激酶通常以非活性形式存在，并且仅在与光激发视紫红质相互作用后被激活。目前的工作加强了这一结论，也表明，激活后，激酶优先磷酸化光激发视紫红质，但也可以对未漂白视紫红质。两种可能的机制激活的激酶被认为是。根据不同漂白水平下的视紫红质和视紫红质磷酸化分布，计算了光激发视紫红质（k（R）*）和视紫红质（k（R））磷酸化的相对速率。得到膜系统的k（R）*/k（R）值为71 +/-20，溶解系统的k（R）*/k（R）值为80 +/-19。用于获得这些值的代数方程突出了这样一个事实，即样品中两种底物（光激发视紫红质和视紫红质）的浓度比决定了漂白和未漂白视紫红质之间磷酸盐的最终分布。这一结论可能有助于理解以前观察到的“高增益”磷酸化。
A protocol for the separation of phosphorhodopsin from phospho-opsin has been developed. The method takes advantage of the finding that, while 0.5% N,N-dimethyldodecylamine-N-oxide completely solubilises membrane-embedded phosphorhodopsin, at this concentration of detergent, phospho-opsin is only sparingly soluble. Phosphorhodopsin solubilised in this manner may be freed from contaminant phospho-opsin by chromatography on hydroxyapatite. Using this method, the rhodopsin-kinase-catalysed phosphorylation of photoexcited rhodopsin and native rhodopsin was studied in rod outer-segment membranes at different levels of bleaching. Prior to analysis of the phosphorylation mixture, the phosphorylated form of photoexcited rhodopsin was converted into phospho-opsin by treatment with NH2OH. It was found that, while at a 5% bleach level the amount of phosphorhodopsin produced was 15% that of phospho-opsin, at 60% bleaching the phosphorhodopsin was less than 1% of phospho-opsin. The phosphorylation reaction under different bleaching conditions was also studied in a completely soluble system (using 2% dodecyl maltoside) and the pattern of phosphate incorporation into rhodopsin versus opsin was identical to that in the membrane system. We have previously proposed that rhodopsin kinase normally exists in an inactive form and is only activated following interaction with photoexcited rhodopsin. The present work strengthens this conclusion and also shows that, following activation, the kinase preferentially phosphorylates photoexcited rhodopsin but can also act upon unbleached rhodopsin. Two possible mechanisms for the activation of the kinase are considered. From the distribution of phosphorhodopsin and phosphoopsin at different bleaching levels, the relative rates of the phosphorylation of photoexcited rhodopsin (k(R)*) and rhodopsin (k(R)) were calculated. k(R)*/k(R) values for the membrane system of 71 +/- 20 and, for the solubilised system, of 80 +/- 19 were obtained. The algebraic equation used to obtain these values highlights the fact that the ratio of the concentrations of the two substrates, photoexcited rhodopsin and rhodopsin, in a sample, determines the final distribution of phosphate between bleached and unbleached rhodopsin. This conclusion may contribute to the understanding of 'high-gain' phosphorylation observed previously.