Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate.
Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate.
复制标题
来自牛杆外节的鸟苷酸环化酶：溶解、部分纯化和无机焦磷酸盐的调节。
作者：
S. Hakki;A. Sitaramayya
尽管鸟苷酸环化酶在视觉信号转导中起着关键作用，但对视网膜感光细胞鸟苷酸环化酶的研究却很少。该酶尚未被纯化，主要是因为在solubilizingit. We在这里报告一个简单的方法溶解67%的环化酶活性从视网膜杆盘膜（RDM）的困难。以Nonidet P-40为去污剂，高浓度KCl和排除锰有利于环化酶的增溶。溶解的和残留的不溶性酶都是高度不稳定的，但可以部分稳定的二硫苏糖醇。它们对钙、钙调素和心钠素均不敏感。它们也对改变测定中的锰浓度作出类似的反应。对于这两种组分中的活性，GTP的Km约为230 μ M，Line-weaver-Burk图显示底物结合是协同的，Hill图表明存在两个底物结合位点。累积起来，这些观察结果表明，虽然整个活性不能溶解，溶解和残留的不溶性活性可能属于同一种酶。部分纯化将溶解的酶分解成两种活性，称为酶1和2。两者的底物饱和动力学与增溶酶相似，并且受到环化酶反应产物之一无机焦磷酸盐的竞争性抑制。酶1的PPi的Ki为70-100 μ M，酶2的Ki为150-200 μ M。cGMP在浓度高达800 μ M时对两种酶的活性都没有影响。（250字处删节）
In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
影响因子：
2.9
作者：
Schnetkamp,PP;Kaupp,UB
通讯作者：
Kaupp,UB
影响因子：
2.9
作者：
B. Springs;K. Welsh;B. Cooperman
通讯作者：
B. Springs;K. Welsh;B. Cooperman
弱漂白时视紫红质的磷酸化和 ATP 对环 GMP 磷酸二酯酶活化的猝灭。
DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Sitaramayya,A;Liebman,PA
通讯作者：
Liebman,PA