Characterization of guanylate cyclase activity in single retinal rod outer segments.
Characterization of guanylate cyclase activity in single retinal rod outer segments.
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DOI：
10.1085/jgp.106.5.863
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发表时间：
1995-11
期刊：
影响因子：
--
通讯作者：
Yau KW
中科院分区：
文献类型：
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作者：
Koutalos Y;Nakatani K;Tamura T;Yau KW
cGMP通过直接控制光感受器外段质膜上的阳离子通道介导脊椎动物的光传导。第二信使由鸟苷酸环化酶产生，并由光活化的cgmp -磷酸二酯酶水解。这两种酶的活性都对Ca2+敏感，鸟苷酸环化酶活性受到抑制，光活化磷酸二酯酶活性受到增强。由于光诱导细胞内Ca2+的减少，这些活动的变化与光感受器对背景光的适应有关。我们在这里描述的实验，表征鸟苷酸环化酶活性及其调节Ca2+使用截断棒外段制备，以评估酶在光适应中的作用。将虎蝾螈棒的外部部分拉入吸液管中，以记录膜电流，并用探针剪掉细胞的其余部分，以进行内部透析。表面膜上的cGMP门控通道用于监测由浴液供应的GTP通过外段的鸟苷酸环化酶转化为cGMP。在名义0 Ca2+条件下，1.6 mM游离Mg2+存在时，环化酶活性Km为250 microM MgGTP， Vmax为25 microM cGMP s-1；在0.5 mM游离Mg2+存在下，Km为310 μ m MgGTP， Vmax为17 μ m cGMP s-1。Mg2+对MgGTP在0.5 mM处的EC50为0.2 mM， Ca2+抑制环化酶活性。在K+细胞内溶液中，含有0.5 mM游离Mg2+和2.0 mM GTP，在名义0 Ca2+下，环化酶活性为13微米cGMP s-1；Ca2+降低了这种活性，IC50约为90 nM， Hill系数约为2.0。
cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.