Pugh E N, Lamb T D
Department of Psychology, University of Pennsylvania, Philadelphia 19104.
Biochim Biophys Acta. 1993 Mar 1;1141(2-3):111-49. doi: 10.1016/0005-2728(93)90038-h.
We can summarize our investigation of amplification in the activation steps of vertebrate phototransduction as follows. (1) A theoretical analysis of the activation steps of the cGMP cascade shows that after a brief flash of phi photoisomerizations the number of activated PDE molecules should rise as a delayed ramp with slope proportional to phi, and that, as a consequence, the cGMP-activated current should decay as a delayed Gaussian function of time (Eqn. 20). (i) Early in the response to a flash, the normalized response R(t) can be approximated as rising as 1/2 phi At2 (after a short delay), where A is the amplification constant characteristic of the individual photoreceptor. (ii) The delayed ramp behavior of PDE activation and the consequent decline of current in the form of the delayed Gaussian are confirmed by experiments in a variety of photoreceptors; the analysis thus yields estimates of the amplification constant from these diverse photoreceptors. (iii) Eqn. 20 further predicts that the response-intensity relation at any fixed time should saturate exponentially, as has been found experimentally. (2) The amplification constant A can be expressed as the product of amplification factors contributed by the individual activation steps of phototransduction, i.e., A = nu RG cGP beta sub n (Eqns. 9 and 21), where (i) nu RG is the rate of G* production per Rh*; (ii) cGP is the efficiency of the coupling between G* production and PDE* production; (iii) beta sub is the increment in hydrolytic rate constant produced by one PDE*, i.e., a single activated catalytic subunit of PDE; and (iv) n is the Hill coefficient of opening of the cGMP-activated channels. (3) The amplification factor beta sub includes the ratio kcat/Km, which characterizes the hydrolytic activity of the PDE in vivo where cG << Km. Two different analyses based upon photocurrents were developed which provide lower bounds for kcat/Km in vivo; these analyses establish that kcat/Km probably exceeds 10(7) M-1 s-1 (and is likely to be higher) in both amphibian and mammalian rods. Few biochemical studies (other than those using trypsin activation) have yielded such high values. A likely explanation of many of the relatively low biochemical estimates of kcat/Km is that Km may have been overestimated by a factor of about 4 in preparations in which stacks of disks are left intact, due to diffusion with hydrolysis in the stacks.(ABSTRACT TRUNCATED AT 400 WORDS)
我们可以将对脊椎动物光转导激活步骤中放大作用的研究总结如下。(1) 对cGMP级联激活步骤的理论分析表明，在短暂的视黄醛异构化闪光之后，激活的磷酸二酯酶（PDE）分子数量应呈延迟斜坡上升，其斜率与视黄醛异构化次数（phi）成正比，因此，cGMP激活电流应呈时间的延迟高斯函数衰减（方程20）。(i) 在对闪光的响应早期，归一化响应R(t) 可近似为（经过短暂延迟后）以1/2 phi At2上升，其中A是单个光感受器的特征放大常数。(ii) 在各种光感受器中的实验证实了PDE激活的延迟斜坡行为以及随之而来的以延迟高斯形式的电流下降；因此，该分析得出了来自这些不同光感受器的放大常数估计值。(iii) 方程20进一步预测，在任何固定时间的响应 - 强度关系应呈指数饱和，这已通过实验发现。(2) 放大常数A可表示为光转导各个激活步骤所贡献的放大因子的乘积，即A = nu RG cGP beta sub n（方程9和21），其中 (i) nu RG是每个视紫红质*（Rh*）产生G的速率；(ii) cGP是G产生与PDE产生之间的耦合效率；(iii) beta sub是由一个PDE（即PDE的单个激活催化亚基）产生的水解速率常数的增量；(iv) n是cGMP激活通道开放的希尔系数。(3) 放大因子beta sub包括kcat/Km的比值，它表征了体内cG << Km时PDE的水解活性。基于光电流开展了两种不同分析，为体内kcat/Km提供了下限；这些分析确定，在两栖动物和哺乳动物的视杆细胞中，kcat/Km可能超过10(7) M-1 s-1（并且可能更高）。很少有生化研究（除了那些使用胰蛋白酶激活的研究）得出如此高的值。许多对kcat/Km的相对较低生化估计的一个可能解释是，在完整保留盘状堆叠的制剂中，由于堆叠中的扩散与水解作用，Km可能被高估了约4倍。（摘要截断于400字）
