Wolbring G, Schnetkamp P P
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Biochemistry. 1995 Apr 11;34(14):4689-95. doi: 10.1021/bi00014a024.
cGMP and Ca2+ are intracellular messengers in vertebrate rod photoreceptors. cGMP is the excitatory messenger, while intracellular free Ca2+ has been implied to be (one of) the messenger(s) in the process of light adaptation in vertebrate rod photoreceptors. The enzyme guanylyl cyclase (GC, EC 4.6.1.2.) catalyzes the reaction GTP-->cGMP + PPi. Bovine retinal rod outer segments (ROS) contain a particulate GC which is inhibited by an increase in free Ca2+ in the submicromolar range, although the precise molecular mechanism underlying this inhibition is unclear. We have developed an optical enzyme-coupled assay to study regulation of the particulate GC endogenous to bovine ROS. The particulate GC exhibited a Ca(2+)-inhibited (IC50 83-144 nM) activity of 13-23 nmol of PPi/(min-(mg of rhodopsin)). ATP increased the maximal velocity of GC by about 2-fold, and this increase was inhibited by the specific PKC inhibitors chelerythrine and the pseudosubstrate-based peptide inhibitor PKC R10-31N. When the factor that mediated the ATP-dependent increase in GC rate was removed by washing, the ATP-dependent increase in GC rate could be reestablished by addition of purified, constitutively active PKC.
环磷酸鸟苷（cGMP）和钙离子（Ca2+）是脊椎动物视杆光感受器中的细胞内信使。cGMP是兴奋性信使，而细胞内游离Ca2+被认为是脊椎动物视杆光感受器光适应过程中的信使（之一）。鸟苷酸环化酶（GC，EC 4.6.1.2.）催化反应GTP→cGMP + 焦磷酸（PPi）。牛视网膜视杆外段（ROS）含有一种颗粒性GC，它在亚微摩尔范围内会被游离Ca2+的增加所抑制，尽管这种抑制的精确分子机制尚不清楚。我们开发了一种光学酶偶联测定法来研究牛ROS内源性颗粒性GC的调节。颗粒性GC表现出Ca2+抑制（IC50为83 - 144 nM）活性，为13 - 23 nmol焦磷酸/（分钟 - （毫克视紫红质））。三磷酸腺苷（ATP）使GC的最大速度增加约2倍，这种增加被特异性蛋白激酶C（PKC）抑制剂白屈菜红碱和基于假底物的肽抑制剂PKC R10 - 31N所抑制。当通过洗涤去除介导ATP依赖性GC速率增加的因子后，通过添加纯化的、组成型活性PKC可以重新建立ATP依赖性GC速率的增加。
