Artemyev N O, Hamm H E
Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago 60680.
Biochem J. 1992 Apr 1;283 ( Pt 1)(Pt 1):273-9. doi: 10.1042/bj2830273.
Light-activated cyclic GMP-phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells which regulates the level of the internal transmitter cyclic GMP. PDE consists of catalytic P alpha and P beta subunits, and two copies of inhibitory P gamma subunit. The two P gamma subunits block the enzyme's activity in the dark and are removed by the alpha-subunit of transducin (alpha 1) upon light-activation of photoreceptor cells. Here we have examined the role of various regions of P gamma, the N-terminal, the central cationic and the C-terminal regions, in interaction with the catalytic subunits of PDE. N-Terminal truncation of P gamma (12-87-P gamma) did not change the potency of PDE inhibition, and thus we conclude that the P gamma N-terminal region is not critical for P gamma-P alpha beta interaction. The central region, 24-46-P gamma, participates in interaction with the catalytic P alpha beta subunits. A synthetic peptide corresponding to this site inhibited approximately 50% of trypsin-activated PDE (tPDE) (Ki approximately 15 microM) and competed with P gamma for inhibition of tPDE. We demonstrated, by using h.p.l.c. gel filtration, that 125I-Tyr-24-46-P gamma peptide bound with high affinity to tPDE, but not to P alpha beta gamma 2. The C-terminal region of 46-87-P gamma was found to be the major region involved in inhibition of PDE. It fully inhibited tPDE with a Ki of approximately 0.8 microM. It also bound to tPDE, but not P alpha beta gamma 2, in h.p.l.c. gel-filtration experiments. In addition, P gamma was cross-linked by p-phenylenedimaleimide to both P alpha and P beta, as was shown by using subunit-specific anti-P alpha, -P beta and -P gamma antibodies. Cys68 of P gamma, which presumably participates in cross-linking, is located near the P gamma C-terminus. These data provide evidence for two regions of P gamma that interact with, and inhibit, P alpha beta. The central region, 24-46 P gamma, is important in binding, but inhibits PDE only weakly, whereas the C-terminal region is most important for PDE inhibition. These results help to explain the well-known fact that P gamma trypsin-activation and C-terminal truncation both lead to PDE activation. Furthermore, our findings on the mechanism of PDE inhibition of P gamma are relevant for understanding the mechanism of PDE activation by transducin.
光激活环鸟苷酸磷酸二酯酶（PDE）是脊椎动物光感受器细胞的关键效应酶，可调节细胞内递质环鸟苷酸的水平。PDE由催化性的Pα和Pβ亚基以及两个抑制性Pγ亚基拷贝组成。两个Pγ亚基在黑暗中会阻断该酶的活性，在光感受器细胞光激活时会被转导素的α亚基（α1）移除。在此，我们研究了Pγ的各个区域，即N端、中央阳离子区域和C端区域，在与PDE催化亚基相互作用中的作用。Pγ的N端截短（12 - 87 - Pγ）并未改变PDE抑制的效力，因此我们得出结论，Pγ的N端区域对于Pγ - Pαβ相互作用并非关键。中央区域24 - 46 - Pγ参与与催化性Pαβ亚基的相互作用。对应于该位点的合成肽可抑制约50%的胰蛋白酶激活的PDE（tPDE）（Ki约为15μM），并与Pγ竞争对tPDE的抑制作用。我们通过高效液相色谱凝胶过滤证明，125I - Tyr - 24 - 46 - Pγ肽与tPDE具有高亲和力结合，但不与Pαβγ2结合。发现46 - 87 - Pγ的C端区域是参与PDE抑制的主要区域。它以约0.8μM的Ki完全抑制tPDE。在高效液相色谱凝胶过滤实验中，它也与tPDE结合，但不与Pαβγ2结合。此外，如使用亚基特异性抗Pα、抗Pβ和抗Pγ抗体所示，Pγ通过对苯二马来酰亚胺与Pα和Pβ都发生了交联。Pγ的Cys68可能参与交联，位于Pγ C端附近。这些数据为Pγ与Pαβ相互作用并抑制Pαβ的两个区域提供了证据。中央区域24 - 46 Pγ在结合中很重要，但对PDE的抑制作用较弱，而C端区域对PDE抑制最为重要。这些结果有助于解释一个众所周知但的事实即Pγ的胰蛋白酶激活和C端截短都会导致PDE激活。此外，我们关于Pγ对PDE抑制机制的发现对于理解转导素对PDE激活的机制具有重要意义。
