2-SITE HIGH-AFFINITY INTERACTION BETWEEN INHIBITORY AND CATALYTIC SUBUNITS OF ROD CYCLIC-GMP PHOSPHODIESTERASE
2-SITE HIGH-AFFINITY INTERACTION BETWEEN INHIBITORY AND CATALYTIC SUBUNITS OF ROD CYCLIC-GMP PHOSPHODIESTERASE
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DOI：
10.1042/bj2830273
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发表时间：
1992-04-01
影响因子：
4.1
通讯作者：
HAMM, HE
中科院分区：
文献类型：
--
作者：
ARTEMYEV, NO;HAMM, HE
光激活环磷酸鸟苷磷酸二酯酶（PDE）是脊椎动物感光细胞的关键效应酶，调节体内递质环磷酸鸟苷的水平。PDE由催化性P-α和P-β亚基以及两个拷贝的抑制性P-γ亚基组成。两个P-γ亚基在黑暗中阻断酶的活性，并在光激活感光细胞时被转导素的α亚基（α（t））去除。在这里，我们已经研究了P-γ，N-末端，中央阳离子和C-末端区域，在与PDE的催化亚基的相互作用中的各个区域的作用。P-γ（12-87-P-γ）的N-末端截短没有改变PDE抑制的效力，因此我们得出结论，P-γ N-末端区域对于P-γ-P-α-β相互作用不是关键的。中心区域24-46-P-γ参与与催化性P-α-β-亚基的相互作用。对应于该位点的合成肽抑制约50%的trypsin激活的PDE（tPDE）（K（i）约15 μ M），并与P-γ竞争抑制tPDE。我们用高压液相色谱法证明了。凝胶过滤，I-125-Tyr-24-46-P-γ肽以高亲和力结合tPDE，但不结合P-α-β-γ（2）。发现46-87-P-γ的C-末端区域是参与抑制PDE的主要区域。它完全抑制tPDE，K（i）约为0.8 μ M。在h.p.l.c.中，它也与tPDE结合，但不与P-α-β-γ结合（2）。凝胶过滤实验。此外，P-γ通过对亚苯基二马来酰亚胺与P-α和P-β交联，如通过使用亚基特异性抗P-α、P-β和P-γ抗体所示。推测参与交联的P-γ的Cys 68位于P-γ C-末端附近。这些数据为P-γ的两个区域与P-α-β相互作用并抑制其作用提供了证据。中心区域，24-46-P-γ，在结合中是重要的，但是仅微弱地抑制PDE，而C-末端区域对于PDE抑制是最重要的。这些结果有助于解释众所周知的事实，即P-γ-胰蛋白酶激活和C-末端截短均导致PDE激活。此外，我们的研究结果的PDE抑制P-γ的机制是相关的了解PDE激活转导的机制。
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(t)) 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 tryspin-activated PDE (tPDE) (K(i) approximately 15-mu-M) and competed with P-gamma for inhibition of tPDE. We demonstrated, by using h.p.l.c. gel filtration, that I-125-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 K(i) of approximately 0.8-mu-M. 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.