Catty P, Deterre P
Départment de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires, Grenoble, France.
Eur J Biochem. 1991 Jul 15;199(2):263-9. doi: 10.1111/j.1432-1033.1991.tb16119.x.
The cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory gamma subunit (2 copies/PDE molecule), leaving intact the alpha beta catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90-99]. Studying the time course of this solubilization, we were able to purify a trypsin-solubilized PDE still completely inhibited (i.e. with its two gamma subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the gamma subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C-terminal fragment of the alpha subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, O. C., Ota, I. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238-9242]. We present the following evidence indicating that the C-terminus of the PDE beta subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin-solubilized PDE alpha beta gamma 2 has intact blocked N-termini. (b) It is still methylated on PDE alpha. (c) The C-terminus of PDE beta can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the alpha and beta polypeptides can also be detected by high-resolution gel electrophoresis: the first cleavage appears on the beta subunit and is completed when cleavage of the alpha subunit begins. The time course for cleavage of the gamma subunits appears to be slower than for the beta subunit and comparable to that of the alpha subunit. Upon longer trypsinization, a 70-kDa polypeptide appears which seems to be a degradation product of PDE beta. Gel-filtration analysis, however, shows that this 70-kDa fragment does not dissociate from the catalytic core.
脊椎动物视网膜视杆细胞外段（ROS）中的环鸟苷酸特异性磷酸二酯酶（PDE）是一种在体内由转导素激活的外周酶。在体外，可以使用胰蛋白酶实现人工激活。据描述，这是由于抑制性γ亚基（每个PDE分子有2个拷贝）降解，而αβ催化核心保持完整所致。然而，有人观察到，在激活之前，胰蛋白酶可诱导PDE从ROS膜中释放（或溶解）[温塞尔，T. G. & 斯特里尔，L.（1986年）《蛋白质结构、功能与遗传学》1，90 - 99]。在研究这种溶解的时间进程时，我们能够纯化出一种仍被完全抑制的胰蛋白酶溶解的PDE（即其两个γ亚基仍结合着）。因此，在γ亚基发生任何功能性降解之前，PDE的胰蛋白酶溶解就已完成。最近有人提出，这种溶解可能与α亚基C末端片段的切割同时发生，该片段可通过末端半胱氨酸残基的甲基化进行标记[翁，O. C.，太田，I. M.，克拉克，S. & 冯，B. K. K.（1989年）《美国国家科学院院刊》86, 9238 - 9242]。我们提供以下证据表明，PDEβ亚基的C末端主要负责PDE锚定到ROS膜上。（a）胰蛋白酶溶解的PDEαβγ2具有完整的封闭N末端。（b）它在PDEα上仍被甲基化。（c）PDEβ的C末端也可通过甲基化进行标记，其胰蛋白酶切割与PDE溶解非常吻合。（d）α和β多肽的顺序切割也可通过高分辨率凝胶电泳检测到：第一次切割出现在β亚基上，当α亚基开始切割时完成。γ亚基的切割时间进程似乎比β亚基慢，但与α亚基相当。长时间用胰蛋白酶处理后，会出现一条70 kDa的多肽，它似乎是PDEβ的降解产物。然而，凝胶过滤分析表明，这条70 kDa的片段不会与催化核心解离。
