Gillespie P G, Prusti R K, Apel E D, Beavo J A
Department of Pharmacology, University of Washington, Seattle 98195.
J Biol Chem. 1989 Jul 25;264(21):12187-93.
A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.
相当一部分（20%-30%）牛视杆外段磷酸二酯酶（PDE）活性与用中等离子强度缓冲液制备的外段膜无关；这种PDE活性似乎代表一种独特的可溶性同工酶。尽管这种PDE同工酶可以证明存在于密封的视杆外段中，但在大多数标准的视杆外段制备过程中会被去除。开发了一种方法，使用单克隆抗体柱（ROS-1a）可将可溶性视杆PDE快速纯化2600倍，达到表观均一性。可溶性视杆PDE同工酶除了有分子量为88,000（αsol）、84,000（βsol）和11,000（γsol）的亚基外，还有一个新的分子量为15,000的亚基（δ）。δ亚基与视锥PDE 15-kDa亚基共迁移，可能与之相同。通过反相色谱法分离了可溶性视杆PDE和膜相关视杆PDE的小亚基。γsol亚基是胰蛋白酶激活的视杆PDE的有效抑制剂，在浓度为11 pM时抑制1 pM PDE活性的50%。该浓度与膜相关视杆PDE的γ亚基所观察到的浓度相似。纯化的δ亚基似乎不影响PDE活性；然而，该亚基极难保持在溶液中。到目前为止测试的可溶性视杆PDE的所有动力学和物理性质与膜相关形式的相似，除了存在δ亚基，这表明这个独特的亚基可能介导可溶性视杆PDE和视锥PDE在完整光感受器中的溶解性。
