Bondarenko V A, Yamazaki M, Hayashi F, Yamazaki A
Kresge Eye Institute, Department of Ophthalmology, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Biochemistry. 1999 Jun 15;38(24):7755-63. doi: 10.1021/bi990106a.
Our previous study has shown that P gamma, the regulatory subunit of cGMP phosphodiesterase (PDE), is ADP-ribosylated by endogenous ADP-ribosyltransferase when P gamma is free or complexed with the catalytic subunits of PDE in amphibian rod photoreceptor membranes. The P gamma domain containing ADP-ribosylated arginines was shown to be involved in its interaction with T alpha, a key interaction for PDE activation. In this study, we describe a possible function of the P gamma ADP-ribosylation in the GTP/T alpha-dependent PDE activation. When rod membranes were preincubated with or without NAD and washed with a buffer containing GTP, the PDE activity of NAD-preincubated membranes was increased by the GTP-washing only to approximately 50% of that of membranes preincubated without NAD. The P gamma release by the GTP-washing from these NAD-preincubated membranes was also suppressed to approximately 50% of that preincubated without NAD. Taking into consideration that approximately 50% of P gamma is ADP-ribosylated under these conditions, these observations suggest that the ADP-ribosylated P gamma cannot interact with GTP/T alpha. We have also shown that a soluble fraction of ROS contains an enzyme(s) to release the radioactivity of [32P]ADP-ribosylated P gamma in concentration- and time-dependent manners, suggesting that the P gamma ADP-ribosylation is reversible. Rod ADP-ribosyltransferase solubilized from membranes by phosphatidylinositol-specific phospholipase C was separated into two fractions by ion-exchange columns. Biochemical characterization of these two fractions, including measurement of the Km for NAD and P gamma, estimation of their molecular masses, ADP-ribosylation of P gamma arginine mutants, effects of ADP-ribosyltransferase inhibitors on the P gamma ADP-ribosylation, and effects of salts and pH on the P gamma ADP-ribosylation, indicates that rod ADP-ribosyltransferase contains two isozymes, and that these two isozymes have similar properties for the P gamma ADP-ribosylation. Our observations strongly suggest that the negative regulation of PDE through the reversible P gamma ADP-ribosylation may function in the phototransduction mechanism.
我们之前的研究表明，环鸟苷酸磷酸二酯酶（PDE）的调节亚基Pγ，在两栖动物视杆光感受器膜中，当Pγ游离或与PDE的催化亚基结合时，会被内源性ADP - 核糖基转移酶进行ADP - 核糖基化修饰。含有ADP - 核糖基化精氨酸的Pγ结构域被证明参与其与Tα的相互作用，这是PDE激活的关键相互作用。在本研究中，我们描述了Pγ的ADP - 核糖基化修饰在GTP/Tα依赖性PDE激活中的一种可能功能。当视杆膜在有无NAD的情况下预孵育，并用含有GTP的缓冲液洗涤时，经NAD预孵育的膜的PDE活性仅通过GTP洗涤增加到未经NAD预孵育的膜的约50%。从这些经NAD预孵育的膜中通过GTP洗涤释放的Pγ也被抑制到未经NAD预孵育的膜的约50%。考虑到在这些条件下约50%的Pγ被ADP - 核糖基化修饰，这些观察结果表明，ADP - 核糖基化的Pγ不能与GTP/Tα相互作用。我们还表明，视网膜外段（ROS）的可溶部分含有一种酶，能以浓度和时间依赖性方式释放[³²P]ADP - 核糖基化Pγ的放射性，这表明Pγ的ADP - 核糖基化修饰是可逆的。通过磷脂酰肌醇特异性磷脂酶C从膜中溶解的视杆ADP - 核糖基转移酶通过离子交换柱被分离成两个部分。对这两个部分的生化特性进行表征，包括测定对NAD和Pγ的米氏常数（Km）、估计它们的分子量、对Pγ精氨酸突变体的ADP - 核糖基化修饰、ADP - 核糖基转移酶抑制剂对Pγ的ADP - 核糖基化修饰的影响以及盐和pH对Pγ的ADP - 核糖基化修饰的影响，表明视杆ADP - 核糖基转移酶含有两种同工酶，并且这两种同工酶对Pγ的ADP - 核糖基化修饰具有相似的特性。我们的观察结果强烈表明，通过可逆的PγADP - 核糖基化修饰对PDE的负调节可能在光转导机制中起作用。
