Choe H G, Ghalayini A J, Anderson R E
Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030.
Exp Eye Res. 1990 Aug;51(2):167-76. doi: 10.1016/0014-4835(90)90069-7.
Previous studies have shown that vertebrate rod outer segments (ROS) have a light activated phospholipase C which hydrolyzes phosphatidylinositol-4,5-bisphosphonate (PIP2). Three different experimental approaches have been used to test the hypothesis that the phosphatidylinositol (PI) biosynthetic cycle is present in ROS and that PIP2 can be regenerated from DG independent of rod inner segments. In the first study, enzyme activities of the PI cycle were assayed simultaneously in the presence of CTP, myo-inositol and [gamma-32P]ATP using endogenous lipids as substrates. Under these conditions, broken (leaky) ROS prepared by continuous sucrose gradient centrifugation showed PI, PIP and DG kinase activities similar to those found in intact ROS and non-ROS membranes, whereas PI synthetase activity was much lower in the leaky ROS than in the other two fractions. The relative distribution of PI synthetase specific activity in the three membrane preparations was similar to that of the microsomal enzyme marker cytochrome c reductase. ROS prepared by discontinuous sucrose gradient centrifugation showed only 2-3% of whole homogenate PI synthetase or phosphatidyl: cytidyl transferase activities, and the distribution of activities was the same as for microsomal and mitochondrial marker enzymes. In the second study, whole retinas were incubated with myo-[2-3H]inositol or [2-3H]glycerol in vitro, and the time course of incorporation of radioactivity into PI and other phospholipids was determined for ROS and three other retinal fractions. Over a 10-hr period, the rate of incorporation of myo-[2-3H]inositol or [2-3H]glycerol into PI in ROS was lowest among the various retinal fractions. In the third study, chemical analysis of the molecular species composition of PI, DG and phosphatidic acid (PA) from ROS shows that PA is substantially different from PI and DG, the latter two being quite similar. These results are consistent with a precursor-product relationship between PI and DG, but not with the conversion of DG to PA or of PA to PI. Taken together, these three studies indicate that ROS do not have PI synthetase or phosphatidyl: cytidyl transferase activities, but do have DG, PI and PIP kinase activities. Thus, the PI in ROS lost through rapid turnover must be replaced with molecules derived from de novo synthesis in the inner segment of the photoreceptor cell.
以往的研究表明，脊椎动物的视杆细胞外段（ROS）具有一种光激活的磷脂酶C，它能水解磷脂酰肌醇-4,5-二磷酸（PIP2）。已采用三种不同的实验方法来检验以下假设：ROS中存在磷脂酰肌醇（PI）生物合成循环，且PIP2可独立于视杆细胞内段从二酰甘油（DG）再生。在第一项研究中，以内源性脂质为底物，在CTP、肌醇和[γ-32P]ATP存在的情况下，同时测定PI循环的酶活性。在这些条件下，通过连续蔗糖梯度离心制备的破碎（渗漏）ROS显示出与完整ROS和非ROS膜中相似的PI、PIP和DG激酶活性，而渗漏ROS中的PI合成酶活性远低于其他两个组分。三种膜制剂中PI合成酶比活性的相对分布与微粒体酶标志物细胞色素c还原酶的相似。通过不连续蔗糖梯度离心制备的ROS仅显示出全匀浆PI合成酶或磷脂酰：胞苷转移酶活性的2 - 3%，且活性分布与微粒体和线粒体标志物酶相同。在第二项研究中，将整个视网膜在体外与肌醇-[2-3H]或[2-3H]甘油一起孵育，并测定ROS和其他三个视网膜组分中放射性掺入PI和其他磷脂的时间进程。在10小时的时间段内，ROS中肌醇-[2-3H]或[2-3H]甘油掺入PI的速率在各个视网膜组分中是最低的。在第三项研究中，对ROS中PI、DG和磷脂酸（PA）的分子种类组成进行化学分析表明，PA与PI和DG有很大不同，后两者非常相似。这些结果与PI和DG之间的前体-产物关系一致，但与DG向PA或PA向PI的转化不一致。综合起来，这三项研究表明，ROS不具有PI合成酶或磷脂酰：胞苷转移酶活性，但具有DG、PI和PIP激酶活性。因此，通过快速周转而损失的ROS中的PI必须由感光细胞内段从头合成的分子来替代。
