PROTEIN COMPLEMENT OF ROD OUTER SEGMENTS OF FROG RETINA
PROTEIN COMPLEMENT OF ROD OUTER SEGMENTS OF FROG RETINA
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DOI：
10.1021/bi00364a010
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发表时间：
1986-08-12
期刊：
影响因子：
2.9
通讯作者：
BOWNDS, MD
中科院分区：
文献类型：
--
作者：
HAMM, HE;BOWNDS, MD
从青蛙视网膜的杆外段（ROS）已纯化的Percoll密度梯度离心，一个程序，保持其形式和完整性。一维和二维电泳分析揭示了比在许多细胞器中观察到的蛋白质数量更少，并允许对20种最丰富的多肽进行定量。视紫红质占总蛋白的70% (3 .；109个拷贝/外段)，大约有70个其他多肽出现在6倍以上。104个拷贝/外段。g蛋白占总蛋白质的17%（3倍）。108个拷贝/外段)，连接视紫红质漂白和环GMP磷酸二酯酶（PDE）的激活。磷酸二酯酶占蛋白质的1.5%（1.5倍）。107个拷贝/外段)，在光下与膜结合的48000道尔顿成分占2.6%。外段约90%的总蛋白的功能是已知的，三分之二的非视紫红质蛋白是由与环GMP代谢相关的酶活性决定的。视紫红质、g蛋白和PDE的相对丰度为100:10:1。除了这些主要的膜相关蛋白外，大多数其他蛋白都是细胞质蛋白。另外13个多肽在每1000个视紫红质中有一个或多个拷贝，9个是可溶性的，4个是膜结合的，它们相对于视紫红质的丰度已经被定量了。活性氧已被分离成亚细胞组分，可分解三类可溶性、外源性膜蛋白和整体膜蛋白。列出了被磷酸化的蛋白质及其亚细胞定位。检测到大约25个磷酸肽，大多数在可溶性部分。与粗活性氧相比，与纯化的外段相关的磷酸化蛋白较少。不同的磷酸化模式与[32P]Pi孵育的完整棒和[.gamma.-32P]ATP孵育的断裂棒相关。
Rod outer segments (ROS) from frog retina have been purified by Percoll density gradient centrifugation, a procedure that preserves their form and intactness. One- and two-dimensional electrophoretic analysis reveals a smaller number of proteins than is observed in many cell organelles and permits quantitation of the 20 most abundant polypeptides. Rhodopsin accounts for 70% of the total protein (3 .times. 109 copies/outer segment), and approximately 70 other polypeptides are present at more than 6 .times. 104 copies/outer segment. Another 17% of the total protein is accounted for by the G-protein (3 .times. 108 copies/outer segment) that links rhodopsin bleaching and the activation of cyclic GMP phosphodiesterase (PDE). The phosphodiesterase accounts for 1.5% of the protein (1.5 .times. 107 copies/outer segment), and a 48,000-dalton component that binds to the membrane in the light accounts for a further 2.6%. The function of approximately 90% of the total protein in the outer segment is known, and two-thirds of the non-rhodopsin protein is accounted for by enzyme activities associated with cyclic GMP metabolism. The relative abundance of rhodopsin, G-protein, and PDE is 100:10:1. Apart from these major membrane-associated proteins, most of the other proteins are cytosolic. Thirteen other polypeptides are found at an abundance of one or more copies per 1000 rhodopsins, nine soluble and four membrane-bound, and their abundance relative to rhodopsin has been quantitated. ROS have been separated into subcellular fractions which resolve three classes of soluble, extrinsic membrane, and integral membrane proteins. A listing of the proteins that are phosphorylated and their subcellular localization is given. Approximately 25 phosphopeptides are detected, and most are in the soluble fraction. Fewer phosphorylated proteins are associated with the purified outer segments than with crude ROS. Distinct patterns of phosphorylation are associated with intact rods incubated with [32P]Pi and broken rods incubated with [.gamma.-32P]ATP.