Ryba N J, Hoon M A, Findlay J B, Saibil H R, Wilkinson J R, Heimburg T, Marsh D
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochemistry. 1993 Apr 6;32(13):3298-305. doi: 10.1021/bi00064a012.
Treatment of outer segment membranes from Loligo forbesi with endoprotease-V8 from Staphylococcus aureus results in cleavage of the C-terminal extension of the squid rhodopsin, with accompanying reduction of the apparent molecular weight from 47,000 to 36,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Negative-stain electron microscopy of the intact membranes shows that small clusters of the rhodopsin C-termini form structures extending from the membrane surface and that these are absent after protease treatment. Fourier transform infrared spectra of the amide I band of the protein indicate that removal of the C-terminal extension increases the relative alpha-helical content of squid rhodopsin to a level comparable to that for bovine rhodopsin in disk membranes, and to an extent which suggests that the alpha-helical structure lies mainly in the M(r) 36,000 (transmembrane) section of the protein. Saturation-transfer electron spin resonance (ESR) spectroscopy of the spin-labeled protein reveals that the rotational diffusion of squid rhodopsin in outer segment membranes that have been extensively washed with urea to remove peripheral proteins is much slower than that of bovine rhodopsin in rod outer segment disk membranes. This reduction in rotational mobility is also found with purified squid rhodopsin reconstituted in egg phosphatidylcholine and in urea-washed outer segment membranes which have been treated with endoprotease-V8 to remove the C-terminal extension of squid rhodopsin. In the latter case, the saturation-transfer ESR spectra are virtually identical to those of the non-proteolyzed membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
用来自金黄色葡萄球菌的内切蛋白酶V8处理福布斯氏枪乌贼的外段膜，会导致鱿鱼视紫红质C末端延伸部分被切割，在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上，其表观分子量随之从47,000降至36,000。完整膜的负染色电子显微镜显示，视紫红质C末端的小簇形成从膜表面延伸的结构，蛋白酶处理后这些结构消失。蛋白质酰胺I带的傅里叶变换红外光谱表明，去除C末端延伸部分会使鱿鱼视紫红质的相对α-螺旋含量增加到与盘膜中牛视紫红质相当的水平，且增加程度表明α-螺旋结构主要位于该蛋白质的M(r) 36,000（跨膜）部分。自旋标记蛋白质的饱和转移电子自旋共振（ESR）光谱显示，用尿素广泛洗涤以去除外周蛋白的外段膜中，鱿鱼视紫红质的旋转扩散比杆状外段盘膜中的牛视紫红质慢得多。在卵磷脂中重构的纯化鱿鱼视紫红质以及用内切蛋白酶V8处理以去除鱿鱼视紫红质C末端延伸部分的尿素洗涤外段膜中，也发现了旋转迁移率的降低。在后一种情况下，饱和转移ESR光谱与未进行蛋白酶解的膜的光谱几乎相同。（摘要截于250字）
