Bayramashvili D I, Drachev A L, Drachev L A, Kaulen A D, Kudelin A B, Martynov V I, Skulachev V P
Eur J Biochem. 1984 Aug 1;142(3):583-90. doi: 10.1111/j.1432-1033.1984.tb08326.x.
Photoreceptor discs from rod outer segments of cattle retina were treated with (a) papain, (b) thermolysin or (c) trypsin, the procedures resulting in the cleavage of the rhodopsin polypeptide chain between (a) 323 and 324, 236 and 237, 241 and 242, (b) 327 and 328, 240 and 241, or (c) 339 and 340 amino acid residues, respectively. In all the cases, partially digested rhodopsins proved to be competent in generating photoelectric potential and increasing membrane conductance of the discs adsorbed onto phospholipid-impregnated collodion film. The kinetics of generation and dissipation of photopotential as well as of formation of metarhodopsin II and of the light-induced rhodopsin protonation were found to be the same in the partially digested preparations and in the intact one. Incubation of papain-treated or thermolysin-treated discs at pH 6.0 induced formation of inside-out vesicles which, when incorporated into the collodion film, generated an oppositely directed photopotential. Treatment of such vesicles with papain gave rise to further cleavages of the polypeptide localized between 30 and 31, 186 and 187 amino acid residues. One more proteinase-sensitive site, localized between 104 and 105 residues, has been discovered in the inside-out vesicles treated with thermolysin. This fact consistent with the scheme of the 'seven column' arrangement of the visual rhodopsin [Ovchinnikov, Yu. A. (1982) FEBS Lett. 148, 179-191]. Rhodopsin, when treated with papain on both sides, was deprived of sixty amino acid residues being split in two sites in the middle part of the polypeptide, but was still active as a photoelectric energy transducer. The main specific feature inherent in the photoelectric response of the papain-treated or thermolysin-treated rhodopsin and absent from the native protein is that the former survives addition of long trains of saturating flashes when the response of the intact preparation becomes negligible. This effect was shown to be due to conversion of partially digested rhodopsin to a photolytic product that at room temperature lived for minutes even in the presence of NH2OH. A 532-nm laser flash effectively converted this product back to rhodopsin.
牛视网膜视杆外段的光感受器圆盘分别用（a）木瓜蛋白酶、（b）嗜热菌蛋白酶或（c）胰蛋白酶处理，这些处理会导致视紫红质多肽链在（a）第323和324、236和237、241和242位氨基酸残基之间，（b）第327和328、240和241位氨基酸残基之间，或（c）第339和340位氨基酸残基之间发生裂解。在所有情况下，部分消化的视紫红质被证明能够产生光电势并增加吸附在磷脂浸渍火棉胶膜上的圆盘的膜电导。发现部分消化制剂和完整制剂中光电势的产生和消散动力学以及视紫红质II的形成和光诱导视紫红质质子化的动力学是相同的。在pH 6.0条件下孵育木瓜蛋白酶处理或嗜热菌蛋白酶处理的圆盘会诱导形成内翻囊泡，当将这些囊泡掺入火棉胶膜中时，会产生相反方向的光电势。用木瓜蛋白酶处理这种囊泡会导致多肽在第30和31、186和187位氨基酸残基之间进一步裂解。在用嗜热菌蛋白酶处理的内翻囊泡中还发现了另一个蛋白酶敏感位点，位于第104和105位氨基酸残基之间。这一事实与视觉视紫红质的“七柱”排列模式[奥夫钦尼科夫，尤·A.（1982年）《欧洲生物化学学会联合会快报》148，179 - 191]相符。视紫红质在两侧用木瓜蛋白酶处理时，在多肽中部的两个位点被裂解，缺失了60个氨基酸残基，但仍作为光电能量转换器发挥作用。木瓜蛋白酶处理或嗜热菌蛋白酶处理的视紫红质的光电响应所固有的主要特定特征，而天然蛋白质中不存在，是当完整制剂的响应变得可忽略不计时，前者在添加一连串饱和闪光后仍能存活。结果表明，这种效应是由于部分消化的视紫红质转化为一种光解产物，该产物在室温下即使在存在羟胺的情况下也能存活数分钟。532纳米的激光闪光有效地将该产物转化回视紫红质。
