Bacteriorhodopsin's M412 intermediate contains a 13-cis, 14-s-trans, 15-anti-retinal Schiff base chromophore.
Bacteriorhodopsin's M412 intermediate contains a 13-cis, 14-s-trans, 15-anti-retinal Schiff base chromophore.
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细菌视紫红质的 M412 中间体含有 13-顺式、14-s-反式、15-抗视网膜希夫碱发色团。
DOI：
10.1021/bi00435a009
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Mathies,RA
中科院分区：
文献类型：
--
作者：
Ames,JB;Fodor,SP;Gebhard,R;Raap,J;vandenBerg,EM;Lugtenburg,J;Mathies,RA
材料和方法样品制备。培养盐生H.halobium(ET 1001)，并按照先前发表的程序提纯紫色膜(Braiman&Mathies，1980)。天然紫膜在1M氢氧化铵中、pH 7.8、37℃的黑暗中漂白12-14小时(Fodor等人，1988a)。然后将漂白的膜制粒，并用pH为7的10 mM HEPES缓冲液洗涤三次，以去除未反应的NH_2OH。漂白的膜用等量的等量同位素标记的全反式视网膜溶解在最小体积的乙醇中滴定。用吸光度增加568 nm来监测再生情况。用2%不含脂肪酸的牛血清白蛋白反复洗涤视网膜，直到在350 nm(~10次)处检测不到视网膜的血氧吸光度。拉曼实验中使用的样品包括悬浮在0.2M氯化钠(低盐)或3Mkc1(高盐)中的紫膜碎片，以及在pH为7的10 mM磷酸盐或高pH下的10 mM的硼酸盐。全视网膜同位素衍生物(15-13C；14，15-13C，15-D；14，15-D2；和15-D)是按照以前发表的程序合成的(Pardoen等人，1984)。[e-，3C]赖氨酸BR是按照Argade等人(1981)的方法制备的。同位素纯度分别为~gt；98%和~gt；95%。
Materials and MethodsSample Preparation. Cultures of H. halobium (ET 1001) were grown and the purple membrane was purified according to previously published procedures (Braiman & Mathies, 1980). Native purplemembrane was bleached in 1M NH2OH at pH 7.8 in the dark for 12-14 h at 37 C (Fodor et al, 1988a). The bleached membrane was then pelleted and washed with pH 7, 10 mM HEPES buffer three times to remove unreacted NH2OH. The bleached membrane was titrated with aliquots of isotopically labeled all-trans-retinal dissolved in a minimum volume of ethanol. The regeneration was monitored by the absorbance increaseat 568 nm. Excess retinal and residual oximes were removed by repeated washes in 2% fatty acid free bovine serum albumin until no detectable retinal oximeabsorbance remained at 350 nm (~ 10 washes). Samples used in the Raman experiments consisted of purple membrane fragments suspended in 0.2 M NaCl (low salt) or 3 Mkc1 (high salt) with 10 mM phosphate at pH 7 or 10 mM borate at high pH. The all-trans-retinal isotopic deriv-atives (15-13C; 14, 15-13C, 15-D; 14, 15-D2; and 15-D) were synthesized according to previously published procedures (Pardoen et al., 1984). The [e-, 3C] lysine BR was prepared following the procedure of Argade et al.(1981). The isotopic purity was> 98% for each deuterium derivativeand> 95% for the l3C derivatives.