Stanton J Brett, Goldberg Andrew F X, Hoppe George, Marmorstein Lihua Y, Marmorstein Alan D
Department of Ophthalmology and Vision Science, University of Arizona, Tucson, AZ 85711, and Cole Eye Institute, Cleveland Clinic Foundation, OH 44195, USA.
Biochim Biophys Acta. 2006 Feb;1758(2):241-7. doi: 10.1016/j.bbamem.2006.01.024. Epub 2006 Mar 6.
Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl- channel and/or a regulator of Ca++ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S20,w) of 4.9, and partial specific volume (nu) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be approximately 138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (Vo) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.
贝斯特罗芬-1（Best-1）是一种整合膜蛋白，其缺陷会导致贝斯特卵黄样黄斑营养不良。据推测，Best-1作为一种氯离子通道和/或钙离子通道的调节剂发挥作用。已有报道称重组Best-1的化学计量为四聚体（或五聚体）。我们结合凝胶排阻色谱法和速度沉降法研究了天然Best-1的四级结构，发现它以单一物种形式迁移，斯托克斯半径为7.3 nm，沉降系数（S20,w）为4.9，比容（ν）为0.80 ml/g。蛋白质-去污剂复合物的质量经计算为206 kDa，蛋白质成分估计约为138 kDa。鉴于单体质量为68 kDa，我们得出结论，用Triton X-100溶解的天然Best-1是同型二聚体。通过凝胶排阻色谱法比较重组Best-1和组织来源的Best-1，研究了这一观察结果与先前报告之间的差异。与从视网膜色素上皮（RPE）细胞中提取的Best-1不同，许多重组Best-1在柱空体积（Vo）部分洗脱。我们得出结论，Best-1的最小功能单位是二聚体。这种化学计量与先前测量的重组Best-1的化学计量不同，这表明有必要进一步研究以确定RPE膜中功能性Best-1的化学计量。
