Ortwerth B J, Olesen P R
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Exp Eye Res. 1989 May;48(5):605-19. doi: 10.1016/0014-4835(89)90003-1.
The water-insoluble fraction from mature bovine lens was solubilized to the same extent either by extraction with 6.0 M urea, by sonication of the suspended proteins or by a brief adjustment of the pH to 3.0 or 11.0. Sonication gave soluble protein levels of 50 mg ml-1 or greater with water or dilute buffers, but the presence of salt markedly diminished the solubility of the sonicated proteins. The sonicated proteins remained soluble upon storage at 5 degrees C, but were readily precipitated by either freezing or by the addition of salt. These re-precipitated proteins were once again insoluble when suspended in dilute aqueous buffers. Water-soluble alpha-crystallin at the same concentrations was unaffected by either high salt or freezing. The sonication-solubilized proteins were shown to be similar in aggregate size and polypeptide composition to the water-soluble HMW fraction isolated from the same lenses. An [125I]-labeled soluble HMW fraction was precipitated to the same extent as [125I]-labeled sonication-solubilized proteins upon freezing. The distribution of HMW aggregated protein between water-soluble aggregates and the water-insoluble fraction was unaltered by the presence of either dithiothreitol (DTT) or high levels of salt during the homogenization. The presence of either [125I]-labeled water-soluble HMW aggregates or [125I]-labeled water-insoluble sonicate supernatant during lens homogenization did not result in a significant incorporation of radioactivity into the water-insoluble fraction. These data argue that the water-insoluble fraction represents coalesced HMW aggregates which had already formed in the lens prior to homogenization. When the sonication-solubilized fraction was disaggregated in 6.0 M urea and then reaggregated by urea removal, the proteins no longer precipitated on freezing, and 85-90% of the protein eluted in the region of alpha-crystallin from an Agarose A-5m column. Only 3-6% of the original protein remained as a void volume peak, and was composed almost exclusively of highly crosslinked proteins. The limited solubility of the HMW proteins may therefore reflect the aggregate state of the alpha-crystallin rather than an inherent insolubility of the subunits.(ABSTRACT TRUNCATED AT 400 WORDS)
成熟牛晶状体的水不溶性部分，通过用6.0 M尿素提取、对悬浮蛋白质进行超声处理或短暂将pH值调至3.0或11.0，均可在相同程度上溶解。超声处理在用水或稀缓冲液时可得到50 mg/ml或更高的可溶性蛋白质水平，但盐的存在会显著降低超声处理后蛋白质的溶解度。超声处理后的蛋白质在5℃储存时仍可溶，但通过冷冻或加盐很容易沉淀。这些重新沉淀的蛋白质再次悬浮于稀水性缓冲液中时又变得不溶。相同浓度的水溶性α-晶状体蛋白不受高盐或冷冻的影响。经超声处理溶解的蛋白质在聚集大小和多肽组成上与从相同晶状体中分离出的水溶性高分子量部分相似。冷冻时，[125I]标记的可溶性高分子量部分与[125I]标记的经超声处理溶解的蛋白质沉淀程度相同。在匀浆过程中，二硫苏糖醇（DTT）或高浓度盐的存在并未改变高分子量聚集蛋白在水溶性聚集体和水不溶性部分之间的分布。在晶状体匀浆过程中，[125I]标记的水溶性高分子量聚集体或[125I]标记的水不溶性超声处理上清液的存在，并未导致放射性显著掺入水不溶性部分。这些数据表明，水不溶性部分代表了在匀浆前已在晶状体中形成的聚结高分子量聚集体。当经超声处理溶解的部分在6.0 M尿素中解聚，然后通过去除尿素重新聚集时，蛋白质在冷冻时不再沉淀，并且85 - 90%的蛋白质从琼脂糖A - 5m柱上以α-晶状体蛋白区域洗脱。只有3 - 6%的原始蛋白质保留为空体积峰，且几乎完全由高度交联的蛋白质组成。因此，高分子量蛋白质的有限溶解度可能反映的是α-晶状体蛋白的聚集状态，而非亚基的固有不溶性。（摘要截短于400字）
