Studies on the nature of the water-insoluble fraction from aged bovine lenses

Abstract

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°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 α-crystallin at the same concentrations was unaffected by either high salt or freezing. The sonication-solublized 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 α-crystalli 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 α-crystallin rather than an inherent insolubility of the subunits. The formation of the HMW proteins may be due to only a small number of crosslinked subunits, however, the possibility of a dialyzable aggregating molecule cannot be eliminated.