Studies on the nature of the water-insoluble fraction from aged bovine lenses.
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)
Ortwerth
B J
BJ
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Olesen
P R
PR
D000375:Aging / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003459:Crystallins; D008970:Molecular Weight; D011189:Potassium Chloride; D011487:Protein Conformation; D011489:Protein Denaturation; D012995:Solubility; D013010:Sonication; D014508:Urea
D003459:Crystallins; D011189:Potassium Chloride; D014508:Urea