The broad objective of this research is to determine the molecular basis of cataracts produced by inhibitors of lens cholesterol biosynthesis. Since development of the U18666A and human senile cataract both likely involve early damage to lens membrane and increased crystallin binding to this membrane, describing the mechanism of the U18666A cataract could contribute to improved understanding of human cataracts. The specific objective is to determine the mechanism by which changing lens membrane sterol composition leads to increased membrane association of crystallins and to protein insolubilization. The work is divided into four phases. 1. We will identify the specific soluble polypeptides which give rise to the insoluble protein that accumulates in the U18666A cataract and their spacial location in the lens at the time of insolubilization. Lens crystallins will be pulse labeled in vivo from 3H-leucine in rats before onset of treatment with U18666A. Label polypeptides will be identified by coupling flatbed isoelectric focusing with fluorography and immunoblotting. 2. The association of crystallins with lens plasma membrane fractions during cataract development could provide nucleation points for insolubilization of cytosolic proteins. Membrane fractions will be recovered with their full complement of extrinsic proteins. These proteins will be identified by SDS-PAGE, IEF and immunoblotting. 3. Fatty acid acylation of lens proteins could be a means for attachment of extrinsic proteins to the lens plasma membrane and could also participate in the control of lens cell proliferation and differentiation. The capacity of the intact lens and cultured lens epithelial cells to acylate membrane and cytoplasmic proteins with 3H-labeled fatty acids will be determined. Proteins will be identified by electrophoretic and immunological methods. The structure of the proteins will be studied by amino acid sequencing. 4. The link between inhibition of lens cholesterol synthesis and disorganization of lens membrane structure will be addressed by examining U18666A effects on the coordination of lens membrane synthesis. Increased exposure of hydrophobic domains of intrinsic proteins due to altered membrane structure could be the basis of the increased crystallin binding to cataractous membrane. The order of assembly of the molecular components of the fiber cell plasma membrane in control and U18666A exposed lenses will be determined by measuring the lens regional distribution of synthesis of the membrane's principal molecular components (cholesterol, phospholipid and MIP26) using 3H2O as substrate.
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