Selective association of crystallins with lens 'native' membrane during dynamic cataractogenesis.
Plasma membrane with its associated extrinsic proteins was isolated from normal and cataractous rat lenses by centrifugation of the total water insoluble fraction from homogenized lenses on a discontinuous sucrose gradient. Membrane, which we call "native" membrane, was recovered mainly from the 25/45% sucrose interface. Development of the experimental U18666A cataract resulted in plasma membrane shifting to higher density (the 50/55% sucrose fraction) and great increases in the urea soluble protein content of the lens. At early stages of cataract development, most of the increased urea soluble protein was membrane associated, presumably as extrinsic protein. With advancing cataract, most of the urea soluble protein appeared in an essentially membrane-free pellet fraction. The urea soluble protein associated with the cataract membrane was shown by combined IEF, SDS-PAGE, Western blotting, amino acid compositional analysis and protein sequence determinations to be mainly composed of modified alpha- and beta-crystallins. Alpha A-crystallin truncated by not more than 27 residues from the carboxyl terminus plus beta b1 crystallin truncated by 49 residues from the amino terminus were conclusively identified. In addition to beta b1, a population of six alpha-crystallin derived polypeptides were specifically enriched in the cataract membrane fraction. Four of these six alpha-crystallins appear to be truncated from their carboxyl terminus, a modification which should have increased their hydrophobicity. The pellet fraction, which accumulated in the lens nucleus as the cataract advanced, was enriched in urea soluble gamma-crystallin derived polypeptides. We suggest that protein insolubilization in this experimental cataract involves the selective and tight association of principally modified alpha-crystallins to the fiber cell plasma membrane.
Cenedella
R J
RJ
Department of Biochemistry, Kirksville College of Osteopathic Medicine, MO 63501.
Fleschner
C R
CR
D000595:Amino Acid Sequence; D000736:Androstenes; D000818:Animals; D000924:Anticholesteremic Agents; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003459:Crystallins / Q000032:analysis / Q000378:metabolism*; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D007908:Lens, Crystalline / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility
D000736:Androstenes; D000924:Anticholesteremic Agents; D003459:Crystallins; D008565:Membrane Proteins; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one