alpha-Crystallin, the major protein component of vertebrate lenses, forms a large complex comprised of two homologous subunits, alphaA- and alphaB-crystallin. It has the ability to suppress stress-induced protein aggregation in vitro, bind saturably to lens plasma membranes, and aid in light refraction through short-range ordering. Recently, a missense mutation in alphaA-crystallin that changes arginine 116 to a cysteine residue (R116C) was genetically linked to one form of autosomal dominant congenital cataracts. This point mutation is reported to cause structural alterations at many levels as well as a 4-fold reduction in chaperone-like activity. To extend these findings, we examined the quaternary stability of the alphaA R116C mutant protein and its effect on chaperone-like activity, subunit exchange, and membrane association. Homocomplexes of mutant subunits become highly polydisperse following incubation at 37 degrees C, reflecting the likely in vivo distribution of the complexes. Chaperone-like activity of the alphaA R116C mutant is approximately 4-fold lower than wild type, whether measured before or after conversion to a polydisperse population with incubation. alphaA R116C complexes also have a 4-fold reduced ability to exchange subunits with wild-type complexes. Finally, membrane binding capacity measurements of mutant subunits showed a 10-fold increase over wild type. Our results, in conjunction with previous reports, suggest that the changes in complex polydispersity, the reduction of subunit exchange, and increased membrane binding capacity are all potential factors in the pathogenesis of alphaA R116C associated congenital cataracts.
Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a variety of aldehydes and carbonyls, including monosaccharides. Intense interest in the discovery and characterization of inhibitors has developed since the action of this enzyme has been linked to the pathogenesis of some diabetic complications. Since past studies indicated that most inhibitors act noncompetitively or uncompetitively versus substrate in the direction of aldehyde reduction, it was assumed that they bind at one or more sites distinct from the active site. However, the crystal structure of aldose reductase complexed with inhibitor [Wilson et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9847-9851] unambiguously revealed the inhibitor bound at the active site. The present study was undertaken to address this apparent discrepancy. Using a fluorometric assay, it was determined that zopolrestat, an acetic acid-type inhibitor, bound to aldose reductase complexed with either NADPH or NADP+. In contrast, the spirohydantoin-type inhibitor sorbinil demonstrated preferential binding to the binary enzyme.NADPH complex. Prior incubation of the enzyme.NADPH complex with zopolrestat prevented subsequent sorbinil binding. These results, together with the published structure of the ternary enzyme.NADPH.zopolrestat complex, are consistent with the conclusion that both sorbinil and zopolrestat bind at the active site. We propose that mixed inhibition patterns previously observed with sorbinil are due to inhibitor binding to both structural isomers of the enzyme.NADPH complex. Similar patterns for inhibition by zopolrestat are due to tight binding of the inhibitor. Substrate inhibition in the direction of aldehyde reduction occurs as a result of substrate binding to the enzyme.NADP+ complex.
The action of aldose reductase has been implicated in the etiology of a variety of diabetic complications affecting the visual system. However, very little is known regarding the structure and functional organization of the genes encoding this key enzyme. In the present study, we have isolated and characterized complementary DNA clones encoding bovine lens aldose reductase. Nucleotide sequencing of four independently isolated clones was used to establish a 1154 nucleotide composite cDNA sequence. The cDNA sequence encodes 296 amino acids of the aldose reductase primary structure, and contains an additional 261 nucleotides of apparently untranslated sequence downstream from the coding region. No nucleotide sequence differences were found among the four independently isolated aldose reductase cDNA clones. The aldose reductase amino acid sequence deduced from the cDNA shows high homology to that reported for aldose reductase of the rat lens. Significant similarities are also evident between bovine lens aldose reductase and both human liver aldehyde reductase and frog lens rho-crystallin.
Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized.
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