Characterization of alpha-crystallin-plasma membrane binding.
Alpha-crystallin, a large lenticular protein complex made up of two related subunits (alphaA- and alphaB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human alphaA- and alphaB-crystallins conjugated to a small fluorescent tag (Alexa350). Both alphaA and alphaB homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of alpha-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that alpha-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3. 45 +/- 0.11 ng/microg of membrane and 4.57 +/- 0.50 x 10(-4) microg(-1) of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed alpha-crystallin complex may hold particular relevance for the function of alpha-crystallin within the lens.
Cobb
B A
BA
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Petrash
J M
JM
D000085:Acetates / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002867:Chromones / Q000737:chemistry; D003459:Crystallins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007908:Lens, Crystalline / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D000085:Acetates; C400304:Alexa 350; D002867:Chromones; D003459:Crystallins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012965:Sodium Chloride; D014357:Trypsin