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PMID: 10692476 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Characterization of alpha-crystallin-plasma membrane binding.

The Journal of biological chemistry ·Vol. 275 ·No. 9 ·2000-03-03 ·Pages 6664-72

Cobb BA, Petrash JM

Abstract

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.

MeSH Terms
Acetates/chemistry Animals Cattle Cell Membrane/metabolism Chromones/chemistry Crystallins/metabolism Fluorescent Dyes/chemistry Humans Hydrogen-Ion Concentration Lens, Crystalline/metabolism Protein Binding Recombinant Proteins/metabolism Sodium Chloride/pharmacology Temperature Trypsin/metabolism
Chemicals
Acetates Alexa 350 Chromones Crystallins Fluorescent Dyes Recombinant Proteins Sodium Chloride Trypsin
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Cobb B A
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Petrash J M
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.275.9.6664
Published
2000-03-03
Pages
6664-72
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2902967
Subset
IM
Grants
NEI NIH HHS · EY50673 · United States
NEI NIH HHS · P30 EY002687 · United States
NEI NIH HHS · F31 EY006901-03 · United States
NEI NIH HHS · F31 EY006901 · United States
NEI NIH HHS · EY02687 · United States
NEI NIH HHS · EY06901 · United States
NIDDK NIH HHS · P60 DK020579 · United States
NIDDK NIH HHS · P30 DK020579 · United States
Corrections
ErratumIn
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External Links
PubMed source DOI: 10.1074/jbc.275.9.6664 PMC Full Text View journal details
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