J Biol Chem. 2000 Mar 3;275(9):6664-72.

Characterization of alpha-crystallin-plasma membrane binding.

Source

Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

Erratum in

J Biol Chem 2002 Jan 11;277(2):1628.

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.

PMID:: 10692476; [PubMed - indexed for MEDLINE]
PMCID:: PMC2902967

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Fig. 2

Binding capacity for α-crystallin membrane binding

Panel A, binding capacity curves. Assays were performed using 500 μg of membranes and a varied amount of α-crystallin. The amount bound was calculated using the fluorescence of the membrane pellet and the specific activity for each α-crystallin complex. The binding capacity (B) was calculated using the asymptote of each curve and was 6.59 ± 0.13 ng/μg of membrane for αA-crystallin (●), 4.23 ± 0.17 ng/μg of membrane for αB-crystallin (○), and 3.45 ± 0.11 ng/μg of membrane for the 3:1 heteropolymer (×). (n = 3) Panel B, effects of BSA and γD-crystallin. Control assays were performed with 500 μg of plasma membranes and 25 μg of conjugated α-crystallin with and without 25 μg of control protein (either BSA or γD-crystallin). Samples A–C show the effect of the control proteins on the binding of αA-crystallin; samples D-F show their effects on αB-crystallin binding. Data were normalized to the “no addition” data. (n = 3).

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Fig. 4

Effect of NaCl on αB-crystallin binding at pH 7.3 and 5.0

Assays were performed with 8 μg of αB-crystallin and 500 μg of membranes in Tri-buffer at either pH 5.0 (□) or 7.3 (

) for 6 h with a range of NaCl concentrations between 0 and 1 M, with 0 M NaCl serving as the reference. All points are the average of at least three repetitions.

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Fig. 6

Affinity plots of α-crystallin membrane binding

Binding assays were performed using 8 μg of conjugated α-crystallin with a varied amount of membranes under standard conditions. The apparent binding constant K at each point was calculated as described under “Experimental Procedures.” For αA-crystallin (●), a linear horizontal plot was observed with a binding constant of 5.89 ± 0.27 × 10⁻⁴ μg⁻¹ of membrane (n = 5). For αB-crystallin (○), a sigmoidal curve was found, with two binding constants, K_high = 14.9 ± 2.5 × 10⁻⁴ μg⁻¹ of membrane and K_low = 5.60 ± 0.50 × 10⁻⁴ μg⁻¹ of membrane (n = 5). The 3:1 heteropolymer (×) has a linear horizontal plot and a binding constant of 4.57 ± 0.50 × 10⁻⁴ μg⁻¹ of membrane (n = 3).

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Fig. 8

Effects of membrane trypsinization on α-crystallin binding affinity

Plasma membranes were treated with trypsin for 3 h at 37 °C. These membranes were then used in binding assays in which the amount of membrane was varied between 80 and 5,000 μg, and the α-crystallin concentration was held constant at 8 μg. For all three graphs n = at least 3; ○, trypsinized membranes; and ●, nontreated membranes. Panel A, αA-crystallin. Both the trypsinized and nontrypsinized affinity plots are roughly linear and horizontal and indicate binding constants of 5.89 ± 0.27 × 10⁻⁴ μg⁻¹ and 2.88 ± 0.75 × 10⁻⁴ μg⁻¹ of membrane, respectively, as well as an overall 2-fold loss of affinity. Panel B, αB-crystallin. With nontreated membranes αB-crystallin shows marked positive cooperativity, with a K_low of 5.60 ± 0.50 × 10⁻⁴ μg⁻¹ of membrane, but with trypsinized membranes, a linear plot was observed with a binding constant of 2.69 ± 0.24 × 10⁻⁴ μg⁻¹ of membrane, which is a 2-fold reduction. Panel C, 3:1 heteromeric complex. The binding constant with trypsinized membranes, 1.78 ± 0.26 × 10⁻⁴ μg⁻¹ of membrane, versus nontreated membranes, 4.57 ± 0.50 × 10⁻⁴ μg⁻¹ of membrane, represents roughly a 2-fold reduction in affinity.

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Fig. 1

SDS-PAGE of membrane and protein preparations

Fractionated plasma membranes (100 μg, lane 1) as well as the αA- and αB-crystallins (0.8 μg, lanes 2 and 3) were resolved by SDS-PAGE. The membrane sample mainly shows MP26, whereas the amount of endogenous α-crystallin is essentially undetectable (labeled arrows indicated along gel). Both αA- and αB-crystallin subunits were essentially homogeneous. Lanes 4 and 6 show 33% of a pellet resulting from binding assays in which 500 μg of membrane was incubated with 16 μg of either nonconjugated homopolymeric complex. Lanes 5 and 7 show an equivalent fraction of the pellet resulting from similar binding assays containing Alexa350^®-conjugated homopolymers. Based on digital image analysis of multiple samples, the amount of α-crystallin bound was not affected significantly by Alexa350^® conjugation, although the protein bands for each conjugate are somewhat more diffuse.

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Fig. 3

Characterization of α-crystallin membrane binding

All binding assays were incubated with an equivalent amount of membranes (500 μg) and α-crystallin (8 μg) for 6 h at 37 °C in PBS supplemented with 5 mM MgCl₂, except as indicated. For the bar graphs ■, αA-crystallin; □, αB-crystallin; and

, the 3:1 heteropolymer. For the line graph ●, αA-crystallin; ○, αB-crystallin; and ×, the 3:1 heteropolymer. All data points shown are the average of at least three replicates. Panel A, time dependence of membrane association. Assays were incubated for a range of time points between 0 and 16 h at 37 °C. For all assays presented each data point is the average of at least three replicates. Panel B, salt dependence of membrane association. All assays were carried out in phosphate buffer, pH 7.3, containing a range of NaCl concentrations from 0 to 1 M, with 0 M NaCl serving as the reference. Panel C, pH dependence of membrane association. Assays were incubated for 6 h in Tri-buffer at a pH range of 5.0–9.0, with pH 7.0 as the reference binding activity. Panel D, temperature dependence of membrane association. Assays were performed in the standard buffer at a range of temperatures from 4 to 45 °C for 6 h, with the 37 °C measurements serving as the reference.

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Fig. 5

Reversibility of α-crystallin membrane binding

Binding assays were performed using a constant membrane concentration and 8 μg of Alexa350^®-conjugated α-crystallin protein under standard conditions. Once formed, the α-crystallin-membrane complexes were resuspended in binding buffer. For competition experiments, membrane-bound α-crystallin was incubated for >24 h at 37 °C with 100 μg of nonconjugated α-crystallin and 100 μg of control protein (BSA or γD-crystallin) or buffer alone (n = 3). After centrifugation, the α-crystallin-membrane complex (pellet) and supernatant were analyzed for fluorescence and compared with the amount bound during the first incubation. There was no observable effect of either BSA or γD-crystallin on αA-(samples A–C) and αB- (samples D–F) crystallin reversibility. In titration experiments (inset), a maximum of 44 ± 2% and 25 ± 1% of bound αA- (●) and αB- (○) crystallins, respectively, could not be removed after treatment of membranes with up to a 50-fold molar excess of the corresponding nonconjugated protein subunit (n = 2).

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Fig. 7

Effect of membrane trypsinization on α-crystallin binding capacity

Plasma membranes were treated with trypsin for 3 h at 37 °C. These membranes were then used in binding assays in which the amount of membrane was held constant at 500 μg, and the α-crystallin concentration was varied from 0 to 64 μg. For all three graphs, n = 3; ○, trypsinized membranes; and ●, non-treated membranes. Panel A, αA-crystallin; panel B, αB-crystallin; panel C, 3:1 heteropolymer.

Characterization of α-Crystallin-Plasma Membrane Binding

J Biol Chem. J Biol Chem;275(9):6664-6672.

Publication Types, MeSH Terms, Substances, Grant Support

Publication Types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

MeSH Terms

Acetic Acids/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

Substances

Acetic Acids
Alexa 350
Chromones
Crystallins
Fluorescent Dyes
Recombinant Proteins
Sodium Chloride
Trypsin

Grant Support

EY02687/EY/NEI NIH HHS/United States
EY06901/EY/NEI NIH HHS/United States
EY50673/EY/NEI NIH HHS/United States
F31 EY006901-03/EY/NEI NIH HHS/United States
etc

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Characterization of alpha-crystallin-plasma membrane binding.

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