Protein area occupancy at the center of the red blood cell membrane - PubMed (original) (raw)

Comparative Study

. 2008 Feb 26;105(8):2848-52.

doi: 10.1073/pnas.0712379105. Epub 2008 Feb 19.

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Comparative Study

Protein area occupancy at the center of the red blood cell membrane

Allison D Dupuy et al. Proc Natl Acad Sci U S A. 2008.

Abstract

In the Fluid Mosaic Model for biological membrane structure, proposed by Singer and Nicolson in 1972, the lipid bilayer is represented as a neutral two-dimensional solvent in which the proteins of the membrane are dispersed and distributed randomly. The model portrays the membrane as dominated by a membrane lipid bilayer, directly exposed to the aqueous environment, and only occasionally interrupted by transmembrane proteins. This view is reproduced in virtually every textbook in biochemistry and cell biology, yet some critical features have yet to be closely examined, including the key parameter of the relative occupancy of protein and lipid at the center of a natural membrane. Here we show that the area occupied by protein and lipid at the center of the human red blood cell (RBC) plasma membrane is at least approximately 23% protein and less than approximately 77% lipid. This measurement is in close agreement with previous estimates for the RBC plasma membrane and the recently published measurements for the synaptic vesicle. Given that transmembrane proteins are surrounded by phospholipids that are perturbed by their presence, the occupancy by protein of more than approximately 20% of the RBC plasma membrane and the synaptic vesicle plasma membrane implies that natural membrane bilayers may be more rigid and less fluid than has been thought for the past several decades, and that studies of pure lipid bilayers do not fully reveal the properties of lipids in membranes. Thus, it appears to be the case that membranes may be more mosaic than fluid, with little unperturbed phospholipid bilayer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Time progression of membrane shaving. (a) Membrane washes. RBC open ghost membranes were washed in 1 M KCl (K), 10 mM NaOH (N), or 100 mM Na2CO3 (O), and then shaved with 3.0 mg/ml proteinase K in the respective wash buffer for 24 h (C, control, membranes were not washed). The membrane samples were then run on 1D SDS/PAGE gels and subjected to standard Coomassie and silver staining procedures. 0, no shaving with proteinase K; 24, membranes were shaved with 3.0 mg/ml proteinase K for 24 h. (b) Elimination of contamination from hemoglobin. RBC open ghost membranes washed (w) in 10 mM NaOH were shaved with 3.0 mg/ml proteinase K in 10 mM NaOH for various lengths of time up to 48 h. The membrane samples were then run on 1D SDS/PAGE gels and subjected to standard immunoblot analysis by using anti-hemoglobin (Sigma, H4890). Lane M, markers; lane H, hemoglobin supernatant (positive control for hemoglobin). (c) Time progression of membrane shaving by using buoyant density measurements from equilibrium centrifugation in linear sucrose gradients. RBC plasma membrane open ghosts were progressively shaved by proteolytic digestion with 3.0 mg/ml proteinase K. The proteolytic digestion was stopped with 5 mM PMSF as a function of time. At each time point, buoyant density measurements were made by using equilibrium centrifugation in continuous, linear sucrose gradients. Membranes were loaded on 5–45% continuous, linear sucrose gradients made by using the BioComp Gradient Master and spun at 215,578 × g for 24 h. Gradients were then fractionated by using the BioComp Gradient Fractionator. The density of sucrose at which the sample band of membranes was found by refractometry (equal to a buoyant density measurement for the membrane itself) was measured by using a temperature-controlled, small-volume, automatic refractometer (Rudolph Research Analytical, J57). The density at each time point was measured independently a total of ten times. (d) Time progression of membrane shaving by using immunoblot analysis. RBC open ghost membranes washed (w) in 10 mM NaOH and unwashed (u) RBC open ghost membranes were shaved with 3.0 mg/ml proteinase K in 10 mM NaOH for various lengths of time up to 24 h. 0, control, no proteinase K; s, shaved for 5 s. The membrane samples were then run on 1D SDS/PAGE gels and subjected to standard immunoblot analysis. Three different antibodies were analyzed in the immunoblot analysis including anti-band 3 (Sigma B9277) (cytoplasmic), anti-glycophorins A and B (Sigma G7650) (extracellular), and anti-spectrin (α and β) (Sigma S3396) (cytoskeleton). Lane M, markers.

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