Study of human erythrocyte membrane protein interactions by selective solubilization of Triton-skeletons.
The membrane skeleton, responsible for shape and mechanical properties of the red cell, was purified by the Triton extraction procedure in presence of 5 mM, 150 mM or 600 mM NaCl. The proportion of spectrin, protein 4.1 and actin present in erythrocyte skeletons does not depend on the molarity of NaCl used. In contrast ankyrin, protein band 3 and protein 4.2 are removed from skeletons as the ionic strength increased. Solubilization assays of membrane skeletons were used to study protein interactions inside the skeleton. Solubilization was performed by Tris, a non-selective disruptive reagent, or by p-mercuribenzene sulfonic acid (PMBS), which principally release spectrin and actin. Tris action was assessed by calculation of the percentage of solubilized proteins, which increased proportionally with Tris molarity. PMBS action was kinetically determined as the decrease in skeleton turbidity. With these two reagents, we observed a lower dissociation of skeletons prepared with high ionic strength buffer. Erythrocyte pretreatment with okadaic acid, an inhibitor of serine-threonine phosphatases, revealed a phosphorylation-induced skeleton gelation and a better resistance to Tris-solubilization.
Navarro-Prigent
M J
MJ
INSERM U409, Hôpital Beaujon, Clichy, France.
Séguin
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I
Boivin
P
P
Dhermy
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D
D000199:Actins / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D017487:Ankyrins / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009479:Neuropeptides; D017830:Octoxynol; D010766:Phosphorylation; D012995:Solubility; D013049:Spectrin / Q000378:metabolism; D014325:Tromethamine
D000199:Actins; D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; C028841:erythrocyte membrane band 4.1 protein; C044791:erythrocyte membrane band 4.2 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D014325:Tromethamine; D013049:Spectrin; D017830:Octoxynol