p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats.
Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.
Basic Medical Sciences Division, National Institute for Environmental Studies, Ibaraki, Japan.
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M
Kunimoto
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K
Shibata
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T
Miura
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002729:Chloromercuribenzoates / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects*; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000648:ultrastructure; D005614:Freeze Fracturing; D006021:Glycophorins / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D051381:Rats; D012795:Sialoglycoproteins / Q000378:metabolism*; D013049:Spectrin / Q000378:metabolism*; D020245:p-Chloromercuribenzoic Acid
D000199:Actins; D002729:Chloromercuribenzoates; D006021:Glycophorins; D012795:Sialoglycoproteins; D013049:Spectrin; D020245:p-Chloromercuribenzoic Acid; D008274:Magnesium; D002118:Calcium