Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups.
1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.
D000596:Amino Acids / Q000032:analysis; D001224:Aspartic Acid; D001665:Binding Sites; D001798:Blood Proteins; D002118:Calcium / Q000378:metabolism*; D002127:Calcium Isotopes; D002462:Cell Membrane / Q000378:metabolism*; D002729:Chloromercuribenzoates; D004912:Erythrocytes / Q000378:metabolism*; D005971:Glutamates; D005998:Glycine / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D009438:Neuraminic Acids; D009439:Neuraminidase; D011485:Protein Binding
D000596:Amino Acids; D001798:Blood Proteins; D002127:Calcium Isotopes; D002729:Chloromercuribenzoates; D005971:Glutamates; D009438:Neuraminic Acids; D001224:Aspartic Acid; D009439:Neuraminidase; D002118:Calcium; D005998:Glycine