"In situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes

Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With [³⁵S]GTPγS this is 0.15±0.2 min^-1. Treatment of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with K_d values of 10.2±2.3 nM [³⁵S]GTPγS, of 18.2±4.3 nM [α-³²P]GTP and of 28.6±3.5 nM [α-³²P]GDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65±7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding K_d values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified K_d values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.