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 Kd 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 Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd 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.
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - Feb 28 1989|
ASJC Scopus subject areas
- Molecular Biology