"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]GTP gamma S 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]GTP gamma S, of 18.2 +/- 4.3 nM [alpha-32P]GTP and of 28.6 +/- 3.5 nM [alpha-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.
Damonte
G
G
Institute of Biochemistry, University of Genoa, Italy.
Morelli
A
A
Piu
M
M
Longone
P
P
De Flora
A
A
D001667:Binding, Competitive; D003902:Detergents / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000097:blood*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000097:blood; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000097:blood; D006801:Humans; D007700:Kinetics; D013873:Thionucleotides / Q000097:blood
D003902:Detergents; D006150:Guanine Nucleotides; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate