Binding characterization of a putative cGMP transporter in the cell membrane of human erythrocytes.
Cyclic GMP is an intracellular signal molecule whose biological and pharmacological role is not well understood. Recent studies with human erythrocytes and other cell types (normal and transformed) have shown that the extrusion of cGMP is an ATP-dependent and saturable process. In this paper, we present our studies on binding of [3H]-cGMP to human erythrocyte ghost and its solubilized extracts. At 4 degrees C, an apparent dissociation constant of 0.15 microM was found in the samples. Maximum specific binding values in ghost and solubilized extracts were 9.0 pmol/mg of protein and 1.0 pmol/mg of protein, respectively. The low dissociation constant was confirmed by kinetic studies with a value of 0.16 microM. Specific [3H]-cGMP binding was inhibited by cAMP, cGMP, and cIMP with KD values of 0.22 microM, 0.09 microM, and 0.17 microM, respectively. Unlabeled cGMP and cIMP inhibited [3H]-cGMP binding completely whereas cAMP inhibited only 70%. The membrane-localized cGMP-binding protein discriminates between cyclic and noncyclic nucleotides, since GMP, IMP, and AMP were unable to displace [3H]-cGMP. A zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate), was able to solubilize a protein with identical binding affinity. The results of this study show that erythrocyte ghosts possess a cGMP-binding protein which is not a kinase (due to a similar affinity for cAMP, cGMP, and cIMP) or phosphodiesterase (due to the inability of IBMX, 3-isobutyl-1-methylxanthine, to inhibit specific [3H]-cGMP binding). We hypothesize that this protein is the cell membrane cGMP transporter.
Boadu
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Department of Pharmacology, Institute of Medical Biology, University of Tromso, N-9037 Tromso, Norway.
Sager
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D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism*; D006801:Humans
D002352:Carrier Proteins; D006152:Cyclic GMP