Abstract
Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.
Highlights
From the Departments of Slnternal Medicine and llPharmacology, University of Texas Medical Schoolat Houston, Houston, Texas 77225
The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, Forskolin and 7-0-hemisuccinyl-7-desacetylforskoliwnere obtained from Behring Diagnostics
Dihydrocytochalasin B, L-rhamnose,L-glucose,D-ga- hemoglobin-freemembranes (“gbosts”)were prepared by the method lactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3- of Dodge et al (6).The membranes were suspended in 5 mM sodium
Summary
Lin to human erythrocyte membraneswas measuredby Material~-[4-~H]Cytochalasin B, [12-3H]forskolin, and [U-14C]. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasinB or [12-3H]forskolin in a glucose-displaceable manner. Binding Assays-Binding of [4-3H]cytochalasin B or [12-3H]forskolin to erythrocyte membranes was determined by a centrifugation method. These results indicate that forskolin, in concentrations [4-3H]cytochalasinB or0.35 pCiof [12-3H]forskolin),nonradioactive routinely employed for stimulation of adenylate cy- competing ligand as appropriate, monosaccharide From 3 to 9% free ligand was trapped in the pellets, as determined by parallel incubations with ['4C]sucrose,which does not enter human erythrocytes (11).All the data have been appropriately corrected for counting efficiency and for trapping, which were measured independently in each day's experiment. The data were analyzed by the "LIGAND" program of Munson and Rodbard (12),kindly provided by Drs Peter Munson and Michael Beveridge of the National Institutes of Health
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