Cytochalasin B binding to rabbit proximal tubular basolateral membranes.
Cytochalasin B binds to the Na+-independent D-glucose transporter in non-renal tissues. We have shown previously that the Na+-independent D-glucose transporter of the rabbit renal proximal tubular cell is localized exclusively in the basolateral membrane. To determine whether cytochalasin B binds to this renal transporter we measured binding of [3H]cytochalasin B to proximal tubular basolateral membranes isolated from rabbit kidneys. A steady state of binding is reached by 15 minutes at 20 degrees C over a concentration range of 0.01 to 50 microM. Non-linear regression analysis of cytochalasin B binding from 0.01 to 20 microM plotted according to Scatchard reveals two classes of binding sites with Kd 5.88 x 10(-8) M, Bmax 16.1 pmol/mg protein; and Kd 5.62 x 10(-5) M, Bmax 2816 pmol/mg protein. [3H]cytochalasin B (0.1 microM) binding to basolateral membranes is a reversible process; it is displacable by excess unlabeled cytochalasin B with a time course similar to binding of [3H]cytochalasin B. Binding of [3H]cytochalasin B is inhibited by 500 mM D-glucose (21%), 2-deoxy-D-glucose (57%) and 3-O-methyl-D-glucose (64%), but not by L-glucose. [3H]cytochalasin B binding is reduced 71% by 0.1 mM phloretin, but only 26% by 0.1 mM phlorizin. Such substrate specificity and inhibitor sensitivity are similar to those previously demonstrated in non-renal tissues by others as well as in rabbit renal proximal tubular basolateral membranes by us. Our data suggest that cytochalasin B binds to the Na+-independent D-glucose transporter or a component of the transporter in the renal proximal tubular basolateral membrane.
Cheung
P T
PT
Renal Division, Washington University School of Medicine, St. Louis, Missouri.
Hammerman
M R
MR
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism*; D003572:Cytochalasins / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000031:analogs & derivatives / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009183:Mycotoxins / Q000494:pharmacology; D010693:Phloretin / Q000494:pharmacology; D010695:Phlorhizin / Q000494:pharmacology; D011817:Rabbits
D003572:Cytochalasins; D009004:Monosaccharide Transport Proteins; D009183:Mycotoxins; C007215:cytochalasin E; D003571:Cytochalasin B; D010695:Phlorhizin; D005947:Glucose; D010693:Phloretin