The binding of D-glucose to the isolated luminal membrane of the renal proximal tubule.

Abstract The specific binding of d-glucose to the isolated luminal cell membrane of the renal proximal tubule, the brush border, was studied. The initial interaction of d-glucose with this membrane was rapid; saturable; dependent on the concentrations of the sugar and the membrane; reversible; selectively influenced by the ionic environment; sensitive to temperature and pH; inhibited by phloridzin, phloretin, and sulfhydryl reagents; and stereospecific. Examination of the relationship between the concentration of d-glucose and the binding of the sugar indicated the presence of two distinct classes of receptor sites for d-glucose in the brush border membrane. One system, having a high affinity class of binding sites, was saturated at about 1 µm, half-saturated at 0.67 µm, and 1.4 x 10-3 nmoles of d-glucose interacted with 1 mg of membrane protein. The other system, having a low affinity class of binding sites, required 50 mm for saturation, and had an affinity for d-glucose at least four-orders of magnitude less than that of the high affinity receptor sites. The time courses of binding of d-glucose in the high and low affinity regions were similar, in both cases approximately 50% of the maximal bindings occurred in the 1st min of incubation and steady state levels were achieved in about 3 min. The specific bindings of d-glucose at both receptor sites were completely reversible. Neither system of binding required Na+. The initial interaction of d-glucose with the brush border membranes was inhibited by Tris ions, but Li+ and choline ions substituted for Na+. Binding of d-glucose to the brush border membrane was markedly dependent on the presence of Ca2+. Binding at the high affinity classes of sites was more sensitive to the removal of Ca2+ than was the binding at the low affinity classes. The deletion of Mg2+ caused a moderate decrease in the binding at both affinity regions. High concentrations of phosphate inhibited binding at the low affinity classes of binding sites. Glutamine was also inhibitory. Phloridzin was found to be a potent inhibitor of the interaction of d-glucose with the receptors on the renal brush border membrane. Kinetics typical of competitive inhibition were observed at the high affinity region of d-glucose binding. The apparent Ki was 2 x 10-4 m. The kinetics of inhibition at the low affinity classes of binding sites were not consistent with competitive inhibition. The inhibition by phloretin at the high affinity sites was noncompetitive. The sulfhydryl reagents, N-ethylmaleimide and mersalyl, blocked the binding of d-glucose at both the high and low affinity sites. The binding of d-glucose was found to differ from that of l-glucose. Neither the binding of d- or l-glucose was affected by the presence of the other stereoisomer. When incubated together, the total sugar bound was the sum of the d-glucose bound alone plus the l-glucose bound alone. The interaction of l-glucose was insensitive to phloridzin. d-Galactose, a competitive inhibitor of d-glucose binding, did not influence the binding of l-glucose. l-Glucose, in contrast to d-glucose, did not reverse the binding of d-glucose. The l-glucose sequestered by the luminal membranes was not releasable by d- or l-glucose. These notable differences between the interactions of d- and l-glucose with the brush border membranes strongly indicated that the two stereoisomers did not share the same carrier transport system on the proximal tubule luminal membrane. It is proposed that the binding of d-glucose to the isolated brush border membranes, as characterized in this report, represents the initial reaction in the active transport of the sugar by the renal tubule.