Glucose transport by lobster hepatopancreatic brush-border membrane vesicles

Abstract

Epithelial brush-border membrane vesicles (BBMV) were made from lobster hepatopancreas by using Mg2+ precipitation. Alkaline phosphatase, Na+-K+-ATPase, and cytochrome c oxidase activities in these vesicles were enriched 15.0-, 1.0-, and 0.19-fold, respectively, compared with activities of a washed original homogenate pellet, indicating a relatively pure apical membrane preparation reduced in basolateral or organelle contamination. Complete vesicular closure was confirmed with electron microscopy and equilibrium [3H]D-glucose uptake experiments using various transmembrane osmotic gradients. Glucose uptake was stimulated by a transmembrane Na+ gradient but not by an identical K+ gradient or by a Na+ gradient in the presence of phloridzin. Electrogenicity of Na+-dependent glucose transport was confirmed in two ways. First, an anion permeability sequence indicated glucose uptake was stimulated in the following order: SCN- greater than Cl- greater than gluconate- greater than SO4(2-). Second, an outwardly directed valinomycin-induced K+ diffusion potential, rendering the vesicle interior electrically negative, enhanced glucose uptake compared with K+-loaded vesicles lacking the ionophore. Glucose influx occurred by a combination of carrier-mediated transfer, illustrating Michaelis-Menten kinetics, and nonsaturable “apparent diffusion.” pH (same on both sides) strongly influenced Na+-dependent glucose uptake according to the sequence: pH 6.0 greater than pH 7.4 greater than pH 8.0. Increased proton concentration lowered the Michaelis constant for glucose transport and increased the apparent diffusional permeability of the membrane to the sugar. Maximal carrier-mediated glucose transport rate was largely unaffected by pH.(ABSTRACT TRUNCATED AT 250 WORDS)