Kinetics of sodium D-glucose cotransport in bovine intestinal brush border vesicles

Abstract

Brush border membrane vesicles ( BBMV ) purified from steer jejunum were used to study the kinetics of sodium D-glucose cotransport under voltage clamped, zero-trans conditions. When the initial rate of glucose transport ( Jgluc ) was measured over a wide range of glucose concentrations ([S] = 0.01-20 mM), curvature of the Woolf - Augustinsson -Hofstee plots was seen, compatible with a diffusional and one major, high capacity (maximal transport rate Jmax = 5.8-8.8 nmol/mg X min) saturable system. Further studies indicated that changes in cis [Na] altered the Kt, but not the Jmax, suggesting the presence of a rapid-equilibrium, ordered bireactant system with sodium adding first. Trans sodium inhibited Jgluc hyperbolically, KCl-valinomycin diffusion potentials, inner membrane face positive, lowered Jgluc , while potentials of the opposite polarity raise Jgluc . At low glucose concentrations ([S] less than 0.05 mM), a second, minor, high affinity transport system was indicated. Further evidence for this second saturable system was provided by sodium activation curves, which were hyperbolic when [S] = 0.5 mM, but were sigmoidal when [S] = 0.01 mM. Simultaneous fluxes of 22Na and [3H]glucose at 1 mM glucose and 30 mM NaCl yielded a cotransport-dependent flux ratio of 2:1 sodium/glucose, suggestive of 1:1 (Na/glucose) high capacity, low affinity system and a approximately 3:1 (Na/glucose) high affinity, low capacity system. Kinetic experiments with rabbit jejunal brush borders revealed two major Na-dependent saturable systems. Extravesicular (cis) Na changed the Kt, but not the Jmax of the major system.