Abstract
The principal aim of the present study was to investigate the effects of variation in proton gradient and membrane potential on the transport of glycyl-L-glutamine (Gly-Gln) by renal brush border membrane vesicles. Under our conditions of transport assay, Gly-Gln was taken up by brush border membrane vesicles almost entirely as intact dipeptide. This uptake was mediated by two transporters shared by other dipeptides and characterized as the high affinity (Kt = 44.1 +/- 11.2 microM)/low capacity (Vmax = 0.41 +/- 0.03 nmol/mg protein/5 s) and low affinity (Kt = 2.62 +/- 0.50 mM)/high capacity (Vmax 4.04 +/- 0.80 nmol/mg protein/5 s) transporters. In the absence of a pH gradient, only the low affinity system was operational, but with a reduced transport capacity. Imposing a pH gradient of 1.6 pH units increased the Vmax of both transporters. Kinetic analysis of the rates of Gly-Gln uptake as a function of external pH revealed Hill coefficients of close or equal to 1, indicating that transporters contain only one binding site for the interaction with external H+. The effects of membrane potential on Gly-Gln uptake were investigated with valinomycin-induced K+ diffusion potentials. The velocity of the high affinity system but not of the low affinity system increased linearly with increasing inside-negative K+ diffusion potentials (p less than 0.01). The Kt of neither system was affected by alterations in either pH gradient or membrane potential. We conclude that (a) the high affinity transporter is far more sensitive to changes in proton gradient and membrane potential than the low affinity transporter and (b) in the presence of a pH gradient, transport of each dipeptide molecule requires cotransport of one hydrogen ion to serve as the driving force.
Highlights
The principal aimof the present study was itnoves- peared to be the Na’ gradient, because it was shown to be tigate the effects of variation in proton gradient and necessary for transport of amino acids and glucose
In theabsence of a pH gradient, only the lation of dipeptide transport in intestinal brush border memlow affinity system was operational, buat rewdiuthced brane vesicles have come to different conclusions
Relevance ofGly-Gln Transport to Other Dipeptides-To investigate whether the existence of two transport systems is unique to Gly-Gln or is common to other dipeptides, we investigated the mechanism of uptake of Gly-Ala exactly as we did that of Gly-Gln
Summary
Preloaded with buffer (pH 8.3), with 160 p1 of medium (pH 6.0), resulted in a final incubation pH of 6.7. Incubation of vesicles was terminated by 5 ml of ice-cold stop buffer and filtration. Effect of Medium Osmolarity onGly-GlnUptake-To distinguish between transport into an osmotically reactive space and binding to themembrane, Gly-Gln uptake was studied in the presence of increasing concentrations of mannitol. Uptake was terminated by ice-cold stop buffer adjusted to the same filterextract were diluted with 6% sulfosalicylic acid to contain approximately 10,000 dpm/ml. Without pHgradient, BBMV preloaded with 45 mM Hepes, 20 mM Tris, 320 mM mannitol (pH 7.4) were incubated for 5 s in 0.05 p M Gly-Gln, 45 mM Hepes, 20 mM Tris (pH 7.4), and either 320 mM mannitol or 1mM glutamine, and 319 mM mannitol, 5mM glutamine, and 315 mM mannitol, 100 mM NaCI, and 120 mM mannitol. The datarepresent mean f S.E. of three membrane preparations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
