The characteristics of taurine uptake in rabbit jejunal brush-border membrane vesicles were investigated. Taurine was transiently accumulated inside the vesicles against a concentration gradient when an inwardly directed NaCl gradient was imposed across the membrane. Uptake of taurine showed an absolute requirement for both Na+ and Cl-. The NaCl gradient-dependent taurine uptake was stimulated by a valinomycin-induced, inside-negative, K+-diffusion potential, suggesting that the uptake process was electrogenic. The uptake system exclusively interacted with beta-amino acids of small size, but had no affinity for alpha-amino acids. Kinetic analysis revealed that the system exhibited high affinity for taurine (Kt = 14.4 +/- 0.5 microM). Taurine uptake was greatly influenced by extravesicular concentrations of Na+ and Cl-. The Cl- stoichiometry was found to be one. In the presence of Cl-, taurine uptake was sigmoidally related to Na+ concentration, and the Na+ stoichiometry was calculated to be three. Thus three Na+ and one Cl- were involved per transport of one taurine molecule. The uptake process was not affected by other transport inhibitors such as amiloride, harmaline, furosemide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonate.
The characteristics of beta-alaline uptake were studied in brush-border membrane vesicles isolated from the proximal small intestine of rabbits and were compared with those of L-alpha-alanine uptake. The uptake of beta-alanine as well as L-alpha-alanine was significantly stimulated by imposing an inwardly directed Na+ gradient. Studies on transstimulation and substrate specificity provide evidence that the transport system serving beta-alanine is distinct from the system serving alpha-alanine. The beta-system also accepts taurine as a substrate. The Na(+)-dependent uptakes of beta-alanine and L-alpha-alanine were differentially influenced by anions. The order in which anions supported uptake was Cl- = SCN- greater than F- greater than NO3- = SO2(-4) for beta-alanine, whereas it was SCN- greater than F- = Cl- = NO3- greater than SO2(-4) for L-alpha-alanine. Cl- appeared to be the preferred anion to support the uptake of beta-alanine. beta-Alanine uptake was greater in the presence of an inwardly directed Cl- gradient than in the presence of Cl- at equal concentrations on both sides of the membrane. The uptake was maximal when a Na+ gradient and a Cl- gradient were present simultaneously. The NaCl gradient-driven beta-alanine uptake was stimulated by an inside-negative K(+)-diffusion potential induced by valinomycin, showing that the uptake process is electrogenic. Stoichiometric analyses suggest that multiple Na+ and one Cl- are associated with the uptake of one beta-alanine molecule. The kinetic study shows that the transporter for beta-alanine is a high-affinity, low-capacity system (Kt = 46 +/- 1 microM; Vmax = 30 +/- 1 pmol.mg protein-1.15 s-1).
The characteristics of guanidine uptake were studied in brush-border membrane vesicles isolated from the rabbit proximal intestine. Guanidine uptake was manyfold greater in the presence of an outward-directed H+ gradient (intracellular pH = 5.5; extracellular pH = 7.2) than in the absence of a H+ gradient (intracellular and extracellular pH = 7.2). The time course of guanidine uptake exhibited an overshoot phenomenon in the presence of the H+ gradient, indicating occurrence of uphill transport. This H+ gradient-stimulated guanidine uptake was not due to an inside-negative H+-diffusion potential because carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, a protonophore, failed to have any effect on guanidine uptake. Moreover, the transient uphill transport of guanidine was observed even in voltage-clamped membrane vesicles. However, under the conditions that effectively dissipated the H+ gradient, there was no active transport of guanidine. This H+ gradient-dependent transport mechanism for guanidine is distinct from the Na+-H+ exchanger, because amiloride did not inhibit guanidine uptake even at a concentration as high as 100 microM. These data provide evidence for the presence of a guanidine-H+ antiport system in the rabbit intestinal brush-border membrane. The presence of a carrier for guanidine in these membranes is further substantiated by the trans-stimulation of the uptake of radiolabeled guanidine by unlabeled guanidine and by the inhibition of guanidine uptake by imipramine under equilibrium exchange conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
We examined the handling of radiolabeled beta-casomorphin, Tyr-Pro-[3H]Phe-Pro-Gly, by rabbit renal brush-border membrane vesicles (BBMV). The uptake of radiolabel into the vesicles was Na+-independent, but an inward-directed H+ gradient stimulated the uptake. The H+ gradient-dependent uptake was further accelerated by an interior-negative membrane potential, but inhibited in the presence of a protonophore. Treatment of the membrane vesicles with diisopropylfluorophosphate (DFP) greatly reduced the uptake of the radiolabel. Control as well as DFP-treated vesicles exhibited H+ gradient-dependent Gly-Sar uptake. Unlabeled beta-casomorphin inhibited Gly-Sar uptake in control vesicles, but the inhibition was significantly reduced in DFP-treated vesicles. DFP inhibited the activity of dipeptidyl peptidase IV in these vesicles and there was a direct correlation between the activity of the enzyme and the capacity of beta-casomorphin to inhibit Gly-Sar uptake. Many di- and tripeptides reduced the uptake of Gly-Sar and the uptake of radiolabel from beta-[3H]casomorphin to a similar extent. We conclude that beta-casomorphin is hydrolyzed by dipeptidyl peptidase IV and the products are transported into the vesicles by the H+ gradient-driven peptide transport system. This conclusion is supported by the results from the analysis of the incubation medium by high-performance liquid chromatography that showed rapid hydrolysis of the pentapeptide by brush-border membranes to di- and tripeptides.
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