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Zusammenfassung:
Glycylsarcosine was found to be very resistant to hydrolysis by brush-border membrane vesicles from rabbit intestine. The dipeptide was transported intact into an osmotically responsive intravesicular space. The initial uptake rate of glycylsarcosine into these vesicles was greater in mannitol medium compared to that in the presence of an inward gradient of either Na+ or other monovalent cations. When vesicles preloaded with glycylsarcosine were incubated in a peptide-free medium, there was a rapid efflux of the dipeptide and the t1/2 for the process was less than 2 min. An inside-negative K+ diffusion potential generated by valinomycin stimulated glycylsarcosine uptake even in the absence of Na+. Experiments with the potential-sensitive dye DiS-C3 (5) showed that glycylsarcosine depolarized the brush-border membrane in the presence and absence of Na+. Imposition of an inward proton gradient stimulated the initial uptake rates of glycylsarcosine while the equilibrium uptake was not affected. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreased this proton gradient-induced stimulation. An inward proton gradient increased the Vmax of the transport system (10.8 ± 0.8 nmol/min/mg of protein when [pH]o = [pH]i = 5.5; and 20.8 ± 2.2 nmol/min/mg of protein when [pH]o = 5.5 and [pH]i = 7.8), without significantly affecting the apparent Kt (17.3 ± 1.4 mM versus 19.5 ± 2.0 mM). Glycyl-L-proline uptake was inhibited by glycylsarcosine and KI for the process was 20.8 ± 3.0 mM. A relatively lower KI (2.8 ± 1.2 mM) was obtained for the inhibition of glycylsarcosine uptake by glycyl-L-proline. The uptake of glycyl-L-proline and glycylsarcosine was strongly inhibited by L-carnosine, glycyl-L-leucine, and L-prolylglycine. With each inhibitory peptide, the KI values for the inhibition of glycyl-L-proline uptake and of glycylsarcosine uptake were comparable. Preloading the vesicles with unlabeled glycylsarcosine stimulated the uptake of labeled glycyl-L-proline. These data suggest that in rabbit intestinal brush-border membrane vesicles (i) glycylsarcosine and proton(s) are co-transported, (ii) this process results in a net transport of positive charge across the membrane and, (iii) a single transport system is involved in the translocation of glycyl-L-proline and glycylsarcosine.
