Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles

With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibri um amino acid uptake and intravesicular volume determined with porcine jeju nal brush border membrane vesicles prepared by Mg2+-aggregation and differe ntial centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occ ur into an osmotically-active space ranging between 1.09-1.58 mu l/mg prote in with little non-specific membrane binding. At different concentrations t in parentheses), the duration of initial glutamine uptake in both Na+ gradi ent and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1 .9 and 9.4 mM), respectively. Substrate concentrations affected the duratio n of initial uptake, with lower substrate concentrations giving shorter dur ation for initial amino acid uptake. At different substrate concentrations tin parentheses), the time required to reach equilibrium glutamine uptake w as 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respecti vely. Thus, substrate concentrations also affected the time required to rea ch equilibrium uptake. The higher the substrate concentration, the longer t he incubation time needed to reach equilibrium amino acid uptake. At the gl utamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intraves icular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, an d 1.36+/-0.21 mu l/mg protein, respectively. Substrate concentrations had n o effect (p>0.05) on the intravesicular volume of membrane vesicles. In con clusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuri ng the initial uptake rate should be determined from the time course experi ments conducted at the lowest substrate concentration used, whereas the int ravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.