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 equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient 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 duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.
Fan
M Z
MZ
Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Adeola
O
O
Turek
J J
JJ
Asem
E K
EK
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002498:Centrifugation; D005973:Glutamine / Q000378:metabolism; D007583:Jejunum / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium; D008871:Microvilli / Q000378:metabolism*; D009994:Osmolar Concentration; D012964:Sodium; D013552:Swine / Q000378:metabolism*
D000596:Amino Acids; D005973:Glutamine; D012964:Sodium; D008274:Magnesium