Estimation
of Apparent L-Amino Acid Diffusion in Porcine
Jejunal Enterocyte Brush Border Membrane Vesicles
M. Z. FAN1,
O. ADEOLA2, E. K. ASEM3
Department
of Animal and Poultry Science1, University of
Guelph, Guelph, Ontario, Canada, Departments of
Animal Sciences2 and Basic Medical Sciences3,
Purdue University, West Lafayette, Indiana, USA
Received August 9, 2000
Accepted November 30, 2000
Summary
There
is an overlap of carrier-mediated L-amino acid
transport and apparent simple diffusion when
measured in intestinal brush border membrane
vesicles. Using L-threonine and L-glutamine as
representative amino acids, this study was
undertaken to estimate apparent simple diffusion
of L-amino acids and to establish the effective
dosage of HgCl2 for completely blocking
carrier-mediated L-amino acid transport in
porcine jejunal enterocyte brush border membrane
vesicles. Jejunal mucosa was scraped from three
pigs weighing 26 kg. Enterocyte brush border
membrane vesicles, with an average enrichment of
24-fold in sucrase specific activity, were
prepared by Mg2+-precipitation and differential
centrifugation. In vitro uptake was measured by
the fast filtration manual procedure. HgCl2
blocked the carrier-mediated initial transport of
L-threonine and L-glutamine under Na+-gradient
condition in a dose-dependent manner. At the
minimal concentration of 0.165 mmol HgCl2 mg-1
protein, carrier-mediated L-threonine and
L-glutamine transport was completely inhibited.
The apparent L-threonine and L-glutamine
diffusion was estimated to be 8.6±0.7 and
12.4±1.0 % of the total uptake at the substrate
concentrations of 5 mM (L-threonine) and 50 mM
(L-glutamine). Therefore, the treatment of
porcine brush border membrane vesicles with a
minimum of 0.165 mmol HgCl2 mg-1 protein
completely blocks carrier-mediated L-amino acid
transport and enables the direct estimation of
apparent L-amino acid diffusion in enterocyte
brush border membrane vesicles.
Key
words
Amino
acids · Enterocyte · Brush border membrane
vesicles · Diffusion · Pigs
Reprint
requests
Ming
Z. Fan, Ph.D., Room 250, #70 Animal
Science/Nutrition Building, Department of Animal
and Poultry Science, University of Guelph,
Guelph, Ontario, Canada N1G 2W1, Fax: (519)
836-9873, E-mail: mfan@uoguelph.ca
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