Leucine transport across the brush border and basal cell membrane surfaces

Brush border (microvillous) plasma membranes (BBM) and the basal surfaces (BCM) from the syncytiotrophoblast of human term placenta were prepared by a method of sonication, dialysis and differential centrifugation in specific buffer systems. Such plasma membranes formed closed, osmotically active, right-side-out vesicles in which amino acid transport could be studied unidirectionally by a carefully designed membrane filtration assay under reduced pressure. In such vesicles, L-leucine (1 mM) was found to be transported in a time-dependent manner, peak accumulation being attained at 45 s in both BBM and BCM. The accumulation of leucine in the vesicles was dependent on an inward NaCl gradient, as replacing the Na+ with K+, Li+ and choline, or replacing the Cl- with SO42- failed to influence the amino acid movement. Leucine transport in the vesicles also appeared to be dependent on the substrate concentration, indicating saturation at a higher concentration. The transport process showed a k(t) (affinity constant) of 3.85 and 6.67 mM, while the values recorded for the J(max) (maximum apparent initial velocity) were 270.27 and 384.62 nmol/mg protein(-1) per min in the BBM and BCM respectively. Leucine transport was inhibited by a number of amino acids, among which amino isobutyric acid (AIB) produced the maximum inhibition. The k(i) (inhibition constant) for different amino acids has also been listed. Lysine showed the least k(i) value, thus showing it to be the most inhibitory compound. These findings are discussed in relation to the mechanism and regulation of transplacental amino acid transfer.