L-proline transport by brush border membrane vesicles prepared from human placenta

Abstract

1. Brush border microvillous plasma membranes were prepared from syncytiotrophoblast of human term placenta by a method of differential centrifugation. 2. Such plasma membranes form closed, osmotically active, right-side-out vesicles into which L-proline (188 microM) is shown to be transported in a time-dependent manner. There is no detectable metabolism of L-proline within the vesicles during 30 min of incubation. 3. Transient accumulation of L-proline to levels of up to three times its equilibrium value occurs in the presence of an inward gradient of sodium chloride. The proline and sodium are shown to have reached electrochemical equilibrium by 30 min, at which stage there is about 100 pmol L-proline mg protein-1. 4. This transient accumulation is abolished by the prior equilibration of the sodium chloride gradient, or by the replacement of sodium by inwardly directed gradients of other cations. Entry of the amino acid into the vesicles is also shown to be influenced by the permeability of the anion in the medium and by an imposed potassium diffusion potential. L-Proline transport across the brush border membrane of human placental syncytiotrophoblast is thus a sodium-dependent, electrogenic process. 5. Studies of the transport processes indicate saturation at higher concentrations of L-proline with a 'Km' of about 1 mM; Vmax averaged about 2 nmol mg protein-1 min-1 varied considerably between preparations. 6. L-Proline (188 microM) transport is inhibited competitively by the presence of many amino acids and by the dipeptide glycyl-L-proline. The Ki for inhibition by methyl AIB is 300 microM. 7. These findings are discussed in relation to the mechanism of transplacental amino acid transfer.