The Na(+)-dependent proline carrier, of eel intestinal brush-border membrane, sequentially binds proline and then Na+

The mechanism of Na+/L-proline cotransport, present on brush-border membrane (BBM) vesicles of the European eel intestine, was studied. Initial cotransport rates, depending on increasing proline and Na+ concentrations in the extravesicular medium (zero-trans conditions), were measured by monitoring the decay of an inside-negative membrane potential, i.e. the fluorescence quenching of the voltage-sensitive cyanine dye 3,3'-diethylthiacarbocyanine iodide (DiS-C2(5)). By simultaneously estimating the substrate-dependent Na(+)-influx (with the fluorescent dye) and the Na(+)-dependent [3H]substrate influx, it was concluded that proline was cotransported with 1 Na+ ion and glucose with 2 Na+ ions. The kinetics of proline/Na+ cotransport were then investigated. Graphical analysis excluded a ping-pong mechanism. Under rapid equilibrium assumptions, by fitting model equations to rate values it was possible to exclude the random and the ordered Na+/proline mechanisms. Therefore, in eel intestinal BBM vesicles, the mechanism of proline/Na+ cotransport is ordered and prolineout binds to the carrier prior to Na+out.