ABSTRACT
Epithelial brush-border membrane vesicles (BBMVs) from the hepatopancreas of the lobster Homarus americanus were prepared using a magnesium precipitation technique and employed in transport experiments designed to demonstrate the effects of external and internal divalent cationic heavy metals on the uptake of L-[3H]proline. When BBMVs were exposed to a high external concentration (2.5 mmol l−1) of Cd2+, Cu2+, Fe2+, Mn2+ or Zn2+, L-[3H]proline (0.5 mmol l−1) uptake was significantly (P<0.05) decreased by each metal. However, if a 30 min pre-incubation period with each metal was used before incubation of the vesicles with amino acid and metal, a significant (P<0.05) enhancement of L-[3H]proline transport occurred. Zinc was the most stimulatory metal of those tested. Proline influxes (1.0 and 2.5 mmol l−1) were hyperbolic functions of bilateral [Zn2+], with a lower apparent zinc half-saturation constant (Km) at the higher amino acid concentration. L-[3H]proline influx was a hyperbolic function of external [L-proline] (Km=2.10±0.26 mmol l−1; Jmax=2290±600 pmol mg−1 protein 10 s−1) (means ± S.E.M., N=3), and bilateral exposure to zinc significantly (P<0.05) increased the maximal rate of influx, Jmax, of proline (Jmax=4890±250 pmol mg−1 protein 10 s−1), but had no effect (P>0.05) on apparent L-[3H]proline binding to the membranes (Km=1.66±0.23 mmol l−1) (means ± S.E.M., N=3). In the presence of 0.5 mmol l−1 l-pipecolate, bilateral zinc-stimulated, carrier-mediated, L-[3H]proline influx was abolished. At low external concentrations of zinc alone (e.g. below 1.0 mmol l−1), L-[3H]proline influx was enhanced by the metal. Enhanced amino acid uptake in the presence of external zinc alone was abolished by L-pipecolate. A model accounting for external and internal zinc enhancements of L-[3H]proline influx by the Na+-dependent L-pipecolate-sensitive IMINO transport system in these membranes is proposed.
