Effects of zinc on L-[3H]proline uptake by lobster (Homarus americanus) hepatopancreatic brush-border membrane vesicles

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-[(3)H]proline. When BBMVs were exposed to a high external concentration (2.5 mmol l(−)(1)) of Cd(2+), Cu(2+), Fe(2+), Mn(2+) or Zn(2+), l-[(3)H]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-[(3)H]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 [Zn(2+)], with a lower apparent zinc half-saturation constant (K(m)) at the higher amino acid concentration. l-[(3)H]proline influx was a hyperbolic function of external [l-proline] (K(m)=2.10+/−0.26 mmol l(−)(1); J(max)=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, J(max), of proline (J(max)=4890+/−250 pmol mg(−)(1)protein 10 s(−)(1)), but had no effect (P>0.05) on apparent l-[(3)H]proline binding to the membranes (K(m)=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-[(3)H]proline influx was abolished. At low external concentrations of zinc alone (e.g. below 1.0 mmol l(−)(1)), l-[(3)H]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-[(3)H]proline influx by the Na(+)-dependent l-pipecolate-sensitive IMINO transport system in these membranes is proposed.