3H-l-histidine and 65Zn2+ are cotransported by a dipeptide transport system in intestine of lobsterHomarus americanus

Abstract

SUMMARYThe tubular intestine of the American lobster Homarus americanuswas isolated in vitro and perfused with a physiological saline whose composition was based on hemolymph ion concentrations and contained variable concentrations of 3H-l-histidine, 3H-glycyl-sarcosine and 65Zn2+. Mucosa to serosa (M→S) flux of each radiolabelled substrate was measured by the rate of isotope appearance in the physiological saline bathing the tissue on the serosal surface. Addition of 1–50 μmol l–1 zinc to the luminal solution containing 1–50 μmol l–13H-l-histidine significantly (P<0.01)increased M→S flux of amino acid compared to controls lacking the metal. The kinetics of M→S 3H-l-histidine flux in the absence of zinc followed Michaelis–Menten kinetics(Km=6.2±0.8 μmol l–1; Jmax =0.09±0.004 pmol cm–2min–1). Addition of 20 μmol l–1 zinc to the luminal perfusate increased both kinetic constants(Km=19±3 μmol l–1; Jmax=0.28±0.02 pmol cm–2min–1). Addition of both 20 μmol l–1 zinc and 100 μmol l–1l-leucine abolished the stimulatory effect of the metal alone (Km=4.5±1.7μmol l–1; Jmax=0.08±0.008 pmol cm–2 min–1). In the absence of l-histidine, M→S flux of 65Zn2+ also followed the Michaelis–Menten relationship and addition of l-histidine to the perfusate significantly (P<0.01)increased both kinetic constants. Addition of either 50 μmol l–1 Cu+ or Cu2+ and 20 μmol l–1l-histidine simultaneously abolished the stimulatory effect of l-histidine alone on transmural 65Zn2+ transport. Zinc-stimulation of M→S 3H-l-histidine flux was significantly(P<0.01) reduced by the addition of 100 μmol l–1 glycyl-sarcosine to the perfusate, as a result of the dipeptide significantly (P<0.01) reducing both l-histidine transport Km and Jmax. Transmural transport of 3H-glycyl-sarcosine was unaffected by the presence of either l-histidine or l-leucine when either amino acid was added to the perfusate alone, but at least a 50% reduction in peptide transport was observed when zinc and either of the amino acids were added simultaneously. These results show that 3H-l-histidine and 65Zn2+ are cotransported across the lobster intestine by a dipeptide carrier protein that binds both substrates in a bis-complex (Zn-[His]2) resembling the normal dipeptide substrate. In addition, the transmural transports of both substrates may also occur by uncharacterized carrier processes that are independent of one another and appear relatively specific to the solutes used in this study.