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First published online January 5, 2005
Journal of Experimental Biology 208, 287-296 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01401

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3H-L-histidine and ⁶⁵Zn²⁺ are cotransported by a dipeptide transport system in intestine of lobster Homarus americanus

Erik M. Conrad and Gregory A. Ahearn^*

Department of Biology, 4567 St Johns Bluff Road, South, University of North Florida, Jacksonville, FL 32224, USA

^* Author for correspondence (e-mail: gahearn{at}unf.edu)

Accepted 22 November 2004

The tubular intestine of the American lobster Homarus americanus was 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 (MS) 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^{–1 3}H-L-histidine significantly (P<0.01) increased MS flux of amino acid compared to controls lacking the metal. The kinetics of MS ³H-L-histidine flux in the absence of zinc followed Michaelis–Menten kinetics (K_m=6.2±0.8 µmol l^–1; J_max =0.09±0.004 pmol cm^–2 min^–1). Addition of 20 µmol l^–1 zinc to the luminal perfusate increased both kinetic constants (K_m=19±3 µmol l^–1; J_max=0.28±0.02 pmol cm^–2 min^–1). Addition of both 20 µmol l^–1 zinc and 100 µmol l^–1 L-leucine abolished the stimulatory effect of the metal alone (K_m=4.5±1.7 µmol l^–1; J_max=0.08±0.008 pmol cm^–2 min^–1). In the absence of L-histidine, MS 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^–1 L-histidine simultaneously abolished the stimulatory effect of L-histidine alone on transmural ⁶⁵Zn²⁺ transport. Zinc-stimulation of MS 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 K_m and J_max. 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.

Key words: L-histidine, zinc, bis-complex, dipeptide, PEPT-1, glycyl-sarcosine, copper, heavy metal, Homarus americanus, intestine, transmural transport, epithelium, L-leucine, cotransport

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