3H-L-histidine and 65Zn(2+) are cotransported by a dipeptide transport system in intestine of lobster Homarus americanus.
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 (3)H-l-histidine, (3)H-glycyl-sarcosine and (65)Zn(2+). 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 micromol l(-1) zinc to the luminal solution containing 1-50 micromol l(-1) (3)H-l-histidine significantly (P<0.01) increased M-->S flux of amino acid compared to controls lacking the metal. The kinetics of M-->S (3)H-l-histidine flux in the absence of zinc followed Michaelis-Menten kinetics (K(m)=6.2+/-0.8 micromol l(-1); J(max) =0.09+/-0.004 pmol cm(-2) min(-1)). Addition of 20 micromol l(-1) zinc to the luminal perfusate increased both kinetic constants (K(m)=19+/-3 micromol l(-1); J(max)=0.28+/-0.02 pmol cm(-2) min(-1)). Addition of both 20 micromol l(-1) zinc and 100 micromol l(-1) l-leucine abolished the stimulatory effect of the metal alone (K(m)=4.5+/-1.7 micromol l(-1); J(max)=0.08+/-0.008 pmol cm(-2) min(-1)). In the absence of l-histidine, M-->S flux of (65)Zn(2+) 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 micromol l(-1) Cu(+) or Cu(2+) and 20 micromol l(-1) l-histidine simultaneously abolished the stimulatory effect of l-histidine alone on transmural (65)Zn(2+) transport. Zinc-stimulation of M-->S (3)H-l-histidine flux was significantly (P<0.01) reduced by the addition of 100 micromol 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 (3)H-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 (3)H-l-histidine and (65)Zn(2+) 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.
Conrad
Erik M
EM
Department of Biology, 4567 St Johns Bluff Road, South, University of North Florida, Jacksonville, FL 32224, USA.
Ahearn
Gregory A
GA
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D004151:Dipeptides / Q000378:metabolism; D005431:Florida; D006639:Histidine / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008121:Nephropidae / Q000378:metabolism* / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D012521:Sarcosine / Q000378:metabolism; D014316:Tritium / Q000378:metabolism; D015037:Zinc Radioisotopes / Q000378:metabolism*
D002352:Carrier Proteins; D004151:Dipeptides; D015037:Zinc Radioisotopes; D014316:Tritium; D006639:Histidine; D012521:Sarcosine