Conrad Erik M, Ahearn Gregory A
Department of Biology, 4567 St Johns Bluff Road, South, University of North Florida, Jacksonville, FL 32224, USA.
J Exp Biol. 2005 Jan;208(Pt 2):287-96. doi: 10.1242/jeb.01401.
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) 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.
美洲螯龙虾（Homarus americanus）的管状肠道被分离出来进行体外实验，并灌注一种基于血淋巴离子浓度的生理盐水，其中含有不同浓度的（3）H - l - 组氨酸、（3）H - 甘氨酰肌氨酸和（65）Zn（2 +）。通过同位素在浆膜表面的生理盐水中出现的速率来测量每种放射性标记底物从黏膜到浆膜（M→S）的通量。向含有1 - 50 μmol l（-1）（3）H - l - 组氨酸的管腔溶液中添加1 - 50 μmol l（-1）锌，与不含该金属的对照组相比，氨基酸的M→S通量显著增加（P<0.01）。在没有锌的情况下，M→S（3）H - l - 组氨酸通量的动力学遵循米氏动力学（K（m）=6.2±0.8 μmol l（-1）；J（max） =0.09±0.004 pmol cm（-2）min（-1））。向管腔灌注液中添加20 μmol l（-1）锌会增加两个动力学常数（K（m）=19±3 μmol l（-1）；J（max）=0.28±0.02 pmol cm（-2）min（-1））。同时添加20 μmol l（-1）锌和100 μmol l（-1）l - 亮氨酸会消除金属单独作用时的刺激作用（K（m）=4.5±1.7 μmol l（-1）；J（max）=0.08±0.008 pmol cm（-2）min（-1））。在没有l - 组氨酸的情况下，（65）Zn（2 +）的M→S通量也遵循米氏关系，向灌注液中添加l - 组氨酸会显著增加两个动力学常数（P<0.01）。同时添加50 μmol l（-1）Cu（+）或Cu（2 +）和20 μmol l（-1）l - 组氨酸会同时消除l - 组氨酸单独对跨膜（65）Zn（2 +）转运的刺激作用。向灌注液中添加100 μmol l（-1）甘氨酰肌氨酸会显著降低锌对M→S（3）H - l - 组氨酸通量的刺激作用（P<0.01），因为该二肽显著降低了l - 组氨酸转运的K（m）和J（max）（P<0.01）。当单独向灌注液中添加l - 组氨酸或l - 亮氨酸时，（3）H - 甘氨酰肌氨酸的跨膜转运不受影响，但当同时添加锌和任何一种氨基酸时，观察到肽转运至少降低50%。这些结果表明，（3）H - l - 组氨酸和（
