Interaction of amino acids with glycyl-glycine transport in the mammalian intestine

In order to investigate a possible interaction between free amino acids and dipeptides during their mucosal uptake in man and monkey, perfusion studiesin vivo and uptake studiesin vitro using labelled and non-labelled dipeptides and amino acids have been carried out. In contrast to the observations of other workers, inhibition of glycyl-glycine uptake was observed with free leucine and methioninc but not with glycine, proline, hydroxyproline or alanine. Leucine and methionine caused inhibition of cytosol glycyl-glycine hydrolase activity, while glycine had no effect. The dipeptide uptake and dipeptide hydrolysis by cytosol enzyme was competitively inhibited by leucine. Although brush border glycyl-glycine hydrolase was also inhibited by leucine, the inhibition was noncompetitive. These data indicate that a few free amino acids can interact with dipeptides during uptake. This interaction might occur either at the transport step or at the stage of intracellular dipeptide hydrolysis. The work reported here was carried out at Wellcome Research Unit, Christian Medical College and Hospital, Vellore 632 004.     

Reduced-folate carrier (RFC) is expressed in placenta and yolk sac,as well as in cells of the developing forebrain,hindbrain, neural tube,craniofacial region,eye, limb buds and heart

Background  

Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively.     

Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na(+)-coupled transporter for short-chain fatty acids

SLC5A8, a tumor suppressor gene down-regulated in human colon cancer, codes for a transporter in the Na(+)/glucose cotransporter gene family, but the definitive functional identity of the transporter protein is not known. Since this gene is expressed abundantly in the colon where short-chain fatty acids are generated by bacterial fermentation, we tested the hypothesis that it codes for a Na(+)-coupled transporter for these fatty acids. The coding region of SLC5A8 mRNA was amplified from human intestine and expressed heterologously in Xenopus laevis oocytes. Transport function was monitored by uptake of radiolabeled substrates and by substrate-induced currents under voltage-clamp conditions. Uptake of short-chain fatty acids (lactate, pyruvate, acetate, propionate, and butyrate) in oocytes expressing SLC5A8 was severalfold higher than in uninjected oocytes. Exposure of SLC5A8-expressing oocytes to these fatty acids induced inward currents under voltage-clamp conditions in a Na(+)-dependent manner. These currents were saturable and the substrate concentrations needed for half-maximal induction of the current were in the range of 0.08-2.5 mm. The substrate-induced currents decreased as the carbon chain length of the substrates increased. The Na(+)-activation kinetics indicated involvement of more than one Na(+) ion in the activation process. Direct measurements of substrate (propionate) and charge transfer showed that three positive charges are transferred into oocytes per substrate molecule. These studies establish the functional identity of SLC5A8 as a Na(+)-coupled transporter for short-chain fatty acids.     

Structure,function, and expression pattern of a novel sodium-coupled citrate transporter (NaCT) cloned from mammalian brain

Citrate plays a pivotal role not only in the generation of metabolic energy but also in the synthesis of fatty acids, isoprenoids, and cholesterol in mammalian cells. Plasma levels of citrate are the highest ( approximately 135 microm) among the intermediates of the tricarboxylic acid cycle. Here we report on the cloning and functional characterization of a plasma membrane transporter (NaCT for Na+ -coupled citrate transporter) from rat brain that mediates uphill cellular uptake of citrate coupled to an electrochemical Na+ gradient. NaCT consists of 572 amino acids and exhibits structural similarity to the members of the Na+-dicarboxylate cotransporter/Na+ -sulfate cotransporter (NaDC/NaSi) gene family including the recently identified Drosophila Indy. In rat, the expression of NaCT is restricted to liver, testis, and brain. When expressed heterologously in mammalian cells, rat NaCT mediates the transport of citrate with high affinity (Michaelis-Menten constant, approximately 20 microm) and with a Na+:citrate stoichiometry of 4:1. The transporter does interact with other dicarboxylates and tricarboxylates but with considerably lower affinity. In mouse brain, the expression of NaCT mRNA is evident in the cerebral cortex, cerebellum, hippocampus, and olfactory bulb. NaCT represents the first transporter to be identified in mammalian cells that shows preference for citrate over dicarboxylates. This transporter is likely to play an important role in the cellular utilization of citrate in blood for the synthesis of fatty acids and cholesterol (liver) and for the generation of energy (liver and brain). NaCT thus constitutes a potential therapeutic target for the control of body weight, cholesterol levels, and energy homeostasis.     

Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA ( approximately 9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na(+)-coupled transport of the system A-specific model substrate alpha-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0. 89 +/- 0.12 mM. The Na(+):amino acid stoichiometry is 1:1 as deduced from the Na(+)-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.