Protein Turnover – Protein and Amino Acids in
2.5 Transport of a mino a cids a cross m embranes
Early studies of amino acid transport used hepatoma cells in culture, everted gut sacs and perfused kidney tubules, as well as observations in patients with genetic defects leading to aminoaciduria (see section 1.7.1 ). These studies showed that amino acids with chemically related side - chains compete with each other for transport. More recent studies have depended on cloning the genes for transport proteins into Xenopus laevis oocytes, and sometimes also inserting the gene products into synthetic lipid membranes in order to study them. Using the gene products, it is possible to raise antibodies and, therefore, to localize different transport proteins to the luminal and systemic sides of epithelial cells in the small intestine and kidney tubule.
In addition to intestinal uptake of dietary amino acids and renal reuptake of fi ltered amino acids, uptake of amino acids into tissues may be rate - limiting for their metabolism. As will be discussed in section 9.4.3 , this is certainly so Figure 2.3 The inter - organ glucose - alanine cycle.
Glucose 6 - phosphatase EC 3.1.3.9.
glycogen
glucose 1-phosphate
glucose 6-phosphate
pyruvate
glycolysis
amino acids muscle protein
keto acids
to citric acid cycle alanine
leucine
keto-isocaproate transaminases
pyruvate
amino acids keto acids transaminases
alanine keto-isocaproate
leucine
glucose 6-phosphate glucose
gluconeogenesis
H2O Pi
glucose 6-phosphatase
muscle liver
glucose
for the synthesis of the neurotransmitter serotonin; changes in the rate of tryptophan uptake into the brain have a considerable effect on the rate of serotonin synthesis.
2.5.1 Families of a mino a cid t ransporters
The classifi cation of amino acid transporters is on the basis of their specifi city for the amino acids transported, and also whether or not they are sodium - or proton- dependent. The amino acid transporters contain 10 – 14 transmem-brane domains, but there are a number of associated proteins with a single transmembrane domain that activate the transporters. Collectrin is one such single transmembrane protein that is essential for activity of large neutral amino acid transport. Knockout mice excrete not only the large neutral amino acids but also proline and glycine, suggesting that collectrin is essential for the function of other transporters as well.
Collectrin is not expressed in the intestinal mucosa, but the angiotensin converting enzyme ACE2 (a carboxypeptidase) serves the same function.
Collectrin and ACE2 show considerable sequence homology, and both are expressed in the kidney, but here ACE2 does not activate amino acid trans-port (Verrey et al. , 2009 ).
In general, the basolateral transporters in the intestinal mucosal cells are the same as those found in other organs, including the blood - brain barrier, and they permit either effl ux of dietary amino acids into the circulation, or uptake of amino acids from the circulation in the fasting state. Several of the apical transporters are unique to the intestinal epithelial cell membrane (Brandsch, 2006 ; Collarini & Oxender, 1987 ; Kilberg et al. , 1993 ). The γ glutamyl cycle of glutathione metabolism (see section 5.4.5 ) may also be involved in membrane transport of amino acids, and 5 - oxoproline formed from glutathione stimulates the activity of sodium - dependent amino acid transporters (Goldberg, 1980 ; Hawkins et al ., 2006 ).
System A preferentially transports amino acids with short, polar or linear side - chains. It is sodium - dependent. While system A transports glycine, there is also a glycine - specifi c sodium - dependent transporter – system Gly. There is de novo synthesis of system A protein when cells are incubated in an amino acid- free medium, and it is subject to trans - inhibition – the presence of the substrates inside the cell inhibits further uptake. However, the synthesis of the system A protein, or its translocation to the cell surface, increases in response to insulin in most tissues, permitting increased amino acid uptake for protein synthesis.
In hepatocytes, system A synthesis and translocation to the cell surface also increase in response to glucagon, permitting increased uptake of the small neutral amino acids that are major substrates for gluconeogenesis. This response to glucagon is in two phases. Initially, there is an increase of 20 – 40
per cent that is not inhibited by cycloheximide, and is mainly due to translo-cation of existing transporter protein to the cell surface. After a time lag, there is a 5 – 10 fold increase in activity due to new protein synthesis. It is not clear whether this is de novo synthesis of the transport protein itself, or a single transmembrane domain - activating protein.
System ASC transports alanine, serine, glycine and proline, and it is subject to trans - stimulation – the presence of the substrates inside the cell increases the rate of uptake from the incubation medium. Again, it is sodium- dependent.
System N has been identifi ed in hepatocytes, muscle and placenta. It trans-ports glutamine, asparagine and histidine, and it is sodium - dependent. There is an intercellular glutamine cycle in the liver; the periportal hepatocytes that receive blood from the hepatic portal vein take up glutamine using the system N transporter and hydrolyze it, releasing ammonium. The perivenous hepa-tocytes (some 10 per cent of the total, localized as a ring of 1 – 3 cells around the terminal central venules) synthesize glutamine from any ammonium remaining, so preventing the entry of ammonium into the peripheral circula-tion (Haussinger, 1990 ; Haussinger et al ., 1992 ). Glutamine effl ux from these cells is sodium - independent.
In the kidney, there is basolateral uptake of glutamine from the circulation in the distal part of the proximal tubule for the production of ammonium to regulate urine pH. In response to acidosis, the activity of this transporter is increased, and it is also localized in the earlier part of the proximal tubule.
The system L neutral amino acid transporter carries the aromatic and branched chain amino acids. It is sodium independent, but it may be proton -dependent; there is some evidence of leucine - proton co - transport. Like the system ASC transporter, the system L transporter is subject to trans stimulation. There are two system L transporters in hepatocytes: a low - affi nity high - capacity transporter and one with high affi nity but low capacity. This latter transporter is presumably important for hepatic uptake of amino acids to meet the liver ’ s needs for protein synthesis.
The system T transporter was originally described in erythrocytes; it is sodium- independent, and it transports aromatic amino acids, with a prefer-ence for tryptophan. It is also present in hepatocytes and in the kidney.
In several tissues, including the brain, intestine and kidney, proline, glycine and alanine are carried by the PAT1 proton - dependent transporter, which has a low affi nity and high capacity. It co - transports one proton and one amino acid, and it also transports short - chain fatty acids (acetate, propionate and butyrate – Metzner et al. , 2006 ).
Positively charged (basic) amino acids are transported by the sodium independent cationic amino acid transporter (sometimes also known as the system y + transporter). Like system A, this transporter is repressed when the intracellular concentrations of lysine and arginine rise; unlike system A, it is
de - repressed when the concentration of any of the essential amino acids (not just its substrates) falls. It is unaffected by limitation of non - essential amino acids. This amino acid responsiveness is regulation by the amino acid - sensing mechanism associated with eIF2 (eukaryotic initiation factor, section 2.2.3 ).
Although this protein catalyzes bidirectional transport, it permits amino acid accumulation against a concentration gradient because of the membrane potential across the plasma membrane. There is very low activity of this transporter in hepatocytes. As noted in section 5.9.1 , net synthesis of arginine occurs in the kidney; in the liver, arginine is an intermediate in the synthesis of urea (section 1.6.2.1 ), and lack of the y + transporter protects extra - hepatic arginine from the action of liver arginase (Hatzoglou et al. , 2004 ).
The main transporter for negatively charged (acidic) amino acids is the sodium- dependent x − ag carrier that transports both aspartate and glutamate.
In foetal cells, there are two separate transporters: the x − a protein preferen-tially transports aspartate and is sodium - dependent, while the x − c protein is a sodium - independent transporter for glutamate and cysteine. Mature hepa-tocytes have no x − a carrier and low activity of the x − c carrier, although this latter is induced in response to insulin.
System b 0,+ is a sodium - independent transporter that carries both neutral and cationic amino acids, so that its specifi city overlaps those of systems L and y + . It shows preference for amino acids with bulky unbranched side chains. System B 0,+ is a sodium - dependent carrier with specifi city similar to that of system b 0,+ , but it also transports branched - chain amino acids. It is found in fi broblasts and endothelial cells, but not hepatocytes.
There are also heterodimeric amino acid transporters that are essentially dimers of two different transport proteins that act in opposite directions, so transporting one amino acid into a cell and another out (Wagner et al. , 2001 ).
These include:
• two system L heterodimers that carry out sodium - independent exchange of large or small neutral amino acids;
• two system y + L heterodimers that exchange dibasic amino acids and large or small neutral amino acids;
• an X − c dimer that exchanges glutamine and cystine;
• a system asc dimer that exchanges small neutral amino acids and also transports d - serine;
• a system b 0,+ dimer that exchanges neutral and dibasic amino acids and is also involved in reabsorption of cysteine, arginine, lysine and ornithine in the kidney.
Mitochondrial uptake of amino acids is indirect. Amino acids are transami-nated on the cytosolic side of the inner mitochondrial membrane, generally linked to pyruvate or 2 - oxoglutarate as the amino acceptor. The oxo - acids enter the mitochondrion and are reaminated by glutamine - dependent transaminases. Net infl ux of oxo - acids and, hence, concentrative uptake of amino acids, is ensured because the product of glutamine transamination – 2 - oxoglutaramate – undergoes non - enzymic cyclization if it is not deamidated byω - amidase (see section 5.3.2 ), so that the substrate for the back reaction is not available (Cooper & Anders, 1990 ).
2.5.1.1 Dipeptide t ransport There are at least two di - and tripeptide trans-porters: PEPT1 in the gut, liver, bile duct, kidney and pancreas; and PEPT2 in kidney, lung, brain, mammary gland and eye. Both are proton symporters and are examples of tertiary active transport. The proton gradient for peptide transport is generated by an Na + /K + ATPase at the basolateral membrane and an Na + /H + exchanger at the apical membrane. These peptide transporters also carry β - lactam antibiotics, ACE inhibitors and other peptidomimetic drugs, but not free amino acids or tetrapeptides. PEPT2 may be involved in glutath-ione metabolism (see section 5.4.4 ) by providing cysteinyl - glycine from extra-cellular glutathione (Biegel et al. , 2006 ).
Further r eading
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