scholarly journals Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa

2018 ◽  
Vol 293 (44) ◽  
pp. 17267-17277 ◽  
Author(s):  
Raghavendar Reddy Sanganna Gari ◽  
Patrick Seelheim ◽  
Brendan Marsh ◽  
Volker Kiessling ◽  
Carl E. Creutz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Outer Membrane ◽  
Amino Acid Transport ◽  
Acid Uptake ◽  
Substrate Uptake ◽  
Acid Transport ◽  
Oligomeric State ◽  
Loop Region

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial infections. The P. aeruginosa outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded β-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake. Pro-92 of OprG is critically important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG may assemble into oligomers in the outer membrane (OM) whose subunit interfaces could form a transport channel. Here, we explored the contributions of the oligomeric state and the extracellular loops to OprG's function. Using chemical cross-linking to determine the oligomeric structures of both WT and P92A OprG in native outer membranes and atomic force microscopy, and single-molecule fluorescence of the purified proteins reconstituted into lipid bilayers, we found that both protein variants form oligomers, supporting the notion that subunit interfaces in the oligomer could provide a pathway for amino acid transport. Furthermore, performing transport assays with loop-deleted OprG variants, we found that these variants also can transport small amino acids, indicating that the loops are not solely responsible for substrate transport. We propose that OprG functions as an oligomer and that conformational changes in the barrel–loop region might be crucial for its activity.

Acute acidosis inhibits liver amino acid transport: no primary role for the urea cycle in acid-base balance

1994 ◽  
Vol 267 (6) ◽  
pp. F1015-F1020 ◽  
Author(s):  
L. Boon ◽  
P. J. Blommaart ◽  
A. J. Meijer ◽  
W. H. Lamers ◽  
A. C. Schoolwerth
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hepatic Vein ◽  
Amino Acid Transport ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Primary Role ◽  
Urea Synthesis ◽  
Acid Transport ◽  
Acid Base

To examine further the role of the liver in acid-base homeostasis, we studied hepatic amino acid uptake and urea synthesis in rats in vivo during acute acidosis and alkalosis, induced by infusion of 1.8 mmol of HCl or NaHCO3 over 3 h. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences of amino acids and urinary urea and NH4+ excretion were measured. In acidosis, urinary urea excretion was reduced 36% (P < 0.01), whereas urinary NH4+ excretion increased ninefold (P < 0.01), but the sum of urea and NH4+ excretion was unchanged. Total hepatic amino acid uptake, as determined from arteriovenous differences, was decreased by 63% (P < 0.01) in acidosis, with the major effect being noted with alanine and glycine. Only glutamine was released in both acidosis and alkalosis but was not significantly different in the two conditions. Since intracellular concentrations of readily transportable amino acids were not different at low pH despite accelerated protein degradation, these results indicate that hepatic amino acid transport was inhibited markedly and sufficiently to explain the observed decrease in urea synthesis. Total hepatic vein amino acid content was greater in acidosis than alkalosis (P < 0.01). Directly or indirectly, by conversion to glutamine elsewhere, these increased amino acids were degraded in kidney and accounted for the ninefold increase in urinary NH4+ excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo muscle amino acid transport involves two distinct processes

2004 ◽  
Vol 287 (1) ◽  
pp. E136-E141 ◽  
Author(s):  
Sharon Miller ◽  
David Chinkes ◽  
David A. MacLean ◽  
Dennis Gore ◽  
Robert R. Wolfe
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Interstitial Fluid ◽  
Muscle Blood Flow ◽  
Amino Acid Uptake ◽  
Transport Systems ◽  
Acid Uptake ◽  
Acid Transport ◽  
Rate Limiting

We have tested the hypothesis that transit through the interstitial fluid, rather than across cell membranes, is rate limiting for amino acid uptake from blood into muscle in human subjects. To quantify muscle transmembrane transport of naturally occurring amino acids, we developed a novel 4-pool model that distinguishes between the interstitial and intracellular fluid compartments. Transport kinetics of phenylalanine, leucine, lysine, and alanine were quantified using tracers labeled with stable isotopes. The results indicate that interstitial fluid is a functional compartment insofar as amino acid kinetics are concerned. In the case of leucine and alanine, transit between blood and interstitial fluid was potentially rate limiting for muscle amino acid uptake and release in the postabsorptive state. For example, in the case of leucine, the rate of transport between blood and interstitial fluid compared with the corresponding rate between interstitial fluid and muscle was 247 ± 36 vs. 610 ± 95 nmol·min−1·100 ml leg−1, respectively ( P < 0.05). Our results are consistent with the process of diffusion governing transit from blood to interstitial fluid without selectivity, and of specific amino acid transport systems with varying degrees of efficiency governing transit from interstitial fluid to muscle. These results imply that changes in factors that affect the transit of amino acids from blood through interstitial fluid, such as muscle blood flow or edema, could play a major role in controlling the rate of muscle amino acid uptake.

Volume-regulatory amino acid transport in erythrocytes of the little skate, Raja erinacea

1993 ◽  
Vol 265 (1) ◽  
pp. R173-R179 ◽  
Author(s):  
J. K. Haynes ◽  
L. Goldstein
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Amino Acid Uptake ◽  
Disulfonic Acid ◽  
Acid Uptake ◽  
Gamma Aminobutyric Acid ◽  
Acid Transport ◽  
Beta Alanine ◽  
Volume Activation

Skate erythrocytes swell in a hypotonic medium and then reduce their volume mainly by releasing the beta-amino acids taurine and beta-alanine. Although these amino acids exhibit a net efflux, Na(+)-independent influx is also increased. Both the reduction in cell volume and increase in amino acid transport are inhibited by several inhibitors of band 3-mediated anion transport, including 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) [L. Goldstein and S. R. Brill, Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R1014-R1020, 1991]. The objective of the present investigation was to further characterize the mechanism of volume-activated amino acid transport. Na(+)-independent amino acid uptake was studied because of the ease in controlling amino acid concentrations. Na(+)-independent taurine uptake was observed to be linear over a range of 0.1-15 mM and was not inhibited by 10 mM beta-alanine, suggesting that the transporter may be a channel rather than a carrier. The uptake of a variety of amino acids was examined to characterize the size of the putative channel. Glycine, beta-alanine, taurine, proline, gamma-aminobutyric acid (GABA), and threonine exhibited volume-activated transport that was DIDS inhibited, whereas aspartic acid, leucine, methionine, and ornithine were not transported. On the basis of the size of these amino acids, it appears that molecules containing eight or fewer major atoms and having a molecular mass of < 125-131 Da are transported during volume activation but larger molecules are not. We estimate the size of the channel to be 5.7-6.3 A in diameter.

Neutral amino acid transport systems of microvillous membrane of human placenta

1988 ◽  
Vol 254 (6) ◽  
pp. C773-C780 ◽  
Author(s):  
L. W. Johnson ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Plasma Membranes ◽  
Competitive Inhibition ◽  
Transport Systems ◽  
Acid Uptake ◽  
Acid Transport ◽  
Independent System ◽  
High Affinity

Placental transport produces concentrations of amino acids in fetal blood greater than those of maternal blood. Competitive inhibition studies of zwitterionic amino acid transport in isolated vesicles from the microvillous (maternal facing) plasma membranes of syncytiotrophoblast defined three transport systems: 1) a sodium-dependent system that supports methylaminoisobutyric acid (MeAIB) transport and has the characteristics of an A system; 2) a sodium-independent system with a high affinity for leucine and other amino acids with branched or aromatic side chains; and 3) a sodium-independent system with a preference for alanine as a substrate. The two sodium-independent systems could be further discriminated by marked specificity for trans stimulation with alanine or with leucine. System ASC, known to be present in whole placenta, and the neutral brush-border or imino systems of other polarized epithelia were apparently absent. Kinetic characteristics of the A system make it the probable primary driving force for concentrative transfer of its substrate amino acids to the fetus. Characteristics of the high-affinity leucine system demonstrated that it is saturated by normal serum leucine concentrations. Regulation of either system has the potential to alter placental amino acid uptake and transfer to the fetus.

Membrane transport of aromatic amino acids by Hymenolepis diminuta (Cestoda)

Parasitology ◽  
1980 ◽  
Vol 81 (2) ◽  
pp. 395-403 ◽  
Author(s):  
P. W. Pappas ◽  
H. R. Gamble
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Aromatic Amino Acid ◽  
Aromatic Amino Acids ◽  
Amino Acid Uptake ◽  
Hymenolepis Diminuta ◽  
Acid Uptake ◽  
Acid Transport ◽  
Concentration Difference

SUMMARYAromatic amino acids (phenylalanine, tryptophan and tyrosine) are absorbed by Hymenolepis diminuta through a combination of mediated (non-Na+-sensitive) transport and diffusion. All 3 amino acids are accumulated against an apparent concentration difference during a 30-min incubation of tapeworms in 0·1 mM 3H-labelled amino acid. Inhibitor studies demonstrate that phenylalanine, tryptophan and tyrosine are mutually competitive inhibitors of the uptake of each other, and the uptake of these amino acids is inhibited by aliphatic amino acids but not by basic or dicarboxylic amino acids. The D- and L-isomers of aromatic amino acids are equally effective in inhibiting aromatic amino acid uptake. The data indicate that at least 3 amino acid transport loci are involved in aromatic amino acid transport by H. diminuta.

Puromycin effect on amino acid transport: differential rates of carrier protein turnover

1975 ◽  
Vol 228 (1) ◽  
pp. 23-26 ◽  
Author(s):  
JM Phang ◽  
DL Valle ◽  
L Fisher ◽  
A Granger
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Half Life ◽  
Transport Systems ◽  
Acid Uptake ◽  
Acid Transport ◽  
Aminoisobutyric Acid ◽  
Fetal Rat ◽  
Protein Components

In fetal rat calvaria, puromycin selectively inhibited the uptake of certain groups of amino acids. Puromycin treatment decreased the uptake of glycine, L-proline, and alpha-aminoisobutyric acid but was without effect on the active uptake of all other amino acids tested. In studies of alpha-aminoisobutyric acid uptake, puromycin decreased the maximal transport velocity by 70% but had no effect on the affinity of the transport system for the amino acid. With puromycin treatment, the fall-off in rates of alpha-aminoisobutyric acid uptake was first order with a half-life of 68 min. Insulin treatment increased this half-life to 118 min. These findings suggest that protein components of specific transport systems are degraded at varying rates after puromycin blockade of protein synthesis. Hormones that stimulate amino acid transport (e.g., insulin) may decrease the rate of degradation of these protein components.

scholarly journals Effect of high altitude on human placental amino acid transport

2020 ◽  
Vol 128 (1) ◽  
pp. 127-133 ◽  
Author(s):  
Owen. R. Vaughan ◽  
Fredrick Thompson ◽  
Ramón. A. Lorca ◽  
Colleen G. Julian ◽  
Theresa L. Powell ◽  
...  
Keyword(s):  
Birth Weight ◽  
Amino Acid ◽  
High Altitude ◽  
Sea Level ◽  
Amino Acid Transport ◽  
Acid Uptake ◽  
Acid Transport ◽  
Transport Capacity ◽  
System A ◽  
Low Altitude

Women residing at high altitudes deliver infants of lower birth weight than at sea level. Birth weight correlates with placental system A-mediated amino acid transport capacity, and severe environmental hypoxia reduces system A activity in isolated trophoblast and the mouse placenta. However, the effect of high altitude on human placental amino acid transport remains unknown. We hypothesized that microvillous membrane (MVM) system A and system L amino acid transporter activity is lower in placentas of women living at high altitude compared with low-altitude controls. Placentas were collected at term from healthy pregnant women residing at high altitude (HA; >2,500 m; n = 14) or low altitude (LA; <1,700 m; n = 14) following planned, unlabored cesarean section. Birth weight, but not placenta weight, was 13% lower in HA pregnancies (2.88 ± 0.11 kg) compared with LA (3.30 ± 0.07 kg, P < 0.01). MVM erythropoietin receptor abundance, determined by immunoblot, was greater in HA than in LA placentas, consistent with lower placental oxygen levels at HA. However, there was no effect of altitude on MVM system A or L activity, determined by Na+-dependent [14C]methylaminoisobutyric acid uptake and [3H]leucine uptake, respectively. MVM abundance of glucose transporters (GLUTs) 1 and 4 and basal membrane GLUT4 were also similar in LA and HA placentas. Low birth weights in the neonates of women residing at high altitude are not a consequence of reduced placental amino acid transport capacity. These observations are in general agreement with studies of IUGR babies at low altitude, in which MVM system A activity is downregulated only in growth-restricted babies with significant compromise. NEW & NOTEWORTHY Babies born at high altitude are smaller than at sea level. Birth weight is dependent on growth in utero and, in turn, placental nutrient transport. We determined amino acid transport capacity in placentas collected from women resident at low and high altitude. Altitude did not affect system A amino acid transport across the syncytiotrophoblast microvillous membrane, suggesting that impaired placental amino acid transport does not contribute to reduced birth weight in this high-altitude population.

scholarly journals Evidence against the participation of the γ-glutamyltransferase-γ-glutamylcylclotransferase pathway in amino acid transport by rabbit erythrocytes

1975 ◽  
Vol 152 (3) ◽  
pp. 713-715 ◽  
Author(s):  
J D Young ◽  
J C Ellory ◽  
P C Wright
Keyword(s):  
Amino Acid ◽  
Amino Acid Transport ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Acid Transport

The GSH concentration of rabbit erythrocytes was monitored under conditions of large net transport of alanine, phenylalane and lysine in the absence of glucose. In no case was there an appreciable alteration in GSH concentration during amino acid uptake. It is suggested that the γ-glutamyltransferase-γ-glutamylcyclotransferase pathway does not participate in amino acid transport by these cells.

scholarly journals Amino acid transport in normal and glutathione-deficient sheep erythrocytes

1976 ◽  
Vol 154 (1) ◽  
pp. 43-48 ◽  
Author(s):  
J D Young ◽  
J C Ellory ◽  
E M Tucker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Cell Types ◽  
The Other ◽  
Acid Transport ◽  
Sheep Erythrocytes ◽  
Uptake Rates

1. Uptake rates for 23 amino acids were measured for both normal (high-GSH) and GSH-deficient (low-GSH) erythrocytes from Finnish Landrace sheep. 2. Compared with high-GSH cells, low-GSH cells had a markedly diminished permeability to D-alanine, L-alanine, α-amino-n-butyrate, valine, cysteine, serine, threonine, asparagine, lysine and ornithine. Smaller differences were observed for glycine and proline, whereas uptake of the other amino acids was not significantly different in the two cell types.

Amino Acid Transport in a Water-mould: The Possible Regulatory Roles of Calcium and N6-(Δ2-isopentenyl)adenine

1975 ◽  
Vol 53 (9) ◽  
pp. 975-988 ◽  
Author(s):  
Danny P. Singh ◽  
Hérb. B. LéJohn
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Osmotic Shock ◽  
Inhibitory Effect ◽  
Transport Systems ◽  
Acid Transport ◽  
Isopentenyl Adenine ◽  
Energy Dependent ◽  
Water Mould

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different transport systems elucidated are: (i) methionine, (ii) cysteine, (iii) proline, (iv) serine–threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine–tyrosine–tryptophan and leucine–isoleucine–valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.Calcium played more than a secondary role in the transport of the amino acids. When bound to the membrane-localized glycopeptide, it permits concentrative transport to take place. However, excess calcium can inhibit transport which can be overcome by chelating with citrate. Calculations show that the concentration of free citrate is most important. At low citrate concentrations (less than 1 mM) in the absence of exogenously supplied calcium, enhancement of amino acid transport occurs. At high concentrations (greater than 5 mM), citrate inhibits but this effect can be reversed by titrating with calcium. Evidently, the glycopeptide acts as a calcium sink to regulate the concentration of calcium made available to the cell for its membrane activities.N6-(Δ2-isopentenyl) adenine (a plant growth 'hormone') and analogues mimic the inhibitory effect of citrate and bind to the glycopeptide as well. Replot data for citrate and N6-(Δ2-isopentyl) adenine inhibition indicate that both agents have no more than one binding constant. These results implicate calcium, glycopeptide, and energy-dependent transport of solutes in some, as yet undefinable, way.

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