DETERMINANTS FOR THE ACTIVITY OF THE NEUTRAL AMINO ACID/K+ SYMPORT IN LEPIDOPTERAN LARVAL MIDGUT

1994 ◽  
Vol 196 (1) ◽  
pp. 145-155 ◽  
Author(s):  
B Giordana ◽  
P Parenti
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Neutral Amino Acid ◽  
Strong Interactions ◽  
Substrate Affinity ◽  
Larval Midgut ◽  
Leucine Uptake

The columnar cells of lepidopteran larvae express, in their apical brush-border membrane, a class of symporters which in vivo couple the intracellularly directed amino acid and K+ fluxes. An analysis of the functional properties of the symporter for neutral amino acids along the anterior, middle and posterior regions of the larval midgut of Bombyx mori demonstrated the ability of a K+ gradient to drive leucine accumulation into brush-border membrane vesicles (BBMV) in all three preparations. However, marked differences are evident between the posterior (P) and the anterior­middle (AM) regions. In P-BBMV, much higher intravesicular accumulations were observed, Vmax was six- to eightfold higher than in AM-BBMV, a lowering of external pH (pHe) from 8.7 to 7.2 caused a tenfold increase of Km, and the absence of a potential difference (delta psi) caused a threefold decrease of Vmax. In contrast, leucine uptake in AM-BBMV was poorly sensitive to both pH and delta psi. The kinetics of leucine uptake as a function of cis K+ concentration were hyperbolic in P-BBMV and sigmoidal in AM-BBMV. More than 50 amino acids and analogues were used in inhibition experiments to characterize the amino acid binding site. Branched-chain amino acids modified on the carboxyl moiety were recognized only by the P-BBMV symporter. In AM-BBMV, substrate affinity was increased by the presence of a heterocyclic sidechain, even in the presence of a modified carboxyl- or alpha-amino group. Together, these results suggest that isoforms of the neutral amino acid/K+ symporter are present. A natural inhibitor of amino acid symport has not yet been identified. However, several lines of evidence suggest that strong interactions exist between the amino acid/K+ symporter and the receptor for the lepidopteran-specific Bacillus thuringiensis delta-endotoxins. CryIA(a) toxin, highly toxic for B. mori larvae, produced a dose-dependent inhibition of leucine uptake into both BBMV populations. The toxin was able to block the symporter in its ternary and leucine-only forms.

Potential differences influence amino acid/Na+ symport rates in larval Manduca sexta midgut brush-border membrane vesicles.

1994 ◽  
Vol 189 (1) ◽  
pp. 55-67
Author(s):  
R Parthasarathy ◽  
W R Harvey
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Rate Determining Step ◽  
Half Saturation Constant

The time-dependent fluorescence intensity of an intravesicular potential-sensitive dye was used to probe the real-time kinetics of potential difference (PD)-dependent amino acid/Na+ symport at pH9 into brush-border membrane vesicles obtained from larval Manduca sexta midgut. Neutral amino acids (alanine, proline) are symported at higher rates as the vesicles are hyperpolarized. The symport rates of acidic (glutamate) and basic (arginine) amino acids are almost PD-independent. The half-saturation constant of alanine is PD-independent between -108 and -78 mV, although the maximal symport velocity increases by half as the voltage is increased. Amino acid throughput is evidently enhanced as the relatively high transmembrane PDs (> 150 mV, lumen positive) measured in vivo are approached. The half-saturation concentrations of Na+ were in the range 15-40 mmol l-1 for most of the amino acids examined and increased with voltage for alanine. The Vmax observed as a function of cation or amino acid concentration increased as the vesicle was hyperpolarized in the case of leucine and alanine. The data support the hypothesis that carrier and substrates are at equilibrium inasmuch as substrate translocation seems to be the rate-determining step of symport.

scholarly journals Intestinal peptidases form functional complexes with the neutral amino acid transporter B0AT1

2012 ◽  
Vol 446 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Stephen J. Fairweather ◽  
Angelika Bröer ◽  
Megan L. O'Mara ◽  
Stefan Bröer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Amino Acid Transporter ◽  
The Body ◽  
Site Directed Mutagenesis ◽  
Substrate Affinity ◽  
Xenopus Laevis Oocytes ◽  
Acid Transporter

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

1999 ◽  
Vol 276 (2) ◽  
pp. R627-R631 ◽  
Author(s):  
Carles Garriga ◽  
Nativitat Rovira ◽  
Miquel Moretó ◽  
Joana M. Planas
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Brush Border ◽  
Synthetic Peptide ◽  
Brush Border Membrane ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Dependent Transport

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

Na+-dependent neutral amino acid transporter ATB0 is a rabbit epithelial cell brush-border protein

2001 ◽  
Vol 281 (3) ◽  
pp. C963-C971 ◽  
Author(s):  
Nelly E. Avissar ◽  
Charlotte K. Ryan ◽  
Vadivel Ganapathy ◽  
Harry C. Sax
Keyword(s):  
Amino Acid ◽  
Hela Cells ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Polyclonal Antibodies ◽  
Neutral Amino Acid ◽  
Rabbit Kidney ◽  
Western Blots ◽  
Amino Acid Transport System ◽  
Rabbit Tissues

System B0 activity accounts for the majority of intestinal and kidney luminal neutral amino acid absorption. An amino acid transport system, called ATB0 (also known as ASCT2), with functional characteristics similar to those of system B0, has been recently cloned. We generated polyclonal antibodies to human and rabbit ATB0 COOH-terminal peptides and used Western blot analysis to detect ATB0 protein in rabbit tissues, rabbit ileal brush-border membrane vesicles (BBMV), and HeLa cells transfected with plasmids containing ATB0 cDNAs. Immunohistochemistry was used to localize ATB0 in rabbit kidney and intestine. In Western blots of rabbit tissues, ATB0 was a broad smear of 78- to 85-kDa proteins. In transfected HeLa cells, ATB0 appeared as a smear consisting of 57- to 65-kDa proteins. The highest expression was found in the kidney. ATB0 was enriched in rabbit ileal BBMV and in HeLa cells transfected with ATB0 cDNAs. In the kidney and in the intestine, ATB0 was confined to the brush-border membrane (BBM) of the proximal tubular cell and of the enterocyte, respectively. Tissue and intracellular distribution of ATB0 protein parallels that of system B0 activity. ATB0protein could be the transporter responsible for system B0in the BBM of epithelial cells.

scholarly journals Characterization of tryptophan transport in human placental brush-border membrane vesicles

1986 ◽  
Vol 238 (1) ◽  
pp. 201-208 ◽  
Author(s):  
M E Ganapathy ◽  
F H Leibach ◽  
V B Mahesh ◽  
J C Howard ◽  
L D Devoe ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Aromatic Amino Acids ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
System 1 ◽  
Tryptophan Uptake ◽  
Tryptophan Transport

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.

Effect of amino acid intake on brush-border membrane uptake of sulfur amino acids

1986 ◽  
Vol 251 (1) ◽  
pp. F125-F131
Author(s):  
R. W. Chesney ◽  
N. Gusowski ◽  
M. Padilla ◽  
S. Lippincott
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Time Course ◽  
Brush Border Membrane ◽  
Sulfur Amino Acids ◽  
Beta Alanine ◽  
Renal Brush Border Membrane ◽  
Amino Acid Intake ◽  
Renal Brush Border

Alterations in the intake of sulfur amino acids (SAA) changes the rat renal brush-border membrane uptake of the beta-amino acid, taurine. A low-SAA diet enhances and a high-taurine diet reduces uptake (Chesney et al., Kidney Int. 24: 588-594, 1983). Neither the low-SAA diet nor the high-taurine diet alters the time course or concentration-dependent accumulation of the sulfur amino acids methionine and cystine or of inorganic sulfate. By contrast the uptake of beta-alanine, another beta-amino acid that competes with taurine, is greater in animals on the low-SAA diet. The high-taurine diet does not change beta-alanine uptake. The plasma levels of taurine are altered by dietary change, but not the values for methionine and cystine. This study indicates that renal adaptation is expressed for beta-alanine, a nonsulfur-containing beta-amino acid. By contrast, methionine, cystine, and sulfate, which participate in a variety of synthetic and conjugative processes, are not conserved by the renal brush-border surface following ingestion of either a low-methionine and -cystine diet or high-taurine diet.

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