A novel proline, glycine: K+ symporter in midgut brush-border membrane vesicles from larval Manduca sexta.

1995 ◽  
Vol 198 (12) ◽  
pp. 2599-2607 ◽  
Author(s):  
A L Bader ◽  
R Parthasarathy ◽  
W R Harvey
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Neutral System ◽  
Aminoisobutyric Acid ◽  
Posterior Midgut

Alkali-cation-dependent uptake of proline and glycine into brush-border membrane vesicles from the midgut of the larval tobacco hornworm Manduca sexta was investigated using rapid filtration assays. Uptake of both amino acids was by electrophoretic symport, with K+ being the favored cation at pH 10. Counterflow accumulation of proline was elicited by glycine and vice versa, suggesting that the two amino acids are transported by a common symporter, which we designate the pro, gly: K+ symporter. L-alpha-Aminoisobutyric acid was the only other amino acid that elicited the accumulation of both proline and glycine. D-Proline was not symported; L-proline, glycine and L-alpha-aminoisobutyric acid appear to be the only substrates of the pro, gly: K+ symporter. Neutral amino acids with relatively short sidechains elicit glycine accumulation, suggesting that glycine may also be symported by the well-established neutral amino acid system. Since proline does not utilize the broad-spectrum, neutral system, its symport appears to be exclusively through the pro, gly: K+ symporter. Proline symport was found mainly in posterior midgut vesicles, suggesting that the pro, gly: K+ symporter may be localized in this region of the midgut.

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 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.

The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism

1997 ◽  
Vol 110 (24) ◽  
pp. 3099-3104
Author(s):  
J. Carroll ◽  
M.G. Wolfersberger ◽  
D.J. Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Aminopeptidase N ◽  
Insecticidal Toxin ◽  
Cry1ac Toxin ◽  
Posterior Midgut

Aminopeptidase N purified from whole Manduca sexta midgut binds the Cry1Ac insecticidal toxin from Bacillus thuringiensis and this binding is inhibited by N-acetylgalactosamine (GalNAc). We have examined the membrane permeabilising activity of the Cry1Ac toxin using brush border membrane vesicles (BBMV) prepared from the anterior (A-BBMV) and posterior (P-BBMV) subregions of the M. sexta midgut. A toxin mixing assay demonstrated a faster rate of toxin activity on P-BBMV than on A-BBMV. In the presence of GalNAc this rapid activity on P-BBMV was reduced to the rate seen with A-BBMV. GalNAc had no effect on the rate of A-BBMV permeabilisation by Cry1Ac. Aminopeptidase N assays of A- and P-BBMV demonstrated that this Cry1Ac binding protein is concentrated in the posterior midgut region of M. sexta. It therefore appears that there are two mechanisms by which Cry1Ac permeabilises the M. sexta midgut membrane: a GalNAc-sensitive mechanism restricted to the posterior midgut region, probably involving aminopeptidase N binding, and a previously undetected mechanism found in both the posterior and anterior regions.

scholarly journals Electroneutral Na+-2 Cl−-Leucine Cotransport by Lobster Hepatopancreatic Brush-Border Membrane Vesicles

1988 ◽  
Vol 136 (1) ◽  
pp. 363-381
Author(s):  
GREGORY A. AHEARN ◽  
LAUREL P. CLAY
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Acidic Ph ◽  
Polar Species ◽  
L System ◽  
Leucine Transport

Uptake of L-[3H]leucine by lobster hepatopancreatic brush-border membrane vesicles was stimulated by a transmembrane NaCl gradient (o>i), but not by identical gradients of NaSCN or other Cl− salts (e.g. K+, Li+, NH4+, Cs+ or choline), suggesting that amino acid transfer depended upon both Na+ and Cl−. In NaCl medium at acidic pH, leucine uptake was largely electroneutral and unresponsive to a transmembrane potential generated by permeable anions; however, in Na+-free medium, amino acid transport was strongly electrogenic under the same conditions. Leucine influx occurred by a combination of two carrier processes at physiologically acidic pH. One exhibited an influx Kt of 0.59 mmol l−1, a JM of 390 pmol mg protein−1 S−1 and a cotransport stoichiometry of 1 Na+: 2 Cl+: 1 leucine. This process was most strongly cis-inhibited by the non-polar amino acids phenylalanine, methionine and isoleucine, and most weakly inhibited by the more polar species methylaminoisobutyric acid (MeAIB), hydroxyproline, glutamate and arginine. The second leucine carrier system showed a very low binding affinity and could not be distinguished from diffusion, was Na+- and Cl−-independent, and was cis-inhibited by more polar amino acids such as lysine, hydroxyproline, MeAIB, alanine and glutamate. These results suggest that brush-border leucine transport in lobster hepatopancreas at acidic pH may occur by a combination of a modified L-system, that includes ion cosubstrates, and either by a second undefined Na+-independent process with a broad structural specificity or by multiple Na+-independent processes.

scholarly journals Substrate structure and amino acid/K+ symport in brush-border membrane vesicles from larval Manduca sexta midgut.

1994 ◽  
Vol 197 (1) ◽  
pp. 237-250
Author(s):  
R Parthasarathy ◽  
T Xie ◽  
M G Wolfersberger ◽  
W R Harvey
Keyword(s):  
Amino Acid ◽  
Charge Distribution ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Isoleucine Leucine ◽  
Substrate Structure ◽  
Molecular Charge

The effects of amino acid sidechain length, substituent position and c chirality on amino acid/K+ symport have been examined in rapid filtration experiments on brush-border membrane vesicles prepared from larval Manduca sexta midgut. Cis-inhibition and trans-stimulation protocols were used to examine the effects of amino acid analogs on the uptake of alanine, phenylalanine, leucine and lysine, which are cotransported with K+ by a zwitterionic symporter at the high pH characteristic of the midgut in vivo. The symporter was found to translocate both L- and D-stereoisomers of alanine, leucine and lysine, but only the L-form of phenylalanine. Alterations to substrate structure that leave the charge distribution unchanged do not affect symport. Thus, moving the methyl group from C-3 to C-5 in the sequence isoleucine, leucine and norleucine has no effect on their ability to inhibit leucine symport. Increasing sidechain length among alanine homologs has little effect on their ability to inhibit alanine uptake, but increasing the sidechain length of lysine homologs from 1 to 3 methylene groups enhances cis-inhibition and trans-stimulation of lysine symport. The substantial difference in molecular charge distribution among aminobutanoic acid isomers has a large impact on alanine symport with only alpha- (or 2-) aminobutanoic acid functioning as an alanine analog. Only those changes in substrate structure that are coupled to the molecular charge distribution seem to affect symport. The tolerance of the symporter may reflect a balance mandated by the conflicting demands of selectivity and throughput.

scholarly journals Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles

2010 ◽  
Vol 1798 (6) ◽  
pp. 1111-1118 ◽  
Author(s):  
Jean-Frédéric Brunet ◽  
Vincent Vachon ◽  
Marc Juteau ◽  
Jeroen Van Rie ◽  
Geneviève Larouche ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Bacillus Thuringiensis Toxin
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