A CYANINE DYE AS INDICATOR OF MEMBRANE ELECTRICAL POTENTIAL DIFFERENCES IN BRUSH BORDER MEMBRANE VESICLES. STUDIES WITH K+ GRADIENTS AND Na+/AMINO ACID COTRANSPORT

1981 ◽  
pp. 409-418 ◽  
Author(s):  
G. Burckhardt ◽  
H. Murer
Keyword(s):  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Cyanine Dye ◽  
Membrane Electrical Potential

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.

Electroneutral Na+/H+exchange in brush-border membrane vesicles fromPenaeus japonicus hepatopancreas

1998 ◽  
Vol 274 (2) ◽  
pp. R486-R493 ◽  
Author(s):  
Sebastiano Vilella ◽  
Vincenzo Zonno ◽  
Laura Ingrosso ◽  
Tiziano Verri ◽  
Carlo Storelli
Keyword(s):  
Kinetic Parameters ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Hill Coefficient ◽  
Uptake Kinetic ◽  
Ph Dependent ◽  
Transmembrane Electrical Potential

An electroneutral Na+/H+exchange mechanism (dimethylamiloride inhibitable, Li+ sensitive, and Ca2+ insensitive) was identified in brush-border membrane vesicles (BBMV) from Kuruma prawn hepatopancreas by monitoring Na+-dependent H+ fluxes with the pH-sensitive dye acridine orange and measuring22Na+uptake. Kinetic parameters measured under short-circuited conditions were the Na+ concentration that yielded one-half of the maximal dissipation rate ( F max) of the preset transmembrane ΔpH ( K Na) = 15 ± 2 mM and F max = 3,626 ± 197 Δ F ⋅ min−1 ⋅ mg protein−1, with a Hill coefficient for Na+ of ∼1. In addition, the inhibitory constant for dimethylamiloride was found to be ∼1 μM. The electroneutral nature of the antiporter was assessed in that an inside-negative transmembrane electrical potential neither affected kinetic parameters nor stimulated pH-dependent (intracellular pH > extracellular pH)22Na+uptake. In contrast, electrogenic pH-dependent22Na+uptake was observed in lobster hepatopancreatic BBMV. Substitution of chloride with gluconate resulted in increasing K Na and decreasing Δ F max, which suggests a possible role of chloride in the operational mechanism of the antiporter. These results indicate that a Na+/H+exchanger, resembling the electroneutral Na+/H+antiporter model, is present in hepatopancreatic BBMV from the Kuruma prawn Penaeus japonicus.

K+-neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions

1998 ◽  
Vol 274 (5) ◽  
pp. R1361-R1371 ◽  
Author(s):  
B. Giordana ◽  
M. G. Leonardi ◽  
M. Casartelli ◽  
P. Consonni ◽  
P. Parenti
Keyword(s):  
Amino Acid ◽  
Bombyx Mori ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Ph Gradient ◽  
Electrical Potential Difference ◽  
Posterior Midgut ◽  
Solution Bathing

The K+-dependent symporter for leucine and other neutral amino acids expressed along the midgut of the silkworm Bombyx mori operates with best efficiency in the presence of a steep pH gradient across the brush-border membrane, with external alkaline pH values up to 11, and an electrical potential difference (Δψ) of ∼200 mV. Careful determinations of leucine kinetics as a function of external amino acid concentrations between 50 and 1,000 μM, performed with brush-border membrane vesicles (BBMV) obtained from the middle and posterior midgut regions, revealed that the kinetic parameter affected by the presence of a ΔpH was the maximal rate of transport. The addition of Δψ caused a further marked increase of the translocation rate. At nonsaturating leucine concentrations in the solution bathing the external side of the brush-border membrane, leucine accumulation within BBMV and midgut cells was not only driven by the gradient of the driver cation K+ and Δψ but occurred also in the absence of K+. The ability of the symporter to translocate the substrate in its binary form allows the intracellular accumulation of leucine in the absence of K+, provided that a pH gradient, with alkaline outside, is present. The mechanisms involved in this accumulation are discussed.

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.

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