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.

Effects of cations on pH gradient-stimulated sulfate transport in rabbit ileal brush-border membrane vesicles

1985 ◽  
Vol 249 (5) ◽  
pp. G614-G621 ◽  
Author(s):  
C. M. Schron ◽  
R. G. Knickelbein ◽  
P. S. Aronson ◽  
J. Della Puca ◽  
J. W. Dobbins
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Disodium Salt ◽  
Brush Border Membrane Vesicles ◽  
Ph Gradient ◽  
Disulfonic Acid ◽  
Rabbit Ileum ◽  
External K

In brush-border membrane vesicles from rabbit ileum, we previously reported pH gradient-stimulated SO4 uptake and presented evidence that this represents carrier-mediated SO4-OH exchange. In the present study inhibitors of SO4-OH exchange (H-SO4 cotransport) were shown not to inhibit Na-SO4 cotransport, suggesting that these are two separate carrier-mediated transport mechanisms. While pH gradient-stimulated SO4 uptake was inhibited 87% by 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, disodium salt (DIDS) and 79% by 1.0 mM furosemide, Na+-stimulated SO4 uptake was only inhibited 11 and 0%, respectively. K+ (20 mM), Cl (5 mM), and oxalate (0.25 mM) inhibited pH gradient-stimulated SO4 uptake (38-65%) but had no effect on Na+-stimulated SO4 uptake. Finally, at Na+ concentrations (10 mM) significantly less than that required for Na+-stimulated SO4 uptake (60-100 mM), external Na+ inhibited pH gradient-stimulated SO4 uptake, suggesting two independent effects of this cation. SO4 uptake was also inhibited by external K+ both in the presence and absence of a pH gradient. A Dixon plot of the DIDS-sensitive SO4 uptake under pH gradient conditions yielded a straight line, indicating a single site of interaction between external K+ and the SO4-OH carrier (apparent Ki = 7.2 mM). In contrast to the inhibition by external K+, internal K+ stimulated SO4 uptake. This effect was DIDS sensitive and not enhanced by valinomycin, suggesting an interaction of internal K+ with the SO4-OH exchanger independent of a K+-induced electrical potential. SO4 uptake and the effects of K+ were pH modulated with less SO4 uptake and less K+ effect at higher pH.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex

1979 ◽  
Vol 182 (1) ◽  
pp. 223-229 ◽  
Author(s):  
Heinrich Lücke ◽  
Gertraud Stange ◽  
Heini Murer
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Kidney Cortex ◽  
Electrical Potential Difference ◽  
Univalent Cations ◽  
Rat Kidney Cortex

Uptake of SO42− into brush-border membrane vesicles isolated from rat kindey cortex by a Ca2+-precipitation method was investigated by using a rapid-filtration technique. Uptake of SO42− by the vesicles was osmotically sensitive and represented transport into an intra-vesicular space. Transport of SO42− by brush-border membranes was stimulated in the presence of Na+, compared with the presence of K+ or other univalent cations. A typical ‘overshoot’ phenomenon was observed in the presence of an NaCl gradient (100mm-Na+ outside/zero mm-Na+ inside). Radioactive-SO42− exchange was faster in the presence of Na+ than in the presence of K+. Addition of gramicidin-D, an ionophore for univalent cations, decreased the Na+-gradient-driven SO42− uptake. SO42− uptake was only saturable in the presence of Na+. Counter-transport of Na+-dependent SO42− transport was shown with MoO42− and S2O32−, but not with PO42−. Changing the electrical potential difference across the vesicle membrane by establishing different diffusion potentials (anion replacement; K+ gradient±valinomycin) was not able to alter Na+-dependent SO42− uptake. The experiments indicate the presence of an electroneutral Na+/SO42−-co-transport system in brush-border membrane vesicles isolated from rat kidney cortex.

scholarly journals Taurocholate–sodium co-transport by brush-border membrane vesicles isolated from rat ileum

1978 ◽  
Vol 174 (3) ◽  
pp. 951-958 ◽  
Author(s):  
Heinrich Lücke ◽  
Gertraud Stange ◽  
Rolf Kinne ◽  
Heini Murer
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Precipitation Method ◽  
Electrical Potential Difference ◽  
Vesicle Membrane ◽  
Brush Border Membranes ◽  
Taurocholate Transport

Uptake of taurocholate into brush-border membrane vesicles isolated from rat small intestine by a Ca2+ -precipitation method was investigated by using a rapid-filtration technique. Uptake of taurocholate by ileal brush-border membranes consisted of three phenomena: binding to the outside of the vesicles, transfer across the vesicle membrane and binding to the intravesicular compartment. The transport of taurocholate across the brush-border membranes was stimulated in the presence of Na+ compared with the presence of K+; stimulation was about 11-fold in the presence of a NaCl gradient (Nao>Nai), where the subscripts refer to ‘outside’ and ‘inside’ respectively, and 4-fold under equilibrium conditions for Na+ (Nao=Nai). In the presence of a Na+ gradient a typical ‘overshoot’ phenomenon was observed. Membranes preloaded with unlabelled taurocholate showed an accelerated entry of labelled taurocholate (tracer exchange) in the presence of Na+ compared with the presence of K+. The stimulation by Na+ was observed only in membrane preparations from the ileum. Addition of monactin, an ionophore for univalent cations, decreased the Na+-gradient-driven taurocholate uptake. The Na+-dependent taurocholate transport showed saturation kinetics and the phenomenon of counterflow and was inhibited by glycocholate. Other cations such as Li+, Rb+ and Cs+ could not replace Na+ in its stimulatory action. When the electrical potential difference across the vesicle membrane was altered by establishing different diffusion potentials (anion replacement; K+ gradient±valinomycin) a more-negative potential inside stimulated Na+-dependent taurocholate transport. These data demonstrate the presence of a rheogenic (potential sensitive) Na+–taurocholate co-transport system in ileal brush-border membranes and support the hypothesis that the reabsorption of bile acids in the ileum is a secondary active uptake.

scholarly journals Evidence for a low-affinity, high-capacity uniport for amino acids in Bombyx mori larval midgut

1998 ◽  
Vol 274 (5) ◽  
pp. R1372-R1375 ◽  
Author(s):  
M. G. Leonardi ◽  
M. Casartelli ◽  
P. Parenti ◽  
B. Giordana
Keyword(s):  
Amino Acids ◽  
Kinetic Analysis ◽  
Bombyx Mori ◽  
Electrical Potential ◽  
High Capacity ◽  
Membrane Vesicles ◽  
Electrical Potential Difference ◽  
Larval Midgut ◽  
Posterior Midgut ◽  
Transmembrane Electrical Potential

We investigated the kinetics of leucine influx as a funtion of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K+-dependent symporter for neutral amino acids, of a K+-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol ⋅ 7 s−1 ⋅ mg protein−1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.

LEUCINE UPTAKE IN BRUSH-BORDER MEMBRANE VESICLES FROM THE MIDGUT OF A LEPIDOPTERAN LARVA, PHILOSAMIA CYNTHIA

1990 ◽  
Vol 149 (1) ◽  
pp. 207-221
Author(s):  
V. FRANCA SACCHI ◽  
BARBARA GIORDANA ◽  
FLAVIA CAMPANINI ◽  
PATRIZIA BONFANTI ◽  
GIORGIO M. HANOZET
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Electrical Potential Difference ◽  
Experimental Conditions ◽  
Leucine Uptake ◽  
Leucine Transport

A potassium- or sodium-activated cotransport of leucine occurs in brush-border membrane vesicles prepared from the midgut of larvae of Philosamia cynthia Drury). The potassium chemical gradient can drive a twofold accumulation of leucine, which is greatly increased under experimental conditions that presumably provide an electrical potential difference (δψ) Kinetic parameters show that leucine transport is improved by these conditions and by a pH gradient similar to that occurring in vivo. However, these gradients cannot drive an intravesicular accumulation of leucine in the absence of potassium. The potassium-dependence of leucine uptake shows that 20% of the transport is potassium-independent and that K50 and Vmax are 30.3± 3.2mmoll−1 and 2584±148pmol 7 s−1mg−1 protein, respectively. The potassium-independent component of leucine transport is also carrier-mediated and some evidence is reported suggesting that the same carrier can cross the membrane as binary carrier and leucine) or ternary (carrier, leucine and potassium) complexes, each having a different mobility

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.

Na-dependent L-glutamate transport by eel intestinal BBMV: role of K+ and Cl-

1989 ◽  
Vol 257 (1) ◽  
pp. R180-R188
Author(s):  
P. M. Romano ◽  
G. A. Ahearn ◽  
C. Storelli
Keyword(s):  
Amino Acid ◽  
Glutamate Uptake ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Anguilla Anguilla ◽  
Competitive Inhibitor ◽  
Glutamate Transport ◽  
Electrical Potential Difference ◽  
Intestinal Brush Border Membrane ◽  
Transmembrane Electrical Potential

L-[3H]glutamate uptake into eel (Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) was a sigmoidal function of extravesicular Na, suggesting that two or more cations accompanied the amino acid during transport. L-[3H]glutamate influx illustrated the following kinetic constants: apparent membrane binding affinity (Kapp) = 0.80 +/- 0.12 mM; influx velocity (Jmax) = 2.61 +/- 0.31 nmol.mg protein-1.min-1; and permeability coefficient (P) = 0.65 +/- 0.10 microliters.mg protein-1. min-1. Results from the imposition of diffusion potentials across vesicle membranes using K-valinomycin or H-carbonyl-cyanide p-chloromethoxyphenylhydrazone suggested that Na-dependent L-glutamate transport was sensitive to transmembrane electrical potential difference. Extravesicular aspartate was a competitive inhibitor of L-[3H]glutamate influx [inhibitory constant (Ki) = 0.28 +/- 0.04 mM]. Intravesicular K and extravesicular Cl ions enhanced maximal amino acid influx and transient L-glutamate accumulation against a concentration gradient (overshoot). Intravesicular K reduced the Kapp of the membrane to L-glutamate, whereas extravesicular Cl increased L-glutamate Jmax. A model for L-[3H]glutamate transport is suggested involving the cotransport of at least two Na and one L-glutamate that is activated by one intravesicular K ion and at least two extravesicular Cl ions.

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