Quantitative studies of cotransport systems: Models and vesicles

Rj, Turner

doi:10.1007/bf01871450

Cited by 107 publications

(48 citation statements)

References 70 publications

(61 reference statements)

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“…Based on these quantitative measurements, symport-dependent proton and sucrose uptake were 2.4 + 0.3 nmol H+/mg min-' and 2.95 ± 0.56 nmol sucrose/mg min-', respectively, and the resultant proton to sucrose stoichiometry was 0.81. Taken together with the data in Figure 6 and Turner's kinetic model (22), it is concluded that the stoichiometry of the protonsucrose symport is 1:1. The apparent underestimation of proton flux may be the result of buffering by the membrane vesicles or the result of proton-glucose efflux from the PMVs as glucose builds up due to invertase activity.…”

Section: Stoichiometry Of the Proton-sucrose Symportsupporting

confidence: 68%

“…Based on the kinetic models describing cotransport systems developed by Turner (22), the hyperbolic dependence of sucrose flux on proton concentration shown in Figure 6 is consistent 8 1/s Figure 6. ApH-dependent sucrose transport as a function of the transport solution proton concentration.…”

Section: Stoichiometry Of the Proton-sucrose Symportsupporting

confidence: 59%

“…If two or more protons were required per sucrose translocated, the data in Figure 6 would exhibit a sigmoidal relationship. Although the data in Figure 6 are consistent with predictions based on Turner's kinetic model (22), it is not valid to use this fit as the only criterion in support of a specific molecular mechanism. Therefore, a direct measurement of both proton and sucrose flux was also used to determine the stoichiometry of the proton-sucrose symport.…”

Section: Stoichiometry Of the Proton-sucrose Symportmentioning

confidence: 63%

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Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

Bush

1990

Plant Physiol.

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The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Westem) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on ApH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V,,, only with no apparent change in Km. Sucrose transport rates driven by membrane potential only, i.e. in the absence of ApH, were comparable to ApH-driven flux. Both membrane potential and ApHdriven sucrose transport were used to examine proton binding to the symport and the apparent Km for H was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.

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Section: Stoichiometry Of the Proton-sucrose Symportsupporting

confidence: 68%

Section: Stoichiometry Of the Proton-sucrose Symportsupporting

confidence: 59%

Section: Stoichiometry Of the Proton-sucrose Symportmentioning

confidence: 63%

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Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

Bush

1990

Plant Physiol.

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“…Although work up to the present time with sealed vesicles has emphasized ATP-dependent primary transport, this system can be extremely useful when utilized to investigate a secondary process such as sucrose transport. Indeed, sealed membrane vesicle systems have proven useful when applied to secondary transport systems present in bacterial (12 and references therein), fungal (9), and animal (28 and references therein) cell membranes.…”

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confidence: 99%

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot

Dp¹,

Wr²,

Re³

1985

Plant Physiol.

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Sealed membrane vesicles were isolated from homogenates of sugarbeet (Beta valgaris L.) taproot by a combination of differential centrifugation, extraction with KI, and dextran gradient centrifugation. Relative to the KI-extracted microsomes, the content of plasma membranes, mitochondrial membranes, and Golgi membranes was much reduced in the final vesicle fraction. A component of ATPase activity that was inhibited by nitrate co-enriched with the capacity of the vesicles to form a steady state pH gradient during the purification procedure. This suggests that the nitrate-sensitive ATPase may be involved in driving H'-transport, and this is consistent with the observation that Hf-transport, in the final vesicle fraction was inhibited by nitrate. Proton transport in the sugarbeet vesicles was substrate specific for ATP, insensitive to sodium vanadate and oligomycin but was inhibited by diethylstilbestrol and N,N'-dicyclohexylcarbodiimide. The formation of a pH gradient in the vesicles was enhanced by halide ions in the sequence I-> Br-> Cl1 while P was inhibitory. These stimulatory effects occur from both a direct stimulation of the ATPase by anions and a reduction in the vesicle membrane potential. In the presence of Cl1, alkali cations reduce the pH gradient relative to that observed with bis-tris-propane, possibly by H'/ alkali cation exchange. Based upon the properties of the HW-transporting vesicles, it is proposed that they are most likely derived from the tonoplast so that this vesicle preparation would represent a convenient system for studying the mechanism of transport at this membrane boundary.Energy dependent, primary proton transport appears to be a ubiquitous property of higher plant cells (21 and references therein). The energy conserved in the electrochemical potential gradient of protons, the proton motive force, can then provide the driving force for the transport of other solutes such as sugars according to the chemiosmotic hypothesis (18). The latter process has been termed 'secondary' transport since the coupling to metabolic energy is indirect via an ion gradient (11 and references therein). Studies mary proton transport can occur both at the plasma membrane (cytoplasm to cell exterior) and at the tonoplast (cytoplasm to vacuole lumen) (27 and references therein). When membrane fractions enriched with plasma membrane (16) or tonoplast (15) are prepared, they are found to contain ATP hydrolytic activity which has been postulated to reflect the presence of ATP-fueled proton pumps responsible for carrying out these primary transport events (16,21,27).A significant advance to the study of membrane transport in higher plants came with the development of the methodology to isolate sealed membrane vesicles by Sze (25). The sealed vesicle system allowed the demonstration that these membrane-associated ATPases isolated from higher plant cells could directly transport protons (27). Several laboratories have since characterized ATPase-mediated proton transport in sealed vesicles thought to be d...

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“…2 were derived, -ln [l -(X,/X,,)] where X, is uptake at time t and X, is uptake at equilibrium (t = a ) , was plotted against t, and used to estimate the half-time ( t I l 2 ) of maximum D-glucose transfer (Hopfer, 2981 ;Turner, 1983). Apparent kinetic constants, V and K,, were calculated from a double-reciprocal plot with a computer-program (Cor- nish- Bowden and Eisenthal, 1978;Kaminski and Domino, 1987).…”

Section: Data Presentation and Analysismentioning

confidence: 99%

1,25‐Dihydroxycholecalciferol‐related Na⁺/D‐glucose transport in brush‐border membrane vesicles from embryonic chick jejunum

Dębiec¹,

Cross

Peterlik

1991

European Journal of Biochemistry

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1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na+-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na+-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ("a' ], = 100 mM; [Na+Ii = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na+-dependent D-glUCOSe transport through changes in the electrochemical Natracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na +/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane.10 nM triiodothyronine, which by itself has no effect on Na+-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na'/D-glucose-carrier activity was increased fourfold above control levels.Triiodothyronine has been shown to facilitate the expression of genomic effects of 1,25-dihydroxycholecalciferol (calcitriol) on calcium, Na f -dependent inorganic phosphate and D-glucose transport in organ-cultured embryonic chick jejunum (Cross et al., 1986;Cross and Peterlik, 1988;Cross and Peterlik, 1991). We had also demonstrated previously that calcitriol can increase Na+-dependent uptake of D-glucose from the intestinal lumen in two ways: by increasing the maximal velocity of the Na+/D-glucose transport system (Peterlik et al., 1981); through appropriate actions on pathways of Na + transfer across the brush-border membrane of enterocytes (Fuchs et al., 1985). In the present study, we utilized brush-border membrane vesicles (BBMV) isolated from organ-cultured embryonic chick jejuna to further characterize the effect of calcitriol on Na+ /D-glucose transport mechanisms and to gain insight into the mode of triiodothyronine interaction. MATERIALS AND METHODS Organ culture of embryonic chick jejunumFertilized eggs were obtained from a local poultry farm and kept in an incubator with forced ventilation at 70% rela- modified medium (Corradino, 1973). Calcitriol or triiodothyronine were added to cultures in ethanol so that the final ethanol concentration did not exceed 0.15%. Ethanol only was added to control cultures. Culture time was 48 h. D-Glucose transport in cultured jejuna was evaluated by tissue accumulation of the radiolabelled non-metabolizable analogue, a-methyl-D-glucoside (0.5 pCi/ml incubation buffer), as described previously (Peterlik et al., 1981 ;Cross and Peterlik, 1982). Preparation of BBMVA modification of a divalent-cation-precipitation method (Schmitz et ...

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Quantitative studies of cotransport systems: Models and vesicles

Cited by 107 publications

References 70 publications

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot

1,25‐Dihydroxycholecalciferol‐related Na⁺/D‐glucose transport in brush‐border membrane vesicles from embryonic chick jejunum

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Quantitative studies of cotransport systems: Models and vesicles

Cited by 107 publications

References 70 publications

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot

1,25‐Dihydroxycholecalciferol‐related Na+/D‐glucose transport in brush‐border membrane vesicles from embryonic chick jejunum

Contact Info

Product

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1,25‐Dihydroxycholecalciferol‐related Na⁺/D‐glucose transport in brush‐border membrane vesicles from embryonic chick jejunum