Na+-gradient-stimulated AIB transport in membrane vesicles from Ehrlich ascites cells

Colombini, Marco; Johnstone, Rose M.

doi:10.1007/bf01870120

Cited by 46 publications

(11 citation statements)

References 28 publications

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“…The maximal iso-Abu uptake observed in Fig. 2A corresponded to 6-fold accumulation above the external iso-Abu concentration, using 3-O-[3H]methylglucose as a marker for sealed plasma membrane intravesicular space (9). Similarly, efflux of accumulated iso-Abu was accelerated when the intravesicular NaCI concentration exceeded the external NaCl concentration, and decreased in the presence of higher external NaCl concentrations (Fig.…”

Section: Methodsmentioning

confidence: 70%

“…Evidence for Na+ gradient-stimulated, electrogenic, concentrative iso-Abu uptake was obtained, as recently described for intestinal brush border membranes (8) and Ehrlich ascites cell membranes (9). By use of transvesicular Na+ gradients maintained independently of endogenous (Na+ + K+)ATPase activity to drive concentrative iso-Abu uptake, the activity of iso-Abu carriers was assayed dissociated from the active Na+ transport system.…”

mentioning

confidence: 85%

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Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Lever¹

1976

Proc. Natl. Acad. Sci. U.S.A.

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Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and from those transformed by simian virus 40 catalyzed carrier-mediated uptake of L-a-aminoisobutyric acid. Concentrative uptake required the presence of a Na+ gradient (external Na+ > internal Na+) and occurred independently of endogenous (Na+ + K+) ATPase activity. This process is electrogenic, since uptake was stimulated by a K+ diffusion gradient (internal > external) in the presence of valinomycin or by the addition of the Na+ salt of a permeant ion, conditions expected to create an interiornegative membrane potential. Both the initial rate of concentrative uptake of L-a-aminoisobutyric acid and its maximal accumulation, driven by a standard Na+ gradient, were decreased in vesicles from density-inhibited, untransformed cells and increased in those from cells transformed by simian virus 40 compared with vesicles from proliferating untransformed cells. An increased maximal velocity ( Vm) of uptake stimulated by Na+ gradient was observed in vesicles from transformed cells compared with those from untransformed cells, suggesting an increase in the number of carriers or in their mobility. Since the relative extent of accumulation of this model amino acid driven by a standard Na+ gradient also differed with growth or transformed status, an additional possibility for cellular regulation of this process could be alteration of membrane Na+ permeability or carrier response to Na+. Alterations in nutrient uptake activity that accompany changes in cellular proliferation rate of untransformed mouse fibroblasts induced by serum, hormones, or viral transformation (1) have been implicated in models of cell cycle control involving regulation of surface membrane functions (2, 3). However, studies of regulation of nutrient uptake using intact cells have been unable to distinguish unambiguously between effects of intracellular metabolic changes, changes associated with the plasma membrane, and effects of surface area and cell overlap on uptake rates. In certain cases, even the use of nonmetabolizable analogues does not permit specific assessment of the locus of regulation of uptake activity. For example, the activity of the Na+ and K+ transport system in fibroblasts, expressed enzymatically as a (Na+ + K+)ATPase, is altered by cell density and proliferation rate (4), serum and purified growth-promoting agents (5), and viral transformation (6). Thus, the rate of Na+-dependent, ouabain-sensitive a-aminoisobutyric acid (iso-Abu) uptake (7) could reflect this observed regulation of the Na+ pump activity rather than alteration of the amino acid carriers.In this report, these possibilities are investigated using sealed membrane vesicles with transport catalytic activity, isolated from mouse fibroblasts. Evidence for Na+ gradient-stimulated, electrogenic, concentrative iso-Abu uptake was obtained, as recently described for intestinal brush border membranes (8) and Ehrlich ascites cell membranes (9). By use of transvesicular Na+ gradients maintai...

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Section: Methodsmentioning

confidence: 70%

mentioning

confidence: 85%

Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Lever¹

1976

Proc. Natl. Acad. Sci. U.S.A.

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“…In vesicles derived from Ehrlich ascites cells, Colombini and Johnstone (28) examined the uptake of AIB and found transport could be stimulated by the existence of a Na+-gradient (high Na+ concentration outside). Also, in vesicles from enterocyte brush border membranes, as shown by Sigrist-Nelson et al (29), L-alanine uptake could be stimulated up to 4-fold by the creation of a Na+-gradient.…”

Section: Membrane Vesicles Accumulate the Amino Acid Intravesicularlymentioning

confidence: 99%

Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells transformed by simian virus 40.

Quinlan¹,

Parnes²,

Shalom³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Mediated uptake of amino acids by membrane vesicles isolated from Balb/c 3T3 cells transformed by simian virus 40 has been demonstrated. Initial rates of transport of radioactively labeled L-leucine and a-aminoisobutyric acid were enhanced by the addition of NaCl (100 mM) to the reaction mixture at the start of the uptake process. This enhancement included a prominent "overshoot" during initial uptake. Slight stimulation of a-aminoisobutyric acid uptake was seen with K+, but none with Li+. The mediated nature of the uptake event for L-leucine was shown by saturation kinetics and by inhibition with L-valine. The transport assay measured predominantly intravesicular amino aciduptake rather than binding, as shown by the variation of uptake in response to changes in extravesicular osmolarity. Electron microscopy confirmed the presence of closed vesicles. Thus, amino acid transport has been characterized in an in vitro membrane vesicle system which should prove useful for studies of growth control. For more than two decades the cell surface membrane has been a focus for the study of differences between tumor cells and normal cells. Studies relating growth regulation to nutrient transport (1-6) have shown that the rate of cellular uptake of certain amino acids, sugars, and nucleosides decreases when growing, untransformed cells reach a confluent, "contact-inhibited" monolayer. Uptake rates have also been shown to increase when the cells are virally transformed. Pardee and coworkers (7,8) and Holley (9) have suggested that these transport variations observed in whole cells reflect primary alterations in the cell membrane, and that malignant growth is made possible by the resulting increase in nutrient supply. However, transport studies on whole cells present the problem of distinguishing between membrane-localized alterations and changes in intracellular metabolism.Attempts have been made to separate uptake from subsequent metabolism by the use of nonmetabolizable analogs.

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“…Many workers propose that these energy-requiring transport processes are mediated by membrane carrier mechanisms that use the inwardly directed free energy (chemical potential) gradients of either sodium ions in animal cells (2,12,19) or hydrogen ions in bacteria, fungi, algae, and higher plants (6, 7, 8, 14, 22). This process, called co-transport (21) or symport (14), is essentially a means for coupling the active transport of one substrate to the passive movement of another substance-both moving in the same direction.…”

Section: Introductionmentioning

confidence: 99%

Evidence for Amino Acid-H⁺ Co-Transport in Oat Coleoptiles

Etherton

Rubinstein²

1978

Plant Physiol.

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Microelectrode and tracer techniques were used to test for possible amino acid-H+ co-transport in coleoptiles of Avena sativa L. cv. "Garry."The amino acid analogue a-aminoisobutyric acid (AIB) caused transient depolarization of the membrane potential. The absolute magnitude of the maximum depolarization was affected by the same factors that affected AIB transport. Both increased with higher concentrations of AIB, increased with higher acidities in the medium, and were enhanced by indoleacetic acid (which hyperpolarized the membrane potential). AIB transport was reduced as K+ concentrations in the medium were increased and by the metabolic inhibitor NaN3, both of which reduce membrane potentials. Our data fit an amino acid-H' co-transport model in which transport is controlled by both the membrane potential and proton concentration components of the chemical potential difference of protons across the coleoptile cell membrane.The cells of many organisms, from bacteria to mammals, accumulate high concentrations of amino acids and carbohydrates from surroundings in which these compounds are dilute (8,11,19,21). Many workers propose that these energy-requiring transport processes are mediated by membrane carrier mechanisms that use the inwardly directed free energy (chemical potential) gradients of either sodium ions in animal cells (2,12,19) or hydrogen ions in bacteria, fungi, algae, and higher plants (6, 7, 8, 14, 22). This process, called co-transport (21) or symport (14), is essentially a means for coupling the active transport of one substrate to the passive movement of another substance-both moving in the same direction.Theory. Figure I shows one of several possible models for cotransport (21). The model assumes a membrane carrier that combines with a substrate (an amino acid in our case) and a proton on one side of the membrane forming a ternary positively charged carrier-substrate-proton complex that moves to the inner membrane surface; at that point the substrate and proton are released and the carrier is allowed to recycle. The direction of the net transport of substrate and its final internal to external concentration ratio depend upon the cytoplasmic and medium substrate and hydrogen ion concentrations, as well as on the potential difference across the membrane. Presumably, the proton concentration on either side of the membrane would affect the rates of proton association or dissociation with the carrier. The membrane potential would act electrophoretically upon the charged membrane species.In the model (Fig. l) (b) membrane depolarizations should be related to substrate transport rates; (c) substrate transport rates and substrate-induced depolarizations should be directly related to proton chemical potential differences caused either by changes in membrane potentials, changes in external to internal proton concentration ratios, or combinations of both; and (d) transport of substrate into the cell with a proton should leave the external solution more alkaline. In the present study, the first th...

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Na+-gradient-stimulated AIB transport in membrane vesicles from Ehrlich ascites cells

Cited by 46 publications

References 28 publications

Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells transformed by simian virus 40.

Evidence for Amino Acid-H⁺ Co-Transport in Oat Coleoptiles

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Na+-gradient-stimulated AIB transport in membrane vesicles from Ehrlich ascites cells

Cited by 46 publications

References 28 publications

Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.

Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells transformed by simian virus 40.

Evidence for Amino Acid-H+ Co-Transport in Oat Coleoptiles

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Evidence for Amino Acid-H⁺ Co-Transport in Oat Coleoptiles