ISSN:
0091-7419
Keywords:
transport mechanisms
;
amino acids
;
mouse fibroblasts
;
plasma membrane vesicles
;
regulation
;
SV40 transformation
;
Life Sciences
;
Molecular Cell Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate.When a Na+ gradient, external Na+ 〉 internal Na+, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na+-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na+-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent Km values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system.Several observations indicated that both a Na+ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na+ applied as a gradient (external 〉 internal) across the membrane. This stimulation was independent of endogenous Na+, K+-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na+. The apparent Km for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na+ concentration. Similarly, in the presence of a standard initial Na+ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K+ diffusion potentials (internal 〉 external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na+ and was blocked by the further addition of either nigericin or external K+. As a corollary, Na+-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na+-coupled active transport of these amino acids but did not affect Na+-independent transport. Translocation of K+, H+, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na+ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na+, amino acid, and membrane component.Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na+-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na+ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jss.400060109
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