Summary
The studies reported here were carried out to characterize further previously described changes in membrane localized amino acid transport associated with simian virus 40 transformation of the mammalian cell line, Balb/c3T3. Membrane vesicles were prepared from confluent cultures of both simian virus 40 transformed Balb/c3T3 (SV3T3) and the untransformed parent line, Balb/c3T3 (3T3). An initial, externally imposed out>in, 100mm Na+ gradient produces acceleration of early ingress of α-aminoisobutyric acid (AIB) in vesicles from both cell lines, but transient, concentrative uptake (overshooting) only in SV3T3 vesicles. Early ingress ofl-leucine is also accelerated in SV3T3 vesicles by a Na+ gradient, and overshooting is also demonstrable.
Na+-gradient independent AIB permeability of SV3T3 and 3T3 membranes was estimated using uptake data, a first order rate equation and measurements of vesicle size derived from quasi-elastic light-scattering studies. AIB permeability of SV3T3 membranes is greater than that of 3T3 membranes (113 Å/min and 43 Å/min, respectively), suggesting that overshooting in 3T3 vesicles is not attenuated by a Na+-independent AIB “leak”. Na+ permeability of the two membranes is similar, ruling out the possibility that a slower rate of Na+ equilibration across the SV3T3 membrane allows development of the overshoot.
In SV3T3 vesicles the height of a Na+-gradient dependent overshoot varies with the initial [Na+] o /[Na+] i ratio, and [Na+] o /[Na+] i is linearly related to ln AIB uptake at overshoot peak/AIB uptake at equilibrium, consistent with the possibility that for [Na+] o /[Na+] i ratios in the range studied, AIB overshoot is energized by a constant proportion of the energy available from the initial electrochemical gradient for Na+.
These results are consistent with the possibility that Na+-gradient dependent overshooting in SV3T3 vesicles is produced by Na+-amino acid carrier interactions resulting in either an increase in maximum transport velocity or an incrase in carrier affinity for AIB.
Similar content being viewed by others
Abbreviations
- 3T3:
-
Balb/c3T3
- SV3T3:
-
simian virus 40 transformed Balb/c3T3
- AIB:
-
α-aminoisobutyric acid
References
Colombini, M., Johnstone, R.M. 1974. Na+-gradient-stimulated AIB transport in membrane vesicles from Ehrlich ascites cells.J. Membrane Biol. 18:315
Fass, S.J., Hammerman, M.R., Sacktor, B. 1977. Transport of amino acids in renal brush border membrane vesicles. Uptake of the neutral amino acidl-alanine.J. Biol. Chem. 252:583
Foster, D.O., Pardee, A.B. 1969. Transport of amino acids by confluent and nonconfluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass cover slips.J. Biol. Chem. 244:2675
Hamilton, R.T., Nilsen-Hamilton, M. 1976. Sodium-stimulated alpha-aminoisobutyric acid transport by membrane vesicles from simian virus-transformed mouse cells.Proc. Nat. Acad. Sci. USA 73:1907
Hammerman, M.R., Sacktor, B. 1977. Transport of amino acids in renal brush border membrane vesicles. Uptake ofl-proline.J. Biol. Chem. 252:591
Hochstadt, J., Quinlan, D.C., Rader, R.L., Li, C.C., Dowd, D. 1975. Use of isolated membrane vesicles in Transport studies.In: Methods in Membrane Biology. E. Korn, editor. Vol V, pp. 117–162. Plenum Press, New York
Holley, R.W. 1972. Increased uptake of amino acids and 2-deoxy-d-glucose by virustransformed cells in culture.Proc. Nat. Acad. Sci. USA 69:2840
Hopfer, U. 1977. Kinetics of Na+-dependentd-glucose transport.J. Supramol. Struct. 7:1
Hopfer, U., Sigrist-Nelson, E., Ammann, E., Murer, H. 1976. Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.J. Cell. Physiol 89:805
Isselbacher, K.J. 1972. Increased uptake of amino acids and 2-deoxy-d-glucose by virus-transformed cells in culture.Proc. Nat. Acad. Sci. USA 69:585
Kinne, R., Murer, H., Kinne-Saffran, E., Thees, M., Sachs, G. 1975. Sugar transport by renal plasma membrane vesicles Characterization of the systems in the brushborder microvilli and basal-lateral plasma membranes.J. Membrane Biol. 21:375
Lever, J.E. 1976. Regulation of active alpha-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts.Proc. Nat. Acad. Sci. USA 73:2614
Lever, J.E. 1976. Regulation of amino acid and glucose transport activity expressed in isolated membranes from untransformed and SV40-transformed mouse fibroblasts.J. Cell. Physiol. 89:779
Lever, J.E. 1977. Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts.J. Biol. Chem. 252:1990
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the folin phenol reagent.J. Biol. Chem. 193:265
Murer, H., Hopfer, U. 1974. Demonstration of electrogenic Na+-dependentd-glucose transport in intestinal brush border membranes.Proc Nat. Acad. Sci. USA 71:484
Murer, H., Hopfer, U., Kinne-Saffran, E., Kinne, R. 1974. Glucose transport in isolated brush border and lateral basal plasma membrane vesicles from intestinal epithelial cells.Biochim. Biophys. Acta 345:170
Nilsen-Hamilton, M., Hamilton, R.T. 1976. Uptake of alpha-aminoisobutyric acid and phosphate by membrane vesicles derived from growing and quiescent fibroblasts.J. Cell. Physiol. 89:795
Nishino, H., Shiller, R.M., Parnes, J.R., Isselbacher, K.J. 1978. Role of Na+ in alphaaminoisobutyric acid uptake by membrane vesicles from mouse fibroblasts transformed by simian virus 40.Proc. Nat. Acad. Sci. USA 75:2329
Oxender, D.L., Christensen, H.N. 1963. Distinct mediating systems for transport of neutral amino acids by Ehrlich cell.J. Biol. Chem. 238:3686
Parnes, J.R., Garvey, T.Q., Isselbacher, K.J. 1976. Amino acid transport by membrane vesicles of virally transformed and nontransformed cells: Effects of sodium gradient and cell density.J. Cell. Physiol. 89:789
Quinlan, D.C., Hochstadt, J. 1974. An altered rate of uridine transport in membrane vesicles isolated from growing and quiescent mouse 3T3 fibroblast cells.Proc. Nat. Acad. Sci. USA 71:5000
Quinlan, D.C., Parnes, J.P., Shalom, R., Garvey, T.Q., Isselbacher, K.J., Hochstadt, J. 1976. Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells,Proc. Nat. Acad. Sci. USA 73:1631
Sigrist-Nelson, K., Murer, H., Hopfer, U. 1975. Active alanine transport in isolated brush border membranes.J. Biol. Chem. 250:5674
Temin, H.M. 1971. Mechanism of cell transformation by RNA tumor viruses.Annu. Rev. Microbiol. 25:609
Turner, R.J., Silverman, M. 1977. Sugar uptake into brush border vesicles from normal human kidney.Proc. Nat. Acad. Sci. USA 74:2825
Tyrakowski, T., Milutinovic, S., Schulz, I. 1978. Studies on isolated subcellular components of cat pancreas. III. Alanine-sodium cotransport in isolated plasma membrane vesicles.J. Membrane Biol. 38:333
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Garvey, T.Q., Babcock, A. Amino acid and22Na+ uptake in membrane vesicles from confluent simian virus 40 transformed Balb/c3T3 and Balb/c3T3. J. Membrain Biol. 49, 139–156 (1979). https://doi.org/10.1007/BF01868722
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01868722