Proc. Natl. Acad. Sci. USA
Vol. 73, No. 6, pp. 1907-1911, June 1976
Biochemistry
Sodium-stimulated a-aminoisobutyric acid transport by membrane vesicles from simian virus-transformed mouse cells (Na+ specificity/amino acid inhibition/K+ inhibition/cell transport/biphasic kinetics)
RICHARD T. HAMILTON AND MARIT NILSEN-HAMILTON Molecular Biology Laboratory, The Salk Institute, Post Office Box 1809, San Diego, California 92112
Communicated by Robert W. Holley, March 22,1976
ABSTRACT Uptake of a-aminoisobutyric acid, by membrane vesicles derived principally from the plasma membrane and endoplasmic reticulum of mouse 3T3 cells transformed by simian virus 40, is stimulated by sodium chloride. Both in the presence and absence of Na+ uptake is time-dependent and osmotically sensitive. The Na+-stimulated uptake is inhibited by other amino acids. The kinetics of transport of a-aminoisobutyric acid are shown to be biphasic both in whole cells and in the membrane vesicles. Only the high affinity system is stimulated by sodium in the membrane vesicles. These results demonstrate that observations made on living cells correlate with observations made on isolated membrane vesicles, and indicate that these membrane vesicles have retained the cellular amino acid transport system functionally intact.
It has been established from a number of studies that transport of some nutrients, such as phosphate (1-4), glucose (5), nucleosides (2, 6), amino acids (7-9), and monovalent cations (10-12), is involved in the control of growth of animal cells in tissue culture. The nature of that involvement is not known. By a determination of the molecular mechanisms of transport for these nutrients before, during, and after growth stimulation, we may be able to determine whether transport of essential nutrients across the cell-surface membrane controls growth. A serious drawback for a determination of the molecular level mechanisms of transport in living cells is that the transported substrate is metabolized after interaction with the transport site. This problem can be overcome by a study of the transport system in isolation. A means of doing this was developed by Kaback (13) who studied nutrient transport by membrane vesicles derived from bacteria. Hochstadt et al. (14-17) have begun to determine mechanisms of transport in membrane vesicles derived from animal cells grown in tissue culture. In addition to the studies reported in this paper, we have completed studies of phosphate uptake by SV3T3 membrane vesicles from simian virus 40-transformed 3T3 cells (SV3T3) (Hamilton and Nilsen-Hamilton, manuscript in preparation). As a prelude to a comparison of the transport properties of membrane vesicles (MV) derived from normal and transformed cells, we report in this paper our studies of a-aminoisobutyric acid (AIB) uptake by a membrane-vesicle population derived principally from the cell-surface membrane and endoplasmic reticulum of SV3T3 cells. We describe the effect of Na+ on transport of AIB by this system. A similar, independent study by Quinlan et al. (18) has recently been reported.
MATERIALS AND METHODS All solutions used for membrane vesicle preparations were sterilized by filtration or by autoclaving. a-Amino[1-14C]isoAbbreviations: AIB, a-aminoisobutyric acid; MV, membrane vesicle; STM, 0.25 M sucrose, 5 mM Tris-HCI (pH 7.3), and 0.2 mM MgSO4; PNS, post nuclear supernatant; HBS, Hanks' Balanced Salt Solution; SV3T3, mouse 3T3 cells transformed by simian virus 40. 1907
butyric acid was purchased from New England Nuclear Corp., and a-amino[methyl-3H]isobutyric acid was purchased from International Chemical Nuclear. The [3H]AIB was shown to chromatograph as a single spot by two dimensional chromatography on thin-layer chromatography plates made with silica gel 60 (Brinkman). The solvent for the first dimension was npropanol:H20 (1:1, vol/vol), and for the second dimension was n-butanol:acetic acid:H20 (4:1:1, vol/vol). AIB was purchased from Calbiochem. All other amino acids were obtained from Sigma Chemical Co. Cell Culture. Simian virus 40-transformed Swiss 3T3 cells, obtained from P. Rudland, were recloned. Stock cultures were maintained in Dulbecco and Vogt's modification of Eagle's medium, supplemented with 10% calf serum. The stock cell cultures were grown as monolayers on plastic petri tissue-culture dishes, at 370, in a water-saturated atmosphere, with 10% CO2 in air. Bimonthly checks for mycoplasma were made by incorporation of [3H]thymidine, and autoradiography of the cells. No mycoplasma was detected. Twenty half-gallon roller bottles (Bellco Glass, Vineland, N.J.) were used per membrane preparation, and each bottle was seeded with 1 to 2 X 107cells in 100 ml of stock culture medium supplemented with 10% calf serum, 0.0146 Ag/ml of d-biotin, and 2.75 jig/ml of vitamin B12. d-Biotin and vitamin B12 minimize loss of SV3T3 cells from the substratum after confluence and overgrowth are reached (our unpublished data). SV3T3 cells were harvested at maximal cell density, and each bottle yielded more than 2 X 108 cells. Preparation of Membrane Vesicles. The preparation was based on the procedure described by Wallach and Kamat (19). We made these modifications: dense cultures were scraped with a rubber spatula into a solution of 0.15 M NaCl and 10 mM potassium phosphate (pH 7.3) at 4°. The suspended cells were washed only once in 0.25 M sucrose, 0.5 mM Tris*HCI (pH 7.4), and 2 mM MgSO4 (STM). Nitrogen pressure was 4.1 MPa. After 5 min under pressure, the homogenate was delivered dropwise from the instrument and no EDTA was added. The membrane pellet from the centrifugation at 110,000 X g was suspended in STM to give a final protein concentration of 10-20 mg/ml (MV). The suspension was stored at -70°. Analytical Methods. Protein was determined by intrinsic protein fluorescence (20). (Na+, K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), a plasma membrane marker, was measured by the method of Yoshikawa et al. (21). The released phosphate was quantitated according to Ames (22). Another plasma membrane marker (23-25), 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), was assayed by the method of Avruch and Wallach (26). The concentration of AMP was 100 gM. NADH oxidase NADH: (acceptor) [oxidoreductase; EC 1.6.99.3], located on the endoplasmic reticulum and the outer mitochondrial membrane (27), was also assayed by the methods of Avruch and Wallach (26). Beta-galactosidase
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Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Hamilton and Nilsen-Hamilton
Table 1. Distribution of marker enzymes after preparation of SV3T3 membranes
Homogenate Post nuclear supernatant Nuclei Pellet (18,000 x g) Membrane vesicles Supernatant (42,000 x g)
Total yield
ATPase
5'-Nucleotidase (pH 8.5)
,B-Galactosidase
NADH oxidase
Cytochrome oxidase
100 94 29 38 30 N.D.
100 71 12 56 29 0.6
(48) 86 14 43 60 6
100 78 13 51 13 3.9
100 55 15 38 24 3
100 75 14 64 3.8 0.02
97
97.6
(123)
80.9
80
81.8
(Na+,K+-
Protein
AIB uptake (15-sec rate)
100 79 17 26 20 32
95
Percent yields expressed relative to the homogenate for all enzymes except 5'-nucleotidase which is expressed as percent of the sum of postnuclear supernatant and nuclear activities. As a result of adenosine binding and loss of the product in the BaSO4 precipitate, the values of 5'-nucleotidase for the homogenate are lower than the real values (our unpublished observations). N.D. = not detectable.
(f3-D-galactoside galactohydrolase; EC 3.2.1.23), a lysosomal enzyme (28), was determined by the method of Ho et al. (29), with the inclusion of 0.1% Triton X-100 for full enzyme expression. Cytochrome oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1), which is located on the inner mitochondrial membrane (30), was assayed by the method of Cooperstein and Lazarow (31). Assay for Uptake of AIB by Membrane Vesicles. For each time point, 90 ,1 aliquots containing from 150 Mig to 350 Mg of membrane protein in 60 mM sucrose, 10 mM Tris*HCl (pH 7.4 at 370), 0.1 mM MgCl2, and 0.1 mM CaCl2, were preincubated at 370 for 10-20 min. At the end of this period, the reaction was started by the addition of 10 Ml of 14C- or 3H-labeled AIR. The reaction was terminated by dilution with 1.4 ml of 0.8 M NaCl, at 37'. Each diluted sample was immediately filtered through a nitrocellulose filter (24 mm diameter and 0.2 Am, average pore size, Schleicher and Schuell, Inc., Keene, N.H.) which had been wetted with sterile water. The filters were then washed twice with the same volume of NaCl. Background adsorption
was determined by addition of radioactively labeled AIB to the membrane suspension. This mixture was diluted and filtered immediately and washed with 0.8 M NaCl at 37'. The nitrocellulose filters were dried by heat and the radioactivity was determined in a Beckman liquid-scintillation counter, with a toluene scintillation mixture (Liquifluor; New England Nuclear). Assay for Uptake of AIB by SV3T3 Cells. The uptake assay is based on a method described by Foster and Pardee (32) with cells grown on glass coverslips. The coverslips were prepared by boiling in an aqueous solution of sodium hexametaphosphate (8.8 g/liter) and sodium metasilicate (70.12 g/liter). Coverslips containing confluent cells were rinsed twice with Hanks' Balanced Salt Solution (HBS) which contained 20 mM Hepes [4(2-hydroxy-ethyl)-1-piperazineethane-sulfonic acid] at pH 7.0, then preincubated for 25-30 min in the same solution in a CO2 incubator at 37'. Uptake was in HBS and Hepes at 370 for 2 min. Coverslips were dipped, into three beakers each of which contained 250 ml of 25 mM Tris, 0.14 M NaCl, 6.7 mM KCI, 0.68 mM CaCl2, 0.49 mM MgCl2, and 0.37 mM Na2HPO4 (pH 7.0) and thereby rinsed three-times before and after uptake. Coverslips were then placed in 1 ml of 0.4 M NaOH and left at 4° overnight to completely dissolve the cells. The solutions were then neutralized with HC1 and aliquots removed for determination of radioactivity in a Beckman liquid scintillation counter, with a Liquifluor scintillation mixture which contained toluene and Triton X-100. Protein concentrations were determined for each point, and five coverslips, taken through the same washing procedures, were trypsinized and the cells counted in a Coulter Counter.
RESULTS
25,
Membrane fractionation The enzymatic analysis of a typical fractionation of membrane 2
4
6
8
TIME (min) FIG. 1. Time-course of uptake of AIB by SV3T3 membrane vesicles in the presence and absence of NaCl. The uptake assay is described in Materials and Methods. At each time point, 150 ,g of membrane-vesicle (MV) protein was used. The concentration of [14C]AIB was 0.5 mM with a specific activity of 4.61 cpm/pmol of AIR. At any time during uptake, the [14CJAIB cpm could be reduced to the background value by a water wash at room temperature, instead of the usual 0.8 M NaCl wash at 370. Uptake of [14C]AIB was measured in the absence of NaCl (0-0); in the presence of 50 mM NaCl preincubated with MV for 20 min at 37°, before the uptake reaction was started by addition of [14C]AIB (A-A); and when 50 mM NaCl was added with [14C]AIB (0-1).
constituents from SV3T3 cells is shown in Table 1. The mem-
brane fraction, MV, was used for the uptake studies described in this paper. This fraction contains 4% of the total mitochondrial activity (range, 1-4%) and 13% of the lysosomal activity (range, 5-14%). Twenty-four percent of the NADH oxidase travels with MV. AIB uptake activity correlates with the plasma membrane markers, (Na+, K+)-ATPase and 5'-nucleotidase. It does not correlate with mitochondrial or lysosomal enzymatic activities. The kinetics of AIB transport in membrane vesicles The uptake of AIB by MV is time dependent (Fig. 1). As evidence that the accumulated [14C]AIB counts represented uptake
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Hamilton and Nilsen-Hamilton
Table 3. Effect of neutral acids on the uptake of AIB by SV3T3 membrane vesicles
Table 2. The effect of cations on uptake of AIB by membrane vesicles derived from SV3T3 cells
% AIB uptake in 15 sec
% Control (-cation)
Cation
15-sec uptake
Li+ Na+ K+ Rb+ Cs+ NH4+ Choline
82 167 67 75 65 82 81
Amino acid added with 0.1 mM AIB
1-min uptake
99
Alanine
182 82
Glycine
91 80 94
Leucine
91
Methionine
Mean values of triplicate determinations are given. The AIB concentration was 0.1 mM and each determination contained 318 ug of membrane protein. The results are expressed as a percentage of the 15-sec and 1-min uptake values obtained in the absence of 50 mM cation added with [3H]AIB. In the absence of cation, the value for 15-sec uptake was 680 pmol/min per mg of protein and that for 1-min uptake was 230 pmol/min per mg of protein. Cation chlorides were used.
of AIB rather than nonspecific binding to the membrane surface, washing the MV with water, at any time during the time-course, reduced the accumulated 14C radioactive material back to the background value. By the use of hyperosmotic conditions to shrink the vesicles, we observed a 2-fold reduction in steady-state levels of AIB transport (data not shown), which is further evidence of intravesicular accumulation of AIB. Sodium ions at the external surface of the membrane of eukaryotic cells are often required for active transport (33). The effect of 50 mM NaCI, added simultaneously with AIB to start the uptake reaction (Fig. 1), is to increase the initial rate of uptake and the maximal level reached from 2- to 4-fold. The response to 50 mM NaCl depends on the concentration of AIB used, and is greater at lower AIB concentrations (our unpublished observations). The stimulatory effect of NaCI most likely results from a transient concentration gradient formed across the membrane, because 50 mM NaCI is nonstimulatory when preequilibrated with the membranes before initiation of uptake. Also, with time, the initial stimulation, seen when NaCl is added with AIB, decreases to the level found when the vesicles are equilibrated with Na+ prior to addition of label. When there is a requirement of eukaryotic active transport systems for monovalent cations at the external surface of the cell, that requirement is generally specific for Na+ (33, 34). We have shown (Table 2) that stimulation of AIB transport into SV3T3 membrane vesicles is also specific for Na+ when compared with the other chlorides of alkali metal ions, NH4C1, and choline chloride. In addition, this experiment provides evidence against transient osmotic or ionic strength effects which might cause stimulation of AIB uptake by the membrane vesicles. The specific Na+-stimulated uptake of AIB is evidence that this uptake is a mediated one. Further evidence for mediated uptake by the membrane vesicles was provided by studies which showed inhibition of AIB uptake by other amino acids (Table 3). No significant inhibition of uptake by these amino acids was observed in the absence of NaCI. To compare the parameters, Km and Vmax, obtained for membrane vesicles with those for cells, we determined the effect of AIB concentration on the initial rates of uptake in SV3T3 cells grown on glass coverslips (Materials and Methods). The use of AIB, an amino acid analog which is not metabolized, in initial studies such as these, has the advantage that cells can be more directly compared with membrane-vesicle studies to
1909
0.1 mM 1.0 mM 0.1 mM 1.0 mM 0.1 mM 1.0 mM 0.1 mM 1.0 mM
Minus NaCl
99 100 95 98 90 89 106 96
(10) (4) (2) (6)
(8) (5) (5) (6)
Plus NaCl 105 80 79 67 81 60 95 59
(11) (2) (2) (6) (6) (3) (3) (3)
A concentration of amino acid equal to the concentration of AIB (0.1 mM) or ten times greater (1 mM) was added with the AIB to start the reaction. Rates of reaction (15 sec) were compared in the absence and presence of 50 mM NaCl added with the AIB. The results are expressed as percentage of the control in the absence or presence of NaCl. Control values were: minus NaCl = 146 pmol/ min per mg of protein; plus NaCl = 381 pmol/min per mg of protein. Values in parentheses are percent of the standard error of the mean. Triplicate determinations for each experiment were made, and the results averaged. Three sets of experiments were made and averaged.
verify that the transport system has been isolated functionally intact. The kinetics of AIB uptake by SV3T3 cells are biphasic
(Fig. 2).
Similar studies were made with the membrane vesicles. Comparisons of the uncorrected, apparent values of Km and Vmax are shown in Table 4. Biphasic uptake kinetics were obtained for cells and membrane vesicles. Good agreement is found for Km values, from cells and membrane vesicles, determined in the low concentration range of 0.1 mM to 2 mM
-0.1
0
0.1
0.2
0.3
0.4
0.5
I/[AIBJ mM FIG. 2. Lineweaver-Burk plot of AIB uptake by SV3T3 cells. Initial rate determinations (Materials and Methods) were made in the concentration range 0.1-100 mM AIR. In the inset is shown the straight line obtained by least-squares analysis in the concentration range 0.1-2.5 mM. The line of the main figure begins to curve off at 2.5 mM and then at approximately 5 mM it straightens to a new line of steeper slope. The values of Km and Vm., obtained by extrapolation, have not been corrected for the contribution to uptake from each of the other systems. We have designated them the uncorrected apparent Km and Vm. values. They are meaningful only for comparisons. These values are given in Table 4. The average protein per coverslip was 353 Ag (167 estimations) and the average number of cells per coverslip was 5.12 X 106.
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Biochemistry: Hamilton and Nilsen-Hamilton
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 4. Summary of uncorrected apparent Km and Vmax values obtained from SV3T3 cells and membrane vesicles
Uncorrected AIB concentration range
Km
System
(mM)
(mM)
SV3T3 cells
0.1-3.3 20-100 0.1-2.0 0.1-2.0 0.1-2.0 0.1-2.0 20-70 20-70
4.4 26 15 3.4 2.6 1.1 143 153
Membrane vesicles
Na+ Vma (nmol/min concenper mg of tration protein) (mM) 18 31 2.1 0.99 1.0 0.51 155 155
140 140 0 25 50 100 0 50
The Lineweaver-Burk plots used to derive these values are Figs. 2, 3, and 4.
AIB. Lack of agreement is found between Km values determined in the high concentration range 5-100 mM AIR. This could result from a higher contribution of simple diffusion to uptake by the membrane vesicles. At high AIB concentrations the uptake does not respond to NaCl (Table 4 and Fig. 3, top and bottom). To test the possibility that 50 mM NaCl was insufficient to cause stimulation at the higher concentrations of AIB, the effect of concentrations greater than 50 mM NaCl, on the initial rate of uptake of 50 mM AIB, was measured. No significant effect was observed up to 300 mM NaCl. However, a pronounced inhibition of uptake was obtained with KC1. At KC1 concentrations of 100 and 200 mM added with 50 mM AIB, 50 and 86% inhibition of AIB uptake was observed. These results
O
0.01 0.02 0.03 I/[AIB], mM
0.04
Q05
FIG. 3. Lineweaver-Burk plots of uptake of high concentrations of AIB in the absence and presence of 50 mM NaCl. Uptake, for 15 sec, was as described in Materials and Methods. For each estimation, 335 Mg of membrane protein was used. The concentration of [3H]AIB was varied from 20 to 70 mM. The [3HJAIB cpm per experiments were 3.24 X 105. Initial rates have the units: nmol/min per mg of protein. Each point is the mean of duplicate determinations. Linear regression analyses were used to obtain the lines of best fit. Uncorrected values of apparent Km and V.,, are given in Table 4.
4
6
I/[AIB] , mM
FIG. 4. The effect of NaCl on Km and K... of SV3T3 membrane vesicles. Uptake, for 15 sec, was as described in Materials and Methods. For each estimation, 335,gg of membrane protein was used. The concentration of [3HJAIB was varied from 0.1 mM to 2 mM. The [3H]AIB cpm per experiment were 4.19 X 105. Initial rates have the units: nmol/min per mg of protein. Each point is the mean oftriplicate determinations. The lines of best fit were derived by least-squares analysis. The two points in parentheses were not included in the least-squares analysis. 0-0, 100 mM choline chloride final concentration added with [3H]AIB. *-*, 25 mM NaCl/75 mM choline chloride. A-*, 50 mM NaCl/50 mM choline chloride. 0-0,100 mM NaCl. Values of apparent Km and Vmax are given in Table 4.
suggest that very little simple diffusion occurs through the membrane vesicles even at 50 mM AIR. However, the possibility that KCI is destroying membrane integrity has not been ruled out. We determined the effect of Na+ on the uncorrected apparent Km and Vmax values for uptake of AIB by the membrane vesicles, in the range of AIB concentration where uptake was sensitive to Na+. We varied the choline/Na+ ratio, and we measured uptake at a constant 100 mM final salt concentration. In the range 25 mM NaCl to 100 mM NaCI, the uncorrected apparent Km value changed from 3.4 mM to 1.1 mM AIB, and the uncorrected apparent V., value was decreased by one-half (Table 4 and Fig. 4).
DISCUSSION Our main finding is that AIB uptake by SV3T3 membrane vesicles is specifically stimulated by Na+, only if sodium chloride is added at the start of the uptake reaction. This implies that the energy of a Na+-electrochemical gradient is used to drive AIB uptake. These findings are in agreement with those of Quinlan et al. (18). Both reports describe the Na+-dependent amino acid uptake into membrane vesicles derived from tissue-culture cells. However, one report (35) described the phenomenon in membrane vesicles derived from Ehrlich Ascites cells and another (36) in membrane vesicles from enterocyte brush border membranes. We have further shown that the Na+-stimulation is accompanied by alterations in apparent Km values. This would indicate an increase in the affinity of the carrier for AIB in the presence of Na+ according to the models of Heinz and others (33, 34). Our results indicate that AIB is take up by the SV3T3 cells and membrane vesicles by two kinetically distinguishable components: one with a low apparent affinity for AIB which is Na+ independent, and the other with a high apparent affinity for AIB which is Na+ dependent. The advantage of membrane-vesicle studies of uptake is that the two systems can be studied independently in the presence and absence of Na+.
Biochemistry: Hamilton and Nilsen-Hamilton We are deeply indebted to Dr. R. W. Holley for his encouragement and support. We thank Drs. J. Hochstadt, D. Quinlan, R. Willis, and R. W. Holley for their criticisms of the manuscript and gratefully acknowledge the excellent technical assistance of Andrew Oesterle. This work was supported by grants from the American Cancer Society (BC-30), The National Science Foundation (GB-32391X), and the National Institutes of Health (CA11176). 1. Blade, E., Harel, L. & Hanania, N. (1966) Exp. Cell Res. 41, 473-482. 2. Cunningham, D. D. & Pardee, A. B. (1969) Proc. Natl. Acad. Sci. USA 64,1049-1056. 3. Weber, M. J. & Edlin, G. (1971) J. Biol. Chem. 246,1828-1833. 4. Jimenez de Asua, L. & Rozengurt, E. (1974) Nature 251,624-626. 5. Hatanaka, M. (1974) Biochim. Biophys. Acta 355, 77-104. 6. Quinlan, D. C. & Hochstadt, J. (1974) Proc. Natl. Acad. Sci. USA 71,5000-5003. 7. Christensen, H. N. & Hendersen, M. E. (1952) Cancer Res. 12, 229-231. 8. Foster, D. 0. & Pardee, A. B. (1969) J. Biol. Chem. 244, 26752681. 9. Otsuka, H. & Moskowitz, M. (1975) J. Cell. Physiol. 85,665-674. 10. Kasarov, L. B. & Friedman, H. (1974) Cancer Res. 34,1862-1865. 11. Rozengurt, E. & Heppel, L. (1975) Proc. Natl. Acad. Sci. USA
72,4492-4495. 12. Kimelberg, H. K. & Mayhew, E. (1975) J. Biol. Chem. 250, 100-104. 13. Kaback, H. R. (1972) Biochim. Biophys. Acta 265, 367-416. 14. Hochstadt, J., Quinlan, D. C., Rader, R. L., Li, C. & Dowd, D. (1975) in Methods in Membrane Biology, ed. Korn, E. D. (Plenum Press, New York), Vol. 5, pp. 217-261. 15. Quinlan, D. C. & Hochstadt, J. (1976) J. Biol. Chem. 251, 344-354. 16. Li, C.-C., & Hochstadt, J. (1976) J. Biol. Chem. 251, 1175-1180. 17. Li, C.-C. & Hochstadt, J. (1976) J. Biol. Chem. 251, 1181-1187. 18. Quinlan, D. C., Parnes, J. R., Shalom, R., Garvey, T. Q., Isselbacher, K. J. & Hochstadt, J. (1976), Proc. Nati. Acad. Sci. USA 73, 1631-1635. 19. Wallach, D. F. H. & Kamat, V. B. (1966) in Methods in Enzymology, eds. Neufeld, E. F. & Ginsburg, V. (Academic Press,
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New York), Vol. 8, pp. 164-172. 20. Udenfriend, S. (1962) in Fluorescence Assay in Biology and Medicine (Academic Press, New York), Vol. 3, p. 206. 21. Yoshikawa-Fukada, M. & Nojima, T. (1973) J. Cell. Physiol. 80, 421-430. 22. Ames, B. (1966) in Methods in Enzymology, eds. Neufeld, E. F. & Ginsburg, V. (Academic Press, New York), Vol. 8, pp. 115-11& 23. Essner, E., Novikoff, A. B. & Masek, B. (1958) Biophys. Biochem.
Cytol. 4,711-716. 24. Wallach, D. F. H. & Winzler, R. J. (1974) in Evolving Strategies and Tactics in Membrane Research (Springer-Verlag, New York), p. 21. 25. De Pierre, J. W. & Karnovsky, M. L. (1974) Science 183, 1096-1098. 26. Avruch, J. & Wallach, D. F. H. (1971) Biochim. Blophys. Acta 233,334-347. 27. Sottocasa, G. L. (1971) in Advances in Experimental Medicine and Biology, eds. Porcellati, G. & Jeso, F. (Plenum Press, New York), Vol. 4, p. 229. 28. Sellinger, 0. Z., Beaufay, H., Jacques, P., Doyen, A. & de Duve, C. (1960) Biochem. J. 74, 450-456. 29. Ho, M. W., Seck, J., Schmidt, D., Veath, M. L., Johnson, W., Brady, R. 0. & O'Brien, J. S. (1972) Am. J. Hum. Genet. 27, 37-45. 30. Sottocasa, G. L., Kuylenstierna, B., Ernster, L. & Bergstrom, A. (1967) in Methods in Enzymology, eds. Estabrook, R. W. & Pullman, M. E. (Academic Press, New York), Vol. 10, pp. 448-463. 31. Cooperstein, S. J. & Lazarow, A. (1951) J. Biol. Chem. 189, 665-670. 32. Foster, D. 0. & Pardee, A. B. (1969) J. Biol. Chem. 244, 26752681. 33. Heinz, E. (1972) in Metabolic Pathways, ed. Hokin, L. E. (Academic Press, New York), Vol. VI, pp. 455-501. 34. Heinz, E. (1972) Sodium-linked Transport of Organic Solutes, ed. Heinz, E. (Springer Publication, New York). 35. Colombini, M. & Johnstone, R. M. (1974) J. Membrane Biol. 15, 261-276. 36. Sigrist-Nelson, K., Murer, H. & Hopfer, U. (1975) J. Biol. Chem. 250,5674-5680.
Vol. 73, No. 6, pp. 1907-1911, June 1976
Biochemistry
Sodium-stimulated a-aminoisobutyric acid transport by membrane vesicles from simian virus-transformed mouse cells (Na+ specificity/amino acid inhibition/K+ inhibition/cell transport/biphasic kinetics)
RICHARD T. HAMILTON AND MARIT NILSEN-HAMILTON Molecular Biology Laboratory, The Salk Institute, Post Office Box 1809, San Diego, California 92112
Communicated by Robert W. Holley, March 22,1976
ABSTRACT Uptake of a-aminoisobutyric acid, by membrane vesicles derived principally from the plasma membrane and endoplasmic reticulum of mouse 3T3 cells transformed by simian virus 40, is stimulated by sodium chloride. Both in the presence and absence of Na+ uptake is time-dependent and osmotically sensitive. The Na+-stimulated uptake is inhibited by other amino acids. The kinetics of transport of a-aminoisobutyric acid are shown to be biphasic both in whole cells and in the membrane vesicles. Only the high affinity system is stimulated by sodium in the membrane vesicles. These results demonstrate that observations made on living cells correlate with observations made on isolated membrane vesicles, and indicate that these membrane vesicles have retained the cellular amino acid transport system functionally intact.
It has been established from a number of studies that transport of some nutrients, such as phosphate (1-4), glucose (5), nucleosides (2, 6), amino acids (7-9), and monovalent cations (10-12), is involved in the control of growth of animal cells in tissue culture. The nature of that involvement is not known. By a determination of the molecular mechanisms of transport for these nutrients before, during, and after growth stimulation, we may be able to determine whether transport of essential nutrients across the cell-surface membrane controls growth. A serious drawback for a determination of the molecular level mechanisms of transport in living cells is that the transported substrate is metabolized after interaction with the transport site. This problem can be overcome by a study of the transport system in isolation. A means of doing this was developed by Kaback (13) who studied nutrient transport by membrane vesicles derived from bacteria. Hochstadt et al. (14-17) have begun to determine mechanisms of transport in membrane vesicles derived from animal cells grown in tissue culture. In addition to the studies reported in this paper, we have completed studies of phosphate uptake by SV3T3 membrane vesicles from simian virus 40-transformed 3T3 cells (SV3T3) (Hamilton and Nilsen-Hamilton, manuscript in preparation). As a prelude to a comparison of the transport properties of membrane vesicles (MV) derived from normal and transformed cells, we report in this paper our studies of a-aminoisobutyric acid (AIB) uptake by a membrane-vesicle population derived principally from the cell-surface membrane and endoplasmic reticulum of SV3T3 cells. We describe the effect of Na+ on transport of AIB by this system. A similar, independent study by Quinlan et al. (18) has recently been reported.
MATERIALS AND METHODS All solutions used for membrane vesicle preparations were sterilized by filtration or by autoclaving. a-Amino[1-14C]isoAbbreviations: AIB, a-aminoisobutyric acid; MV, membrane vesicle; STM, 0.25 M sucrose, 5 mM Tris-HCI (pH 7.3), and 0.2 mM MgSO4; PNS, post nuclear supernatant; HBS, Hanks' Balanced Salt Solution; SV3T3, mouse 3T3 cells transformed by simian virus 40. 1907
butyric acid was purchased from New England Nuclear Corp., and a-amino[methyl-3H]isobutyric acid was purchased from International Chemical Nuclear. The [3H]AIB was shown to chromatograph as a single spot by two dimensional chromatography on thin-layer chromatography plates made with silica gel 60 (Brinkman). The solvent for the first dimension was npropanol:H20 (1:1, vol/vol), and for the second dimension was n-butanol:acetic acid:H20 (4:1:1, vol/vol). AIB was purchased from Calbiochem. All other amino acids were obtained from Sigma Chemical Co. Cell Culture. Simian virus 40-transformed Swiss 3T3 cells, obtained from P. Rudland, were recloned. Stock cultures were maintained in Dulbecco and Vogt's modification of Eagle's medium, supplemented with 10% calf serum. The stock cell cultures were grown as monolayers on plastic petri tissue-culture dishes, at 370, in a water-saturated atmosphere, with 10% CO2 in air. Bimonthly checks for mycoplasma were made by incorporation of [3H]thymidine, and autoradiography of the cells. No mycoplasma was detected. Twenty half-gallon roller bottles (Bellco Glass, Vineland, N.J.) were used per membrane preparation, and each bottle was seeded with 1 to 2 X 107cells in 100 ml of stock culture medium supplemented with 10% calf serum, 0.0146 Ag/ml of d-biotin, and 2.75 jig/ml of vitamin B12. d-Biotin and vitamin B12 minimize loss of SV3T3 cells from the substratum after confluence and overgrowth are reached (our unpublished data). SV3T3 cells were harvested at maximal cell density, and each bottle yielded more than 2 X 108 cells. Preparation of Membrane Vesicles. The preparation was based on the procedure described by Wallach and Kamat (19). We made these modifications: dense cultures were scraped with a rubber spatula into a solution of 0.15 M NaCl and 10 mM potassium phosphate (pH 7.3) at 4°. The suspended cells were washed only once in 0.25 M sucrose, 0.5 mM Tris*HCI (pH 7.4), and 2 mM MgSO4 (STM). Nitrogen pressure was 4.1 MPa. After 5 min under pressure, the homogenate was delivered dropwise from the instrument and no EDTA was added. The membrane pellet from the centrifugation at 110,000 X g was suspended in STM to give a final protein concentration of 10-20 mg/ml (MV). The suspension was stored at -70°. Analytical Methods. Protein was determined by intrinsic protein fluorescence (20). (Na+, K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), a plasma membrane marker, was measured by the method of Yoshikawa et al. (21). The released phosphate was quantitated according to Ames (22). Another plasma membrane marker (23-25), 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), was assayed by the method of Avruch and Wallach (26). The concentration of AMP was 100 gM. NADH oxidase NADH: (acceptor) [oxidoreductase; EC 1.6.99.3], located on the endoplasmic reticulum and the outer mitochondrial membrane (27), was also assayed by the methods of Avruch and Wallach (26). Beta-galactosidase
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Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Hamilton and Nilsen-Hamilton
Table 1. Distribution of marker enzymes after preparation of SV3T3 membranes
Homogenate Post nuclear supernatant Nuclei Pellet (18,000 x g) Membrane vesicles Supernatant (42,000 x g)
Total yield
ATPase
5'-Nucleotidase (pH 8.5)
,B-Galactosidase
NADH oxidase
Cytochrome oxidase
100 94 29 38 30 N.D.
100 71 12 56 29 0.6
(48) 86 14 43 60 6
100 78 13 51 13 3.9
100 55 15 38 24 3
100 75 14 64 3.8 0.02
97
97.6
(123)
80.9
80
81.8
(Na+,K+-
Protein
AIB uptake (15-sec rate)
100 79 17 26 20 32
95
Percent yields expressed relative to the homogenate for all enzymes except 5'-nucleotidase which is expressed as percent of the sum of postnuclear supernatant and nuclear activities. As a result of adenosine binding and loss of the product in the BaSO4 precipitate, the values of 5'-nucleotidase for the homogenate are lower than the real values (our unpublished observations). N.D. = not detectable.
(f3-D-galactoside galactohydrolase; EC 3.2.1.23), a lysosomal enzyme (28), was determined by the method of Ho et al. (29), with the inclusion of 0.1% Triton X-100 for full enzyme expression. Cytochrome oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1), which is located on the inner mitochondrial membrane (30), was assayed by the method of Cooperstein and Lazarow (31). Assay for Uptake of AIB by Membrane Vesicles. For each time point, 90 ,1 aliquots containing from 150 Mig to 350 Mg of membrane protein in 60 mM sucrose, 10 mM Tris*HCl (pH 7.4 at 370), 0.1 mM MgCl2, and 0.1 mM CaCl2, were preincubated at 370 for 10-20 min. At the end of this period, the reaction was started by the addition of 10 Ml of 14C- or 3H-labeled AIR. The reaction was terminated by dilution with 1.4 ml of 0.8 M NaCl, at 37'. Each diluted sample was immediately filtered through a nitrocellulose filter (24 mm diameter and 0.2 Am, average pore size, Schleicher and Schuell, Inc., Keene, N.H.) which had been wetted with sterile water. The filters were then washed twice with the same volume of NaCl. Background adsorption
was determined by addition of radioactively labeled AIB to the membrane suspension. This mixture was diluted and filtered immediately and washed with 0.8 M NaCl at 37'. The nitrocellulose filters were dried by heat and the radioactivity was determined in a Beckman liquid-scintillation counter, with a toluene scintillation mixture (Liquifluor; New England Nuclear). Assay for Uptake of AIB by SV3T3 Cells. The uptake assay is based on a method described by Foster and Pardee (32) with cells grown on glass coverslips. The coverslips were prepared by boiling in an aqueous solution of sodium hexametaphosphate (8.8 g/liter) and sodium metasilicate (70.12 g/liter). Coverslips containing confluent cells were rinsed twice with Hanks' Balanced Salt Solution (HBS) which contained 20 mM Hepes [4(2-hydroxy-ethyl)-1-piperazineethane-sulfonic acid] at pH 7.0, then preincubated for 25-30 min in the same solution in a CO2 incubator at 37'. Uptake was in HBS and Hepes at 370 for 2 min. Coverslips were dipped, into three beakers each of which contained 250 ml of 25 mM Tris, 0.14 M NaCl, 6.7 mM KCI, 0.68 mM CaCl2, 0.49 mM MgCl2, and 0.37 mM Na2HPO4 (pH 7.0) and thereby rinsed three-times before and after uptake. Coverslips were then placed in 1 ml of 0.4 M NaOH and left at 4° overnight to completely dissolve the cells. The solutions were then neutralized with HC1 and aliquots removed for determination of radioactivity in a Beckman liquid scintillation counter, with a Liquifluor scintillation mixture which contained toluene and Triton X-100. Protein concentrations were determined for each point, and five coverslips, taken through the same washing procedures, were trypsinized and the cells counted in a Coulter Counter.
RESULTS
25,
Membrane fractionation The enzymatic analysis of a typical fractionation of membrane 2
4
6
8
TIME (min) FIG. 1. Time-course of uptake of AIB by SV3T3 membrane vesicles in the presence and absence of NaCl. The uptake assay is described in Materials and Methods. At each time point, 150 ,g of membrane-vesicle (MV) protein was used. The concentration of [14C]AIB was 0.5 mM with a specific activity of 4.61 cpm/pmol of AIR. At any time during uptake, the [14CJAIB cpm could be reduced to the background value by a water wash at room temperature, instead of the usual 0.8 M NaCl wash at 370. Uptake of [14C]AIB was measured in the absence of NaCl (0-0); in the presence of 50 mM NaCl preincubated with MV for 20 min at 37°, before the uptake reaction was started by addition of [14C]AIB (A-A); and when 50 mM NaCl was added with [14C]AIB (0-1).
constituents from SV3T3 cells is shown in Table 1. The mem-
brane fraction, MV, was used for the uptake studies described in this paper. This fraction contains 4% of the total mitochondrial activity (range, 1-4%) and 13% of the lysosomal activity (range, 5-14%). Twenty-four percent of the NADH oxidase travels with MV. AIB uptake activity correlates with the plasma membrane markers, (Na+, K+)-ATPase and 5'-nucleotidase. It does not correlate with mitochondrial or lysosomal enzymatic activities. The kinetics of AIB transport in membrane vesicles The uptake of AIB by MV is time dependent (Fig. 1). As evidence that the accumulated [14C]AIB counts represented uptake
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Hamilton and Nilsen-Hamilton
Table 3. Effect of neutral acids on the uptake of AIB by SV3T3 membrane vesicles
Table 2. The effect of cations on uptake of AIB by membrane vesicles derived from SV3T3 cells
% AIB uptake in 15 sec
% Control (-cation)
Cation
15-sec uptake
Li+ Na+ K+ Rb+ Cs+ NH4+ Choline
82 167 67 75 65 82 81
Amino acid added with 0.1 mM AIB
1-min uptake
99
Alanine
182 82
Glycine
91 80 94
Leucine
91
Methionine
Mean values of triplicate determinations are given. The AIB concentration was 0.1 mM and each determination contained 318 ug of membrane protein. The results are expressed as a percentage of the 15-sec and 1-min uptake values obtained in the absence of 50 mM cation added with [3H]AIB. In the absence of cation, the value for 15-sec uptake was 680 pmol/min per mg of protein and that for 1-min uptake was 230 pmol/min per mg of protein. Cation chlorides were used.
of AIB rather than nonspecific binding to the membrane surface, washing the MV with water, at any time during the time-course, reduced the accumulated 14C radioactive material back to the background value. By the use of hyperosmotic conditions to shrink the vesicles, we observed a 2-fold reduction in steady-state levels of AIB transport (data not shown), which is further evidence of intravesicular accumulation of AIB. Sodium ions at the external surface of the membrane of eukaryotic cells are often required for active transport (33). The effect of 50 mM NaCI, added simultaneously with AIB to start the uptake reaction (Fig. 1), is to increase the initial rate of uptake and the maximal level reached from 2- to 4-fold. The response to 50 mM NaCl depends on the concentration of AIB used, and is greater at lower AIB concentrations (our unpublished observations). The stimulatory effect of NaCI most likely results from a transient concentration gradient formed across the membrane, because 50 mM NaCI is nonstimulatory when preequilibrated with the membranes before initiation of uptake. Also, with time, the initial stimulation, seen when NaCl is added with AIB, decreases to the level found when the vesicles are equilibrated with Na+ prior to addition of label. When there is a requirement of eukaryotic active transport systems for monovalent cations at the external surface of the cell, that requirement is generally specific for Na+ (33, 34). We have shown (Table 2) that stimulation of AIB transport into SV3T3 membrane vesicles is also specific for Na+ when compared with the other chlorides of alkali metal ions, NH4C1, and choline chloride. In addition, this experiment provides evidence against transient osmotic or ionic strength effects which might cause stimulation of AIB uptake by the membrane vesicles. The specific Na+-stimulated uptake of AIB is evidence that this uptake is a mediated one. Further evidence for mediated uptake by the membrane vesicles was provided by studies which showed inhibition of AIB uptake by other amino acids (Table 3). No significant inhibition of uptake by these amino acids was observed in the absence of NaCI. To compare the parameters, Km and Vmax, obtained for membrane vesicles with those for cells, we determined the effect of AIB concentration on the initial rates of uptake in SV3T3 cells grown on glass coverslips (Materials and Methods). The use of AIB, an amino acid analog which is not metabolized, in initial studies such as these, has the advantage that cells can be more directly compared with membrane-vesicle studies to
1909
0.1 mM 1.0 mM 0.1 mM 1.0 mM 0.1 mM 1.0 mM 0.1 mM 1.0 mM
Minus NaCl
99 100 95 98 90 89 106 96
(10) (4) (2) (6)
(8) (5) (5) (6)
Plus NaCl 105 80 79 67 81 60 95 59
(11) (2) (2) (6) (6) (3) (3) (3)
A concentration of amino acid equal to the concentration of AIB (0.1 mM) or ten times greater (1 mM) was added with the AIB to start the reaction. Rates of reaction (15 sec) were compared in the absence and presence of 50 mM NaCl added with the AIB. The results are expressed as percentage of the control in the absence or presence of NaCl. Control values were: minus NaCl = 146 pmol/ min per mg of protein; plus NaCl = 381 pmol/min per mg of protein. Values in parentheses are percent of the standard error of the mean. Triplicate determinations for each experiment were made, and the results averaged. Three sets of experiments were made and averaged.
verify that the transport system has been isolated functionally intact. The kinetics of AIB uptake by SV3T3 cells are biphasic
(Fig. 2).
Similar studies were made with the membrane vesicles. Comparisons of the uncorrected, apparent values of Km and Vmax are shown in Table 4. Biphasic uptake kinetics were obtained for cells and membrane vesicles. Good agreement is found for Km values, from cells and membrane vesicles, determined in the low concentration range of 0.1 mM to 2 mM
-0.1
0
0.1
0.2
0.3
0.4
0.5
I/[AIBJ mM FIG. 2. Lineweaver-Burk plot of AIB uptake by SV3T3 cells. Initial rate determinations (Materials and Methods) were made in the concentration range 0.1-100 mM AIR. In the inset is shown the straight line obtained by least-squares analysis in the concentration range 0.1-2.5 mM. The line of the main figure begins to curve off at 2.5 mM and then at approximately 5 mM it straightens to a new line of steeper slope. The values of Km and Vm., obtained by extrapolation, have not been corrected for the contribution to uptake from each of the other systems. We have designated them the uncorrected apparent Km and Vm. values. They are meaningful only for comparisons. These values are given in Table 4. The average protein per coverslip was 353 Ag (167 estimations) and the average number of cells per coverslip was 5.12 X 106.
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Biochemistry: Hamilton and Nilsen-Hamilton
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 4. Summary of uncorrected apparent Km and Vmax values obtained from SV3T3 cells and membrane vesicles
Uncorrected AIB concentration range
Km
System
(mM)
(mM)
SV3T3 cells
0.1-3.3 20-100 0.1-2.0 0.1-2.0 0.1-2.0 0.1-2.0 20-70 20-70
4.4 26 15 3.4 2.6 1.1 143 153
Membrane vesicles
Na+ Vma (nmol/min concenper mg of tration protein) (mM) 18 31 2.1 0.99 1.0 0.51 155 155
140 140 0 25 50 100 0 50
The Lineweaver-Burk plots used to derive these values are Figs. 2, 3, and 4.
AIB. Lack of agreement is found between Km values determined in the high concentration range 5-100 mM AIR. This could result from a higher contribution of simple diffusion to uptake by the membrane vesicles. At high AIB concentrations the uptake does not respond to NaCl (Table 4 and Fig. 3, top and bottom). To test the possibility that 50 mM NaCl was insufficient to cause stimulation at the higher concentrations of AIB, the effect of concentrations greater than 50 mM NaCl, on the initial rate of uptake of 50 mM AIB, was measured. No significant effect was observed up to 300 mM NaCl. However, a pronounced inhibition of uptake was obtained with KC1. At KC1 concentrations of 100 and 200 mM added with 50 mM AIB, 50 and 86% inhibition of AIB uptake was observed. These results
O
0.01 0.02 0.03 I/[AIB], mM
0.04
Q05
FIG. 3. Lineweaver-Burk plots of uptake of high concentrations of AIB in the absence and presence of 50 mM NaCl. Uptake, for 15 sec, was as described in Materials and Methods. For each estimation, 335 Mg of membrane protein was used. The concentration of [3H]AIB was varied from 20 to 70 mM. The [3HJAIB cpm per experiments were 3.24 X 105. Initial rates have the units: nmol/min per mg of protein. Each point is the mean of duplicate determinations. Linear regression analyses were used to obtain the lines of best fit. Uncorrected values of apparent Km and V.,, are given in Table 4.
4
6
I/[AIB] , mM
FIG. 4. The effect of NaCl on Km and K... of SV3T3 membrane vesicles. Uptake, for 15 sec, was as described in Materials and Methods. For each estimation, 335,gg of membrane protein was used. The concentration of [3HJAIB was varied from 0.1 mM to 2 mM. The [3H]AIB cpm per experiment were 4.19 X 105. Initial rates have the units: nmol/min per mg of protein. Each point is the mean oftriplicate determinations. The lines of best fit were derived by least-squares analysis. The two points in parentheses were not included in the least-squares analysis. 0-0, 100 mM choline chloride final concentration added with [3H]AIB. *-*, 25 mM NaCl/75 mM choline chloride. A-*, 50 mM NaCl/50 mM choline chloride. 0-0,100 mM NaCl. Values of apparent Km and Vmax are given in Table 4.
suggest that very little simple diffusion occurs through the membrane vesicles even at 50 mM AIR. However, the possibility that KCI is destroying membrane integrity has not been ruled out. We determined the effect of Na+ on the uncorrected apparent Km and Vmax values for uptake of AIB by the membrane vesicles, in the range of AIB concentration where uptake was sensitive to Na+. We varied the choline/Na+ ratio, and we measured uptake at a constant 100 mM final salt concentration. In the range 25 mM NaCl to 100 mM NaCI, the uncorrected apparent Km value changed from 3.4 mM to 1.1 mM AIB, and the uncorrected apparent V., value was decreased by one-half (Table 4 and Fig. 4).
DISCUSSION Our main finding is that AIB uptake by SV3T3 membrane vesicles is specifically stimulated by Na+, only if sodium chloride is added at the start of the uptake reaction. This implies that the energy of a Na+-electrochemical gradient is used to drive AIB uptake. These findings are in agreement with those of Quinlan et al. (18). Both reports describe the Na+-dependent amino acid uptake into membrane vesicles derived from tissue-culture cells. However, one report (35) described the phenomenon in membrane vesicles derived from Ehrlich Ascites cells and another (36) in membrane vesicles from enterocyte brush border membranes. We have further shown that the Na+-stimulation is accompanied by alterations in apparent Km values. This would indicate an increase in the affinity of the carrier for AIB in the presence of Na+ according to the models of Heinz and others (33, 34). Our results indicate that AIB is take up by the SV3T3 cells and membrane vesicles by two kinetically distinguishable components: one with a low apparent affinity for AIB which is Na+ independent, and the other with a high apparent affinity for AIB which is Na+ dependent. The advantage of membrane-vesicle studies of uptake is that the two systems can be studied independently in the presence and absence of Na+.
Biochemistry: Hamilton and Nilsen-Hamilton We are deeply indebted to Dr. R. W. Holley for his encouragement and support. We thank Drs. J. Hochstadt, D. Quinlan, R. Willis, and R. W. Holley for their criticisms of the manuscript and gratefully acknowledge the excellent technical assistance of Andrew Oesterle. This work was supported by grants from the American Cancer Society (BC-30), The National Science Foundation (GB-32391X), and the National Institutes of Health (CA11176). 1. Blade, E., Harel, L. & Hanania, N. (1966) Exp. Cell Res. 41, 473-482. 2. Cunningham, D. D. & Pardee, A. B. (1969) Proc. Natl. Acad. Sci. USA 64,1049-1056. 3. Weber, M. J. & Edlin, G. (1971) J. Biol. Chem. 246,1828-1833. 4. Jimenez de Asua, L. & Rozengurt, E. (1974) Nature 251,624-626. 5. Hatanaka, M. (1974) Biochim. Biophys. Acta 355, 77-104. 6. Quinlan, D. C. & Hochstadt, J. (1974) Proc. Natl. Acad. Sci. USA 71,5000-5003. 7. Christensen, H. N. & Hendersen, M. E. (1952) Cancer Res. 12, 229-231. 8. Foster, D. 0. & Pardee, A. B. (1969) J. Biol. Chem. 244, 26752681. 9. Otsuka, H. & Moskowitz, M. (1975) J. Cell. Physiol. 85,665-674. 10. Kasarov, L. B. & Friedman, H. (1974) Cancer Res. 34,1862-1865. 11. Rozengurt, E. & Heppel, L. (1975) Proc. Natl. Acad. Sci. USA
72,4492-4495. 12. Kimelberg, H. K. & Mayhew, E. (1975) J. Biol. Chem. 250, 100-104. 13. Kaback, H. R. (1972) Biochim. Biophys. Acta 265, 367-416. 14. Hochstadt, J., Quinlan, D. C., Rader, R. L., Li, C. & Dowd, D. (1975) in Methods in Membrane Biology, ed. Korn, E. D. (Plenum Press, New York), Vol. 5, pp. 217-261. 15. Quinlan, D. C. & Hochstadt, J. (1976) J. Biol. Chem. 251, 344-354. 16. Li, C.-C., & Hochstadt, J. (1976) J. Biol. Chem. 251, 1175-1180. 17. Li, C.-C. & Hochstadt, J. (1976) J. Biol. Chem. 251, 1181-1187. 18. Quinlan, D. C., Parnes, J. R., Shalom, R., Garvey, T. Q., Isselbacher, K. J. & Hochstadt, J. (1976), Proc. Nati. Acad. Sci. USA 73, 1631-1635. 19. Wallach, D. F. H. & Kamat, V. B. (1966) in Methods in Enzymology, eds. Neufeld, E. F. & Ginsburg, V. (Academic Press,
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