Proc. Natl. Acad. Sci. USA Vol. 73, No. 8, pp. 2614-2618, August 1976
Biochemistry
Regulation of active a-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts (Na+ gradient/electrogenic/simian virus 40-transformed)
JULIA E. LEVER Department of Cell Regulation, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England
Communicated by Renato Dulbecco, May 14, 1976
Membrane vesicles isolated from untransABSTRACT formed 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+.
+ K+)ATPase activity to drive concentrative iso-Abu uptake, the activity of iso-Abu carriers was assayed dissociated from the active Na+ transport system. It was found that regulation of iso-Abu uptake observed in fibroblasts (10, 11) was expressed in isolated membranes as an altered maximal velocity (Vmax) for iso-Abu carrier activity that mediated concentrative uptake driven by a Na+ gradient.
MATERIALS AND METHODS Swiss and Balb/c 3T3 mouse fibroblasts transformed by simian virus 40 (SV40) and Balb/c tertiary mouse embryo cells were grown at 370 in 9520-cm2 culture vessels (12) in 2 liters of Dulbecco's modified Eagle's medium with 10% calf serum equilibrated with 10% CO2 in air. Swiss 3T3 mouse fibroblasts (13) were grown in this medium with 10% fetal calf serum in 792-cm2 roller bottles. Cell lines were monitored for absence of mycoplasma contamination. For the preparation of membrane vesicles, 1 to 5 X 109 cells were harvested, washed, disrupted; and the postnuclear supernatant was obtained as described by Hochstadt et al. (14), with the exception that 0.2 mM CaCl2 was added during nitrogen cavitation. The postnuclear supernatant (100-150 ml) was layered over a 35% sucrose-0.01 M Tris-HCl, pH 7.5, barrier in 4 X 50 ml tubes, and centrifuged at 20,000 X g for 30 min in a Sorvall HB-4 rotor. The membrane vesicles in the upper fraction were concentrated at 105 X g for 45 min and resuspended at 2-9 mg/ml in 0.25 M sucrose-0.01 M Tris-HCl, pH 7.5. Vesicles were stored in liquid nitrogen. The 5'-nucleotidase (EC 3.1.3.5), NADH diaphorase (EC 1.6.99.3) (15), and succinate-cytochrome c reductase (EC 1.3.99.1) (16) activities of each membrane and soluble fraction were determined. Protein was measured by the method of Lowry et al.
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
(17). Transport assays of membrane vesicles were carried out in 100-,Al volumes at 220 with additions as described. Incubations were terminated by addition of 5 ml of ice-cold 0.8 M NaCl0.01 M Tris-HC1, pH 7.5, and rapid filtration through a 0.45,um, 2.5-cm diameter nitrocellulose filter, followed by a 5-ml wash. Radioactivity of dried filters in toluene-Liquifluor was determined by scintillation counting. Ouabain, valinomycin, a-aminoisobutyric acid, and unlabeled nucleosides were obtained from Sigma. Radioisotopes were purchased from the Radiochemical Centre.
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 maintained independently of endogenous (Na+
RESULTS
Na+-gradient-dependent, electrogenic intravesicular uptake of iso-Abu Several lines of evidence indicate that iso-Abu retention on filters in the presence of membrane vesicles represents carrier-mediated intravesicular uptake rather than binding to fixed sites. Countertransport of iso-Abu could be demonstrated by
Abbreviations: iso-Abu, a-aminoisobutyric acid; SV40, simian virus 40.
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Proc. Natl. Acad. Sci. USA 73 (1976)
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FIG. 1. Evidence for carrier-mediated intravesicular iso-Abu uptake. (A) Countertransport effects on the rate of iso-Abu uptake. Membrane vesicles from Swiss 3T3 cells transformed by SV40 were incubated for 15 min at 4 mg/ml in 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, 0.125 M sucrose, and 50 mM NaCl in the presence (0) or absence (0) of 9 mM unlabeled iso-Abu. Then, vesicles were diluted 10-fold in 100 ,ul of this solution to 1.1 mM extravesicular iso-[14C]Abu (58 fmol/cpm) for the times indicated. (B) Osmotic sensitivity of accumulated iso-Abu. Vesicles were incubated for 15 min with iso[I4C]Abu, as in footnotet, Table 1, and then for another 5 min after the addition of sucrose to obtain the concentrations indicated.
increased uptake of iso414C]Abu when vesicles were first loaded with unlabeled iso-Abu (Fig. 1A). Increasing sucrose concentration in the incubation medium decreased the extent of equilibrium accumulation of iso-Abu without appreciatively affecting the initial rate of iso-Abu uptake (Fig. 1B). Dilution of vesicles in hypotonic solutions caused loss of accumulated iso-Abu. Concentrative uptake of iso-Abu is dependent upon a Na+ gradient and independent of endogenous (Na+ + K+) ATPase
TIME (MIN) Dependence of iso-Abu transport upon Na+ gradient. (A) Aliquots of 180 lg of vesicles from Swiss 3T3 cells transformed by SV40 were incubated for 30 min in the presence (A) or absence (0, 0) of 50 mM NaCl before initiation of uptake in solutions containing 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, and 1.1 mM iso-[14C]Abu (0.061 pmol/cpm), with 50 mM NaCl (0, A) or 50 mM choline chloride (0) added to the extravesicular space at zero time. (B) cis and trans effects of Na+ on iso-Abu efflux. Aliquots of 138 lg of vesicles from Balb/c 3T3 cells transformed by SV40 were incubated for 15 min in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, 1.1 mM iso-[14C]Abu (0.061 pmol/cpm), with 50 mM NaCl (o, *) added to the extravesicular solution at zero time. Then each was diluted 10-fold to 1 ml in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, with 50 mM NaCl (o) or 50 mM choline chloride (-) for the times indicated. FIG. 2.
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0
TIME (MIN) FIG. 3. Evidence for an electrogenic mechanism of concentrative iso-Abu uptake. (A) Effect of a K+ diffusion gradient in the presence of valinomycin. Aliquots of 80 jig of vesicles from Balb/c 3T3 cells transformed by SV40 were incubated for 15 min with 50 mM choline chloride and 1 gug of valinomycin in 2% ethanol (-), 50 mM KCI and 2% ethanol (0), 2% ethanol (A), no additions (A), or 50 mM KCl and 1 ,g of valinomycin in 2% ethanol (-) and then diluted 10-fold into a solution containing 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, 50 mM NaCl, and 0.17 mM iso-[14C]Abu (9.1 fmol/cpm), in 100 gl, for the times indicated. (B) Effect of permeant and impermeant anions on iso-Abu uptake. Aliquots of 138 Aig of vesicles from Balb/c 3T3 cells transformed by SV40 were incubated in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, with 50 mM NaCl (0), choline chloride (0), NaN03 (A), Na2SO4 (A), NaBrO3 (o), NaCl minus phosphate (-), or NaSCN (v) added to the extravesicular solution at zero time with 1.1 mM iso-[14C]Abu (0.061 pmol/cpm).
activity. Preincubation of vesicles with NaCI for 15 min before addition of iso-['4C]Abu reduced the rate and extent of accumulation of iso-Abu (Fig. 2A) compared with uptake observed when NaCI was added externally together with iso-[14C]Abu. Monensin and gramicidin D, ionophores that dissipate a Na+ gradient, reduced Na+-gradient-driven uptake to the level observed after Na+ preincubation (not shown). 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. 2B). Preincubation of vesicles up to 30 min with ouabain concentrations up to 3 mM did not inhibit the rate of iso-Abu uptake, and K+ and ATP did not stimulate Na+-dependent iso-Abu uptake. Li+ but not K+ could stimulate iso-Abu uptake above the level observed with choline in the absence of Na+. Evidence for an electrogenic mechanism of Na+-gradientstimulated iso-Abu uptake in fibroblast membranes was obtained. Creation of a K+ diffusion gradient and an interiornegative membrane potential (18) by dilution of vesicles loaded with KCI in the presence of valinomycin into the standard incubation mixture caused marked stimulation of iso-Abu uptake compared with controls (Fig. 3A). A similar K+ gradient in the presence of 6 ,ug/ml of nigericin (not shown), which mediates a nonelectrogenic K+/H+ exchange (18), did not stimulate iso-Abu uptake. The rate and extent of Na+-dependent iso-Abu uptake was accelerated compared with Cl- in the presence of anions such as N03- and SCN-, which penetrate membranes readily (19), and was decreased by relatively impermeable anions such as S04m (Fig. 3B).
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Proc. Natl. Acad. Sci. USA 73 (1976)
Table 1. Specific activities of solute uptake in isolated membranes*
Uptake activities (pmol/min per mg protein)t§
Source Balb/c tertiary mouse embryo fibroblasts (Confluent) (Subconfluent) Balb/c SV3T3 (Confluent) (Subconfluent) Swiss 3T3K (Confluent) (Subconfluent) Swiss SV3T3 (Confluent) (Subconfluent)
No. of preparations
Na+-independent iso-Abu
Na+-gradientdependent iso-Abut
Adenosine
Uridine
5 3
560 810
440 1300
1.7 2.6
0.10 0.20
3 2
870 930
960 2300
2.9 2.0
0.19 0.19
2 2
210 600
190 440
1.3 1.7
0.04 0.26
2 2
530 687
660 480
2.2 1.9
0.32 0.13
* Recovery of plasma membrane material in vesicle preparations from subconfluent SV 3T3 cells, estimated by 5'-nucleotidase activity, was 32 + 4%, and in all other preparations was 56 to 68 i 15%. Contamination by mitochondrial membranes, estimated by succinate-cytochrome c reductase activity, was less than 2 + 1.5%, and by endoplasmic reticulum, estimated by NADH diaphorase activity, was 20 ± 3% of the total amount in the initial homogenate. Total recovery of each marker enzyme activity in all the fractions was 70-120%, and vesicle preparations were 4- to 11-fold enriched in 5'-nucleotidase specific activity. t Incubations were carried out for 30 s and 1 min in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 50 mM NaCl, and 5 mM MgCl2 with 1 mM iso-[14C]Abu (0.061 pmol/cpm), 0.5 p [3H]adenosine (9 x 10-5 pmol/cpm), or 0.2 uM uridine (3.9 x 10-5 pmol/cpm), using 0.07-0.17 mg of vesicle protein. Initial rates of uptake for each substrate were measured at concentrations below their Km values to minimize nonspecific entry by simple diffusion. + For iso-Abu uptake, values obtained when choline chloride was substituted for NaCl were subtracted, to give Na+-dependent uptake. § Results were averaged from duplicate determinations repeated two to three times on each preparation. Variation was + 10-25% among
duplicates.
Comparison of iso-Abu carrier activity of isolated membranes The iso-Abu uptake activity of vesicles from untransformed and SV40-transformed mouse fibroblasts was determined both in the presence of a standard Na+ gradient (external Na+ > internal Na+) driving force and in the absence of Na+. The specific activity of adenosine uptake in vesicles, relatively invariant in vivo with growth or transformed state (20), was measured as an internal control to indicate the fraction of sealed, transport-competent vesicles in the preparation. The specific activity of the initial rate of uridine uptake was measured as a control for expression of a cell density-dependent membrane transport property in each vesicle preparation (20, 21). Results summarized in Table 1 indicate that iso-Abu carrier specific activity, stimulated by a Na+ gradient, was 56-66% decreased in vesicles from confluent untransformed fibroblasts and up to 1.8-fold increased in vesicles from SV40-transformed fibroblasts, in comparison with the activities observed in vesicles from subconfluent untransformed fibroblasts. The decrease at confluence of Na+-independent iso-Abu carrier activity of membranes from untransformed cells was less marked (2330%) and not appreciably increased (up to 13-23%) after transformation. Fig. 4 indicates that these uptake differences were also expressed in extent of accumulation of iso-Abu after imposition of a standard Na+ gradient. The Na+-independent extent of iso-Abu accumulation did not differ significantly, suggesting similar intravesicular volumes in each case. No overshoot of iso-Abu accumulation was observed within this 15-min interval. The maximal extent of Na+-gradient-driven accumulation of iso-Abu should be independent of the number of iso-Abu carriers, but should reflect the energization of the system by the Na+ gradient. The changes in iso-Abu carrier specific activity after trans-
formation by SV40 were reflected in altered maximal velocities (Vmax) of the initial rate of Na+-gradient-stimulated iso-Abu uptake (Fig. 5). The Km values for Na+-gradient-stimulated iso-Abu uptake at 50 mM NaCl, present externally, of preparations from Swiss or Balb/c transformed or untransformed fibroblasts varied from 0.7 to 1.5 mM. Preliminary experiments indicate that the Km for Na+-dependent iso-Abu uptake decreases at increased Na+ concentrations. The Vm. for iso-Abu uptake in vesicles from subconfluent mouse embryo fibroblasts was 1 + 0.2 nmol/min per mg, and was 2 ± 0.4 nmol/min per mg in vesicles from Balb/c SV40-transformed cells (Fig. 5). Activities in vesicles from confluent untransformed cells were too low to permit accurate Vmax determination. These values agree with Vmax values observed in intact cells (11) expressed per mg of protein, after correction for recovery of membrane protein. A
c
-T
7
A 2 1000
0.
E
,C
:n 0
500
-6
T0~~~~~~~~~~~~ o
10 5 0 15 15 TIME (MIN) FIG. 4. Uptake of iso-Abu into membrane vesicles from Balb/c 3T3 cells transformed by SV40 (A), confluent (B), and subconfluent (C) Balb/c tertiary mouse embryo cells as a function of time. Aliquots of 120,ug (A), 90 Ag (B), and 115 ,g (C) were incubated in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5),5 mM MgCl2, with 0.17 mM iso-[14C]Abu (9.1 fmol/cpm) and 50 mM NaCl (0) or 50 mM choline chloride (0) added externally at zero time. 0
5
10
Biochemistry:
Proc. Natl. Acad. Sci. USA 73 (1976)
Lever
5-
w
In-._
,0
z Z
I I
0E o
5-
Z
0_
I
0f
wc z
(1/iso-Abm CONCENTRATION)
mM 1
I-
FIG. 5. Concentration dependence of the initial rate of Na+dependent iso-Abu uptake. Vesicles from subconfluent (0) Balb/c tertiary mouse embryo fibroblasts, or Balb/c SV40-transformed 3T3 cells (0) were incubated 30 s and 1 min in incubation mixtures as described in Fig. 4, with various concentrations of iso-[14C]Abu at 0.061 pmol/cpm added at zero time. The rate of Na+-dependent uptake was obtained by subtraction of the rate in 50 mM choline chloride from that in 50 mM NaCl, added externally at zero time.
DISCUSSION Assay of vectorial transport activity of vesicles from membranes isolated from mammalian cells in culture provides a system for characterizing functional plasma membrane alterations associated with hormones (22) and cellular proliferative and transformed state in terms of specific parameters related to the transport mechanism. This approach, first applied by Kaback and Stadtman (23) to study transport in bacterial membranes, permits dissociation from intracellular and topographical factors and assay of transport function across compartments of defined composition. The experiments reported here provide evidence for a Na+-
gradient-stimulated, electrogenic mechanism of iso-Abu uptake (24) in membranes from untransformed and SV40-transformed mouse fibroblasts. Observations that iso-Abu uptake against its concentration gradient could be driven by transmembrane Na+ gradients imposed independently of (Na+ + K+)ATPase activity, and that flux of iso-Abu in either direction across the membrane was decreased by Na+ added to the opposite side, support a type of Na+-gradient-coupled mechanism proposed by Crane (25). In this model, the carrier has increased affinity for the solute in the presence of Na+, and concentrative solute uptake is driven by an electrochemical Na+ gradient, external Na+ > internal Na+. In the intact cell, this Na+ gradient is maintained by the (Na+ + K+)ATPase. Observations that Na+driven iso-Abu uptake was enhanced under conditions expected to create an interior-negative membrane potential, such as a K+ diffusion potential, internal K+ > external K+, in the presence of valinomycin, or by using the Na+ salts of highly permeant anions such as SCN- and NO3- provided evidence that the iso-Abu carrier complex moves in cotransport with a cation, but without charge compensation via the same carrier. Thus, among the possibilities for cellular control of iso-Abu carrier function, are regulation of (i) theactiveNa+ pump; (ii) membrane permeability to Na+; (ii) electrogenic membrane processes or membrane depolarization affecting membrane potential; (iv) modification of the Na+-dependent and Na+-
2617
independent affinity for iso-Abu; (v) number of iso-Abu carriers; and (vi) carrier mobility. Most of these alternatives can be distinguished using membrane vesicles. Since iso-Abu uptake changes similar to those observed in intact cells with growth or transformed state (10, 11) were detected in their isolated vesicles when transport was driven by a standard Na+ gradient, these alterations occur independently of regulation of the Na+ pump. Membrane Na+ permeability or carrier response to Na+ could change with growth or transformed state, since membranes from these various cell types showed differences in steady-state accumulation of iso-Abu driven by a Na+ gradient. Although presumably dissipation of the Na+ gradient occurs during the incubation, no efflux of accumulated iso-Abu was observed during a 15-min interval. Since changes in iso-Abu uptake stimulated by Na+ gradient were expressed as changes in Vmax for iso-Abu uptake, with minimal variations in Km, possibly alteration of the number or the mobility of iso-Abu carriers resulted in another component of the observed uptake differences between the cell populations in this comparison. This does not rule out other mechanisms of carrier regulation listed above in more rapid and transient responses to hormonal or metabolic regulation. I thank Dr. R. Dulbecco for generous support and encouragement, N. Neyhard and Mrs. P. Pettican for expert technical assistance, the Cell Production Unit for cell cultures, and C. Dixon and J. Elkington for drawing the graphs. This work was carried out during tenure of a research training fellowship awarded by the International Agency for Research on Cancer and a United States National Institutes of Health Research Service Award CA05174-01 from the National Cancer Institute. 1. Pardee, A. B. & Rozengurt, E. (1974) in Biochemistry of Cell Walls and Membranes, ed. Fox, C. F. (Medical and Technical Publishing Co., London), pp. 155-185. 2. Pardee, A. B. (1974) Proc. Natl. Acad. Sci. USA 71, 12861290. 3. Holley, R. W. (1972) Proc. Natl. Acad. Sci. USA 69, 28402841. 4. Elligsen, J. D., Thompson, J. E., Frey, H. E. & Kruuv, J. (1974) Exp. Cell Res. 87, 233-240. 5. Rozengurt, E. & Heppel, L. A. (1975) Proc. Natl. Acad. Sci. USA 72,4492-4495. 6. Kimelberg, H. K. & Mayhew, E. (1975) J. Biol. Chem. 250, 100-104. 7. Schultz, S. G. & Curran, P. F. (1970) Physiol. Rev. 50, 637718. 8. Sigrist-Nelson, K., Murer, H. & Hopfer, U. (1975) J. Biol. Chem. 250,5674-5680. 9. Columbini, M. & Johnstone, R. M. (1974) J. Membr. Biol. 18, 315-334. 10. Foster, D. 0. & Pardee, A. B. (1969) J. Biol. Chem. 224, 26752681. 11. Isselbacher, K. J. (1972) Proc. Natl. Acad. Sci. USA 69, 585589. 12. House, W., Shearer, M. & Maroudas, N. G. (1972) Exp. Cell Res.
71,293-296. 13. Todaro, G. J., Lazar, G. K. & Green, H. (1965) J. Cell. Physiol. 66,325-334. 14. Hochstadt, J., Quinlan, D. C., Rader, R. L., Li, C. C. & Dowd, D. (1974) in Methods in Membrane Biology, ed. Korn, E. (Plenum Press, New York), Vol. 5, pp. 117-162. 15. Avruch, J. & Wallach, D. F. H. (1971) Biochim. Biophys. Acta
233,334-347. 16. Mackler, B., Collip, P. J., Duncan, H. M., Ras, N. A. & Huennekens, F. M. (1962) J. Biol. Chem. 237, 2968-2974. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193,265-275. 18. Hirata, H., Altendorf, K. H. & Harold, F. M. (1973) Proc. Natl. Acad. Sci. USA 70, 1804-1808.
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19. Gamble, J. G. & Lehninger, A. L. (1973) J. Biol. Chem. 248, 610-618. 20. Cunningham, D. D. & Pardee, A. B. (1969) Proc. Natl. Acad. Sca. USA 64, 1049-1056. 21. Quinlan, D. C. & Hochstadt, J. (1974) Proc. NatI. Acad. Sci. USA
71,5000-5003.
Proc. Natl. Acad. Sci. USA 73 (1976) 22. Holley, R. W. (1975) Nature 258,487-490. 23. Kaback, H. R. & Stadtman, E. R. (1966) Proc. Nati. Acad. Sci. USA 55, 920-927. 24. Inui, Y. & Christensen, H. N. (1966) J. Gen. Physiol. 50, 203224. 25. Crane, R. K. (1965) Fed. Proc. 24, 1000-1006.
Biochemistry
Regulation of active a-aminoisobutyric acid transport expressed in membrane vesicles from mouse fibroblasts (Na+ gradient/electrogenic/simian virus 40-transformed)
JULIA E. LEVER Department of Cell Regulation, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England
Communicated by Renato Dulbecco, May 14, 1976
Membrane vesicles isolated from untransABSTRACT formed 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+.
+ K+)ATPase activity to drive concentrative iso-Abu uptake, the activity of iso-Abu carriers was assayed dissociated from the active Na+ transport system. It was found that regulation of iso-Abu uptake observed in fibroblasts (10, 11) was expressed in isolated membranes as an altered maximal velocity (Vmax) for iso-Abu carrier activity that mediated concentrative uptake driven by a Na+ gradient.
MATERIALS AND METHODS Swiss and Balb/c 3T3 mouse fibroblasts transformed by simian virus 40 (SV40) and Balb/c tertiary mouse embryo cells were grown at 370 in 9520-cm2 culture vessels (12) in 2 liters of Dulbecco's modified Eagle's medium with 10% calf serum equilibrated with 10% CO2 in air. Swiss 3T3 mouse fibroblasts (13) were grown in this medium with 10% fetal calf serum in 792-cm2 roller bottles. Cell lines were monitored for absence of mycoplasma contamination. For the preparation of membrane vesicles, 1 to 5 X 109 cells were harvested, washed, disrupted; and the postnuclear supernatant was obtained as described by Hochstadt et al. (14), with the exception that 0.2 mM CaCl2 was added during nitrogen cavitation. The postnuclear supernatant (100-150 ml) was layered over a 35% sucrose-0.01 M Tris-HCl, pH 7.5, barrier in 4 X 50 ml tubes, and centrifuged at 20,000 X g for 30 min in a Sorvall HB-4 rotor. The membrane vesicles in the upper fraction were concentrated at 105 X g for 45 min and resuspended at 2-9 mg/ml in 0.25 M sucrose-0.01 M Tris-HCl, pH 7.5. Vesicles were stored in liquid nitrogen. The 5'-nucleotidase (EC 3.1.3.5), NADH diaphorase (EC 1.6.99.3) (15), and succinate-cytochrome c reductase (EC 1.3.99.1) (16) activities of each membrane and soluble fraction were determined. Protein was measured by the method of Lowry et al.
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
(17). Transport assays of membrane vesicles were carried out in 100-,Al volumes at 220 with additions as described. Incubations were terminated by addition of 5 ml of ice-cold 0.8 M NaCl0.01 M Tris-HC1, pH 7.5, and rapid filtration through a 0.45,um, 2.5-cm diameter nitrocellulose filter, followed by a 5-ml wash. Radioactivity of dried filters in toluene-Liquifluor was determined by scintillation counting. Ouabain, valinomycin, a-aminoisobutyric acid, and unlabeled nucleosides were obtained from Sigma. Radioisotopes were purchased from the Radiochemical Centre.
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 maintained independently of endogenous (Na+
RESULTS
Na+-gradient-dependent, electrogenic intravesicular uptake of iso-Abu Several lines of evidence indicate that iso-Abu retention on filters in the presence of membrane vesicles represents carrier-mediated intravesicular uptake rather than binding to fixed sites. Countertransport of iso-Abu could be demonstrated by
Abbreviations: iso-Abu, a-aminoisobutyric acid; SV40, simian virus 40.
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Proc. Natl. Acad. Sci. USA 73 (1976)
10 15
FIG. 1. Evidence for carrier-mediated intravesicular iso-Abu uptake. (A) Countertransport effects on the rate of iso-Abu uptake. Membrane vesicles from Swiss 3T3 cells transformed by SV40 were incubated for 15 min at 4 mg/ml in 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, 0.125 M sucrose, and 50 mM NaCl in the presence (0) or absence (0) of 9 mM unlabeled iso-Abu. Then, vesicles were diluted 10-fold in 100 ,ul of this solution to 1.1 mM extravesicular iso-[14C]Abu (58 fmol/cpm) for the times indicated. (B) Osmotic sensitivity of accumulated iso-Abu. Vesicles were incubated for 15 min with iso[I4C]Abu, as in footnotet, Table 1, and then for another 5 min after the addition of sucrose to obtain the concentrations indicated.
increased uptake of iso414C]Abu when vesicles were first loaded with unlabeled iso-Abu (Fig. 1A). Increasing sucrose concentration in the incubation medium decreased the extent of equilibrium accumulation of iso-Abu without appreciatively affecting the initial rate of iso-Abu uptake (Fig. 1B). Dilution of vesicles in hypotonic solutions caused loss of accumulated iso-Abu. Concentrative uptake of iso-Abu is dependent upon a Na+ gradient and independent of endogenous (Na+ + K+) ATPase
TIME (MIN) Dependence of iso-Abu transport upon Na+ gradient. (A) Aliquots of 180 lg of vesicles from Swiss 3T3 cells transformed by SV40 were incubated for 30 min in the presence (A) or absence (0, 0) of 50 mM NaCl before initiation of uptake in solutions containing 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, and 1.1 mM iso-[14C]Abu (0.061 pmol/cpm), with 50 mM NaCl (0, A) or 50 mM choline chloride (0) added to the extravesicular space at zero time. (B) cis and trans effects of Na+ on iso-Abu efflux. Aliquots of 138 lg of vesicles from Balb/c 3T3 cells transformed by SV40 were incubated for 15 min in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, 1.1 mM iso-[14C]Abu (0.061 pmol/cpm), with 50 mM NaCl (o, *) added to the extravesicular solution at zero time. Then each was diluted 10-fold to 1 ml in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, with 50 mM NaCl (o) or 50 mM choline chloride (-) for the times indicated. FIG. 2.
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TIME (MIN) FIG. 3. Evidence for an electrogenic mechanism of concentrative iso-Abu uptake. (A) Effect of a K+ diffusion gradient in the presence of valinomycin. Aliquots of 80 jig of vesicles from Balb/c 3T3 cells transformed by SV40 were incubated for 15 min with 50 mM choline chloride and 1 gug of valinomycin in 2% ethanol (-), 50 mM KCI and 2% ethanol (0), 2% ethanol (A), no additions (A), or 50 mM KCl and 1 ,g of valinomycin in 2% ethanol (-) and then diluted 10-fold into a solution containing 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, 50 mM NaCl, and 0.17 mM iso-[14C]Abu (9.1 fmol/cpm), in 100 gl, for the times indicated. (B) Effect of permeant and impermeant anions on iso-Abu uptake. Aliquots of 138 Aig of vesicles from Balb/c 3T3 cells transformed by SV40 were incubated in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 5 mM MgCl2, with 50 mM NaCl (0), choline chloride (0), NaN03 (A), Na2SO4 (A), NaBrO3 (o), NaCl minus phosphate (-), or NaSCN (v) added to the extravesicular solution at zero time with 1.1 mM iso-[14C]Abu (0.061 pmol/cpm).
activity. Preincubation of vesicles with NaCI for 15 min before addition of iso-['4C]Abu reduced the rate and extent of accumulation of iso-Abu (Fig. 2A) compared with uptake observed when NaCI was added externally together with iso-[14C]Abu. Monensin and gramicidin D, ionophores that dissipate a Na+ gradient, reduced Na+-gradient-driven uptake to the level observed after Na+ preincubation (not shown). 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. 2B). Preincubation of vesicles up to 30 min with ouabain concentrations up to 3 mM did not inhibit the rate of iso-Abu uptake, and K+ and ATP did not stimulate Na+-dependent iso-Abu uptake. Li+ but not K+ could stimulate iso-Abu uptake above the level observed with choline in the absence of Na+. Evidence for an electrogenic mechanism of Na+-gradientstimulated iso-Abu uptake in fibroblast membranes was obtained. Creation of a K+ diffusion gradient and an interiornegative membrane potential (18) by dilution of vesicles loaded with KCI in the presence of valinomycin into the standard incubation mixture caused marked stimulation of iso-Abu uptake compared with controls (Fig. 3A). A similar K+ gradient in the presence of 6 ,ug/ml of nigericin (not shown), which mediates a nonelectrogenic K+/H+ exchange (18), did not stimulate iso-Abu uptake. The rate and extent of Na+-dependent iso-Abu uptake was accelerated compared with Cl- in the presence of anions such as N03- and SCN-, which penetrate membranes readily (19), and was decreased by relatively impermeable anions such as S04m (Fig. 3B).
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Table 1. Specific activities of solute uptake in isolated membranes*
Uptake activities (pmol/min per mg protein)t§
Source Balb/c tertiary mouse embryo fibroblasts (Confluent) (Subconfluent) Balb/c SV3T3 (Confluent) (Subconfluent) Swiss 3T3K (Confluent) (Subconfluent) Swiss SV3T3 (Confluent) (Subconfluent)
No. of preparations
Na+-independent iso-Abu
Na+-gradientdependent iso-Abut
Adenosine
Uridine
5 3
560 810
440 1300
1.7 2.6
0.10 0.20
3 2
870 930
960 2300
2.9 2.0
0.19 0.19
2 2
210 600
190 440
1.3 1.7
0.04 0.26
2 2
530 687
660 480
2.2 1.9
0.32 0.13
* Recovery of plasma membrane material in vesicle preparations from subconfluent SV 3T3 cells, estimated by 5'-nucleotidase activity, was 32 + 4%, and in all other preparations was 56 to 68 i 15%. Contamination by mitochondrial membranes, estimated by succinate-cytochrome c reductase activity, was less than 2 + 1.5%, and by endoplasmic reticulum, estimated by NADH diaphorase activity, was 20 ± 3% of the total amount in the initial homogenate. Total recovery of each marker enzyme activity in all the fractions was 70-120%, and vesicle preparations were 4- to 11-fold enriched in 5'-nucleotidase specific activity. t Incubations were carried out for 30 s and 1 min in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5), 50 mM NaCl, and 5 mM MgCl2 with 1 mM iso-[14C]Abu (0.061 pmol/cpm), 0.5 p [3H]adenosine (9 x 10-5 pmol/cpm), or 0.2 uM uridine (3.9 x 10-5 pmol/cpm), using 0.07-0.17 mg of vesicle protein. Initial rates of uptake for each substrate were measured at concentrations below their Km values to minimize nonspecific entry by simple diffusion. + For iso-Abu uptake, values obtained when choline chloride was substituted for NaCl were subtracted, to give Na+-dependent uptake. § Results were averaged from duplicate determinations repeated two to three times on each preparation. Variation was + 10-25% among
duplicates.
Comparison of iso-Abu carrier activity of isolated membranes The iso-Abu uptake activity of vesicles from untransformed and SV40-transformed mouse fibroblasts was determined both in the presence of a standard Na+ gradient (external Na+ > internal Na+) driving force and in the absence of Na+. The specific activity of adenosine uptake in vesicles, relatively invariant in vivo with growth or transformed state (20), was measured as an internal control to indicate the fraction of sealed, transport-competent vesicles in the preparation. The specific activity of the initial rate of uridine uptake was measured as a control for expression of a cell density-dependent membrane transport property in each vesicle preparation (20, 21). Results summarized in Table 1 indicate that iso-Abu carrier specific activity, stimulated by a Na+ gradient, was 56-66% decreased in vesicles from confluent untransformed fibroblasts and up to 1.8-fold increased in vesicles from SV40-transformed fibroblasts, in comparison with the activities observed in vesicles from subconfluent untransformed fibroblasts. The decrease at confluence of Na+-independent iso-Abu carrier activity of membranes from untransformed cells was less marked (2330%) and not appreciably increased (up to 13-23%) after transformation. Fig. 4 indicates that these uptake differences were also expressed in extent of accumulation of iso-Abu after imposition of a standard Na+ gradient. The Na+-independent extent of iso-Abu accumulation did not differ significantly, suggesting similar intravesicular volumes in each case. No overshoot of iso-Abu accumulation was observed within this 15-min interval. The maximal extent of Na+-gradient-driven accumulation of iso-Abu should be independent of the number of iso-Abu carriers, but should reflect the energization of the system by the Na+ gradient. The changes in iso-Abu carrier specific activity after trans-
formation by SV40 were reflected in altered maximal velocities (Vmax) of the initial rate of Na+-gradient-stimulated iso-Abu uptake (Fig. 5). The Km values for Na+-gradient-stimulated iso-Abu uptake at 50 mM NaCl, present externally, of preparations from Swiss or Balb/c transformed or untransformed fibroblasts varied from 0.7 to 1.5 mM. Preliminary experiments indicate that the Km for Na+-dependent iso-Abu uptake decreases at increased Na+ concentrations. The Vm. for iso-Abu uptake in vesicles from subconfluent mouse embryo fibroblasts was 1 + 0.2 nmol/min per mg, and was 2 ± 0.4 nmol/min per mg in vesicles from Balb/c SV40-transformed cells (Fig. 5). Activities in vesicles from confluent untransformed cells were too low to permit accurate Vmax determination. These values agree with Vmax values observed in intact cells (11) expressed per mg of protein, after correction for recovery of membrane protein. A
c
-T
7
A 2 1000
0.
E
,C
:n 0
500
-6
T0~~~~~~~~~~~~ o
10 5 0 15 15 TIME (MIN) FIG. 4. Uptake of iso-Abu into membrane vesicles from Balb/c 3T3 cells transformed by SV40 (A), confluent (B), and subconfluent (C) Balb/c tertiary mouse embryo cells as a function of time. Aliquots of 120,ug (A), 90 Ag (B), and 115 ,g (C) were incubated in 0.125 M sucrose, 10 mM Tris-phosphate (pH 7.5),5 mM MgCl2, with 0.17 mM iso-[14C]Abu (9.1 fmol/cpm) and 50 mM NaCl (0) or 50 mM choline chloride (0) added externally at zero time. 0
5
10
Biochemistry:
Proc. Natl. Acad. Sci. USA 73 (1976)
Lever
5-
w
In-._
,0
z Z
I I
0E o
5-
Z
0_
I
0f
wc z
(1/iso-Abm CONCENTRATION)
mM 1
I-
FIG. 5. Concentration dependence of the initial rate of Na+dependent iso-Abu uptake. Vesicles from subconfluent (0) Balb/c tertiary mouse embryo fibroblasts, or Balb/c SV40-transformed 3T3 cells (0) were incubated 30 s and 1 min in incubation mixtures as described in Fig. 4, with various concentrations of iso-[14C]Abu at 0.061 pmol/cpm added at zero time. The rate of Na+-dependent uptake was obtained by subtraction of the rate in 50 mM choline chloride from that in 50 mM NaCl, added externally at zero time.
DISCUSSION Assay of vectorial transport activity of vesicles from membranes isolated from mammalian cells in culture provides a system for characterizing functional plasma membrane alterations associated with hormones (22) and cellular proliferative and transformed state in terms of specific parameters related to the transport mechanism. This approach, first applied by Kaback and Stadtman (23) to study transport in bacterial membranes, permits dissociation from intracellular and topographical factors and assay of transport function across compartments of defined composition. The experiments reported here provide evidence for a Na+-
gradient-stimulated, electrogenic mechanism of iso-Abu uptake (24) in membranes from untransformed and SV40-transformed mouse fibroblasts. Observations that iso-Abu uptake against its concentration gradient could be driven by transmembrane Na+ gradients imposed independently of (Na+ + K+)ATPase activity, and that flux of iso-Abu in either direction across the membrane was decreased by Na+ added to the opposite side, support a type of Na+-gradient-coupled mechanism proposed by Crane (25). In this model, the carrier has increased affinity for the solute in the presence of Na+, and concentrative solute uptake is driven by an electrochemical Na+ gradient, external Na+ > internal Na+. In the intact cell, this Na+ gradient is maintained by the (Na+ + K+)ATPase. Observations that Na+driven iso-Abu uptake was enhanced under conditions expected to create an interior-negative membrane potential, such as a K+ diffusion potential, internal K+ > external K+, in the presence of valinomycin, or by using the Na+ salts of highly permeant anions such as SCN- and NO3- provided evidence that the iso-Abu carrier complex moves in cotransport with a cation, but without charge compensation via the same carrier. Thus, among the possibilities for cellular control of iso-Abu carrier function, are regulation of (i) theactiveNa+ pump; (ii) membrane permeability to Na+; (ii) electrogenic membrane processes or membrane depolarization affecting membrane potential; (iv) modification of the Na+-dependent and Na+-
2617
independent affinity for iso-Abu; (v) number of iso-Abu carriers; and (vi) carrier mobility. Most of these alternatives can be distinguished using membrane vesicles. Since iso-Abu uptake changes similar to those observed in intact cells with growth or transformed state (10, 11) were detected in their isolated vesicles when transport was driven by a standard Na+ gradient, these alterations occur independently of regulation of the Na+ pump. Membrane Na+ permeability or carrier response to Na+ could change with growth or transformed state, since membranes from these various cell types showed differences in steady-state accumulation of iso-Abu driven by a Na+ gradient. Although presumably dissipation of the Na+ gradient occurs during the incubation, no efflux of accumulated iso-Abu was observed during a 15-min interval. Since changes in iso-Abu uptake stimulated by Na+ gradient were expressed as changes in Vmax for iso-Abu uptake, with minimal variations in Km, possibly alteration of the number or the mobility of iso-Abu carriers resulted in another component of the observed uptake differences between the cell populations in this comparison. This does not rule out other mechanisms of carrier regulation listed above in more rapid and transient responses to hormonal or metabolic regulation. I thank Dr. R. Dulbecco for generous support and encouragement, N. Neyhard and Mrs. P. Pettican for expert technical assistance, the Cell Production Unit for cell cultures, and C. Dixon and J. Elkington for drawing the graphs. This work was carried out during tenure of a research training fellowship awarded by the International Agency for Research on Cancer and a United States National Institutes of Health Research Service Award CA05174-01 from the National Cancer Institute. 1. Pardee, A. B. & Rozengurt, E. (1974) in Biochemistry of Cell Walls and Membranes, ed. Fox, C. F. (Medical and Technical Publishing Co., London), pp. 155-185. 2. Pardee, A. B. (1974) Proc. Natl. Acad. Sci. USA 71, 12861290. 3. Holley, R. W. (1972) Proc. Natl. Acad. Sci. USA 69, 28402841. 4. Elligsen, J. D., Thompson, J. E., Frey, H. E. & Kruuv, J. (1974) Exp. Cell Res. 87, 233-240. 5. Rozengurt, E. & Heppel, L. A. (1975) Proc. Natl. Acad. Sci. USA 72,4492-4495. 6. Kimelberg, H. K. & Mayhew, E. (1975) J. Biol. Chem. 250, 100-104. 7. Schultz, S. G. & Curran, P. F. (1970) Physiol. Rev. 50, 637718. 8. Sigrist-Nelson, K., Murer, H. & Hopfer, U. (1975) J. Biol. Chem. 250,5674-5680. 9. Columbini, M. & Johnstone, R. M. (1974) J. Membr. Biol. 18, 315-334. 10. Foster, D. 0. & Pardee, A. B. (1969) J. Biol. Chem. 224, 26752681. 11. Isselbacher, K. J. (1972) Proc. Natl. Acad. Sci. USA 69, 585589. 12. House, W., Shearer, M. & Maroudas, N. G. (1972) Exp. Cell Res.
71,293-296. 13. Todaro, G. J., Lazar, G. K. & Green, H. (1965) J. Cell. Physiol. 66,325-334. 14. Hochstadt, J., Quinlan, D. C., Rader, R. L., Li, C. C. & Dowd, D. (1974) in Methods in Membrane Biology, ed. Korn, E. (Plenum Press, New York), Vol. 5, pp. 117-162. 15. Avruch, J. & Wallach, D. F. H. (1971) Biochim. Biophys. Acta
233,334-347. 16. Mackler, B., Collip, P. J., Duncan, H. M., Ras, N. A. & Huennekens, F. M. (1962) J. Biol. Chem. 237, 2968-2974. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193,265-275. 18. Hirata, H., Altendorf, K. H. & Harold, F. M. (1973) Proc. Natl. Acad. Sci. USA 70, 1804-1808.
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19. Gamble, J. G. & Lehninger, A. L. (1973) J. Biol. Chem. 248, 610-618. 20. Cunningham, D. D. & Pardee, A. B. (1969) Proc. Natl. Acad. Sca. USA 64, 1049-1056. 21. Quinlan, D. C. & Hochstadt, J. (1974) Proc. NatI. Acad. Sci. USA
71,5000-5003.
Proc. Natl. Acad. Sci. USA 73 (1976) 22. Holley, R. W. (1975) Nature 258,487-490. 23. Kaback, H. R. & Stadtman, E. R. (1966) Proc. Nati. Acad. Sci. USA 55, 920-927. 24. Inui, Y. & Christensen, H. N. (1966) J. Gen. Physiol. 50, 203224. 25. Crane, R. K. (1965) Fed. Proc. 24, 1000-1006.
