The Isolation and Characterization of Plasma Membranes from Cultured Cells CHICK

SUMMARY Cultured chick embryo fibroblasts accumulate Ca2+ in the presence of Mg2+ and ATP. The uptake is highly specific; Mn2+ inhibits it, and other nucleotide triphosphates are with- out effect. The presence of oxalate, Na+ and/or K+ in- creases the amount of accumulated Ca2+. The cation trans- port is inhibited by mersalate, oligomycin, and hydroxyl- amine. The capacity of fibroblasts to energetically transport and/or bind Ca2+ resides in components of the plasma mem- brane. The function of this Ca2+ uptake may be to control motility in ameboid-like cells. The ionic interaction of Ca2+ with biopolymers such as pectin and mucopolysaccharides, having negative charges (I), and with lipids (2, 3) leads to changes in the physical properties of these molecules. An understanding of the physicochemical changes in these defined systems has suggested insights into the effects Cat+ has on the cell membrane in permeability to ions, water (4), and proteins (5), in nerve transmission (B), in intercellular communication (7), in membrane hardening (4), and in cell adhesion (8, 9). It is thought that Ca2+ enters the cell by an initial binding to the cell surface, followed by a passive influx into the intracellular compartment (10, 11). The low (low5

brane preparations have been reported to bind Ca2+ in the presence of ATP.
These membranes were obtained from the peripheral nerve of the walking leg of the crab (18), the kidney cortex of the rabbit (lQ), and human blood platelets (20).
Cultured chick embryo fibroblasts contain microfilaments, w-80 A in diameter, which have Mg2+-and Ca2f-dependent ATPase activity. ' These microfilaments extend into membrane extensions and constitute the main structural element in the pseudopodia.
A protein having conformational changing properties can be prepared from these cells.2 It was decided to examine the fibroblasts for systems which might transport and regulate the concentration of Ca2+ in a manner analogous to that of the sarcoplasmic reticulum of muscle. These studies indicate that chick embryo fibroblasts can energetically take up Ca2+, and this property is associated with the plasma membrane isolated from these cells. EXPERIMENTAL PROCEDURE Culturing, Harvesting of Cells, and Isolation of Plasma Membrane-primary cultures of chick embryo fibroblasts were prepared from 13-day-old embryonated eggs and cultured at 37" in a humidified CO2 incubator in Eagle's minimal essential medium containing 4% fetal calf serum, according to the method of Temin (21). Five days later, 4 x lo6 cells were transferred to Falcon tissue culture dishes (150 x 15 mm).
These cells became confluent in 3 to 4 days and were transferred once again under the above conditions. Confluent,, tertiary cultures were used for these studies. These cultures consisted of relabively homogeneous populations of spindle-shaped fibroblasts, since other cell types did not survive these cult.ure conditions (22). A uniform suspension of cells was prepared by incubation wit,h 0.1 'i; trypsin in isotonic Tris for 3 min at 37" and scraping with a rubber policeman.
The released cells were centrifuged, washed three times, and resuspended in 0.25 M sucrose. Approximately 90.5 =t 1.3y0 of the cells were viable, as evidenced by permeation measurements with trypan blue in 14 experiments.
The plasma membrane (the A and B bands) and intracellular membranes and organelles (the C and D bands) were prepared by homogenization of a suspension of the cells in 0.16 M NaCI, sedimentation, resuspension of the resulting pellet in sucrose to a final concentration of 6Syc (w/v), and flotation equilibrium centrifugation of the particulate material on a linear 25 to 65% (w/v) sucrose-in-water gradient (23). The layers A through D were removed, sedimented, and the pellets resuspended in 0.25 M sucrose.
Measurement of Cu2f Accumulation-Cultured cells or membrane fractions at a protein concentration of 65 to 150 pg were incubated in medium containing 5 mM buffer,usually Bicine [iV,glycine] at pH 8.4, 3 or 6 m&f MgC12, 20 to 200 PM Nazi-(specific activity 3.6 PCi per pmole), sucrose and water to a final volume of 1.0 ml and 250 mosM; f 3 mM ATP.
The transport studies were carried out for varying lengths of time at 24" unless designated differently in the legends to the tables or figures.
Following incubation, the sample suspension was filtered under vacuum through 13 mm, type HA Millipore filters with an average pore diameter of 0.45 pm (24). The particulate material on the filter was rinsed with 10 ml of 0.16 M NaCl, the washed filter placed in 10 ml of Scintisol (Isolab Inc.), and the radioactivity measured in a Packard Tri-Carb scintillation counter. Preparation and Measurement of Binding of y-labeled ["PI-ATP-ATP with the y position labeled with 32P was prepared according to the procedure of Glynn and Chappell (25), and the ATP was separated from inorganic a21'Pi 011 a Dowes l-chloride column (26). The ATP prepared by this method had a specific acti&)-of 214 $Zi per hmole. The position of the 32P was determined by a i-min hydrolysis in 1 N I-ICI, incubation with rabbit muscle myosin, and paper chromatography (27) in isobutyric acid, NHdOH, EDTA, and II?0 (100:4.2:1.6:55.8).
On the basis of these results, 97% of the label was in the terminal phosphate position.
The labeled ATP was incubated with intact cells, the cells filtered, and the radioactivity determined as previously described for measuring Ca*+ uptake.
Chemical Determinations-Protein was measured by the method of Lowry et al. (28). The ATPase activity (3.6.1. .3) of the intact cells was determined in the same medium and at the substrate and ion concentrations used to measure Ca*f binding (29). The quantity of Pi released from this substrate was determined by the procedure of Martin and Doty (30) as modified by Lindberg and Ernster (31). All reagents used were of analytical reagent grade. The following were purchased from the Sigma Company: the sodium and Tris salts of ATP, CTP, GTP, ITP,

Initial determinations
of the amount of CaX+ bound to isolated membrane fractions were carried out in a KC1 medium. This medium was used to measure Ca*+ uptake in muscle sarcoplasmic reticulum (24). A small but variable quantity of Cazf was bound to the fibioblast membranes following the addition of ATP.
However, more Ca"+ was bound to membranes incubated in an isotonic sucrose medium than was bound to membranes incubated in the salt medium.
This latter medium was used in studies measuring the nucleotide phosphohydrolase activity of intact fibroblasts (29) and was the medium of choice in subsequent Ca2+ transport experiments. Filtration Method for Measurement of Ca2f Accumulation-Filtration under vacuum has been used to measure Ca2f uptake in the sarcoplasmic reticulum (24) and was the method employed in this study.
With this procedure, it was possible to break intact cells during filtration and release their contents, including Ca2f, into the washing medium.
To test for this possibility, cell cultures were incubated for 2 hours in medium containing 2-[1J4C]deoxyglucose; this sugar is transported and phosphorylated, but it is not metabolized (32). The cells were released by trypsin and incubated for 30 min in the isotonic sucrose medium ( Table I). Some of the incubated cells were centrifuged and washed by resuspension and sedimentation.
An equal number of cells were filtered and washed under vacuum.
The difference in counts associated with the filtered cells when compared with those found in the sedimented cells indicates that about 15y0 of the radioactivity of the cells was lost during the filtration process. Accumulation of Ca*+ as Function of Cell Protein Concentration-uniform suspensions of chick fibroblasts passively accumulate Ca2+ in proportion to the cell protein concentration (Fig. 1). In the presence of 3 mM ATP there was a,n increased accumulation of Ca2f. This additional uptake of cation was linear with protein concentration up to about 200 pg. Cells at, concentrations of 50 to 150 pg of protein per assay were used in all other experiments.
Ca2f Accumulation in Heated Cells-.The Ca2f that was accumulated by the fibroblasts in the presence of ATP was lost by heating the cells prior to filtration (Table II). The fibroblasts which were incubated in the absence of ATP and then made permeable by heating also lost CaZf. During this 30.min incubation period, there was no significant increase in the number of dye-permeable cells. Cells which had been released by trypsin,  to trypan blue. Following incubation, approximately 11% of the cells were permeable to the dye. Ca2f in the preseuce of ATP, did not bind to heat-denatured cells (Table II).

EJect of Divalent Cations
on Ca zf Accumu.Zation-In the absence of exogenous n4,g+, a small quantity of Ca2f was taken up by the cells when they were incubated with ATP ( Fig. 2). The addition of Mg 2+ to the medium resulted in a linear increase in the amount of Ca2f accumulated by the cells up to about 6 IYLM. A ratio of ATP :X!Igz+ of 1:2 appears to be optimum for the Ca2+ accumula,tion; a ra.tio of 1:l had previously been found to be optimum for Mg2+-dependent ATPase activity (29). Cardiac muscle microsomes took up Ca2+ at an optimum ATP:lVg2+ ratio of 1: 1 with no increase in cation accumulation occurring at higher ratios (33). Studies with skeletal muscle sarcoplasmic reticulum as well as with membranes from noncontractile sources used Mg2+ in a ratio of 1: 1 with ATP (16, 18-20, 24). Manganese, in contrast, to its observed stimulation of Ca2f transport in muscle reticulum (24, 33), inhibited the uptake of Ca2f in chick fibroblasts (Fig. 2).

EJect
of Nucleotide Phosphate Substrates on Ca2f Accumulation--In the presence of 3 and 6 nlM XIg2+, t,he addition of ATP produced an increased accumulation of Ca2f by t,he cells up to a concentration of 3 m&f (Fig. 3). Higher levels of substrate resulted in no greater amounts of transported Ca*f. Adenosine triphosphate ivas the only nucleotide phosphate substrate which could promote Ca2f transport by fibroblasts (Table III).
The ot.her nucleotide triphosphates, t#wo of which (CTP and UTP) are substrates for phosphat,ases localized specifically on the plasma membrane (29), were unable to promote Ca2+ accumulation by these cells. AMP and ADP, the latter also a substrate for a plasma membrane phosphohydrolase (29), similarly could not increase the amount of Ca2f accumulated by the fibroblasts. This high degree of substrate specificity for the energized-uptake extrusion in resealed red blood cell ghosts (36). However, membrane fragments prepared from red blood cells have the same substrate specificity for Ca 2f binding as was found for fibroblasts (37). Effect of Ca2f Concentrntion on Caz+ Accumulation-The maximum amount of ATP-dependent uptake of Ca"f occurred at a medium concentration of 200 pM CaZf (Fig. 4). Higher levels of medium Ca2f resulted in more accumulation by the cells, but there was no proportional increase in this uptake following the addition of ATP.
A similar plateau in energized uptake can be seen in the data of Palmer and Posey (19). A Ca2+ concentration of 20 pM was used in most of the experiments, since 15% of this label was accumulated by the cells, in contrast to a 3% incorporation of cation at a medium concentration of 200 MM. With an extracellular Ca2+ concentration of 20 pM, the masimum quantity of Ca2f to be taken up was about 12 nmoles per mg of protein (Fig. 7). This level of transport was 1 to 2(r, of the level of transport observed for preparations of skeletal muscle sarcoplasmic reticulum and was similar to the level of Ca2f uptake reported for cardiac muscle reticulum incubated in the absence of oxalate (38).
Accumulation of Ca2+ as Function of pH-The ATP-dependent uptake of Ca2+ occurred over the range of pH values studied from 6.5 to 8.7 (Fig. 5). The maximum accumulation was at pH 7.0. However, a pH of 8.4 was used in many of these studies, since preliminary experiments indicated no significant differences between pH 7.0 and 8.4. B pH of 8.0 was found to be optimum for the hydrolysis of Mgz+ATP stimulated by Na+ + K+ in intact fibroblasts (29). lation of Ca2+ by intact fibroblasts (Fig. 6). At a concentration of 8 InM oxalate, there was a 30% increase in the quantity of Ca2+ accumulated by these cells. However, this enhancement of Cazf uptake was variable, as indicated in Figs. 7 and 8. Oxalate potentiates, markedly, the amount of Ca2+ accumulated by the reticulum, but not that of the mitochondria from muscle (24,33), and appears to be transported stoichiometrically as the diffusible counterion with the divalent cation (24). Intracellular and cell membrane preparations from blood platelets (20), red blood cells (37), nerve (18), and the parotid and submaxillary glands (34) accumulated additional Ca2f in the presence of oxalate; exceptions to these observations were membranes prepared from the renal cortex of the rabbit (19) and brain microsomes (35).
Accumulation of Ca2f a.s Function of Temperatureand Iodoacetate -Chick embryo fibroblasts incubated with ATP or ATP and oxalate accumulated maximum levels of Ca2+ at 20" to 24" (Fig. 7). At higher incubation temperatures the amount of Ca2+ taken up by the cells decreased; this decrease was greater for fibroblasts incubated in medium containing oxalate.
A possible explanation for the decreased Ca2f accumulat,ion at higher incubation temperatures has its origin in the observations of Schatzmann and Vincenzi (12) and Lee and Shin (36) that red blood cell ghosts can energetically extrude Ca2+. Chick fibroblasts incubated at the higher temperatures, 30" and 37", could make ATP through intermediary metabolism, and this substrate would then be used by the cells to extrude the Ca2f accumulated in the presence of extracellular ATP.
If a Ca2f extrusion mechanism is operating, one would expect the Ca2f accumulation to be higher in cells incubated at 37" in the presence of metabolic inhibitors than in untreated fibroblasts.
This was not found to be t,rue. Fibroblasts cultured for 2 hours with 100 ptil iodoacetate, harvested, and incubated in the Ca2f-transport medium which, in some experiments, contained additional iodoacetate (100 P&X) accumulated less cation at 37" than at 15" when compared with untreated cells (Table IV).
The iodoacet,ate-treated cells and cells incubated with additional inhibitors accumulated less Ca2f. This inhibition was particularly evident for cells incubated at 379 Another explanation of the decrease in Ca2f accumulation at The release of 30" and 37" could be that the solubility of Ca2f-oxalate or of Ca2f with an unknown anion may be higher at the elevated temperatures, with a resulting exchange of the cation and extracellular ions. This temperature-binding relationship was offered as an explanation for Ca2f accumulation in muscle reticulum (24).
Accumulation of Ca2f as Function of Time-Intact cells took up significant quantities of Ca2f during 1 min of incubation. This uptake remained linear for about 8 min for fibroblasts incubated with ATP, and for 16 min for cells incubated with ATP and oxalate (Fig. 8). The maximum accumulation of Ca2+ occurred at 30 min of incubation.
Incubation for longer periods of time resulted in lower levels of cation accumulation, with a plateau being reached by 90 min.
Leakage of Accumulated 45Ca2+-Much of the Ca2f that was taken up by the fibroblasts during 30 min of incubation was lost from these cells when they were incubated for an additional 30 min in medium minus ATP or Ca2f (Fig. 9). There was little net loss of Wa2f when these cells were incubated in 45Ca2+-and ATPcontaining medium, and a significant quantity of labeled cation was retained when the cells were reincubated with this Ca*+ alone. The loss or turnover of accumulated 45Ca2+ was not increased by the addition of @Ca2+ to the medium. ATP, with or without Waz+, promoted some retention of 45Ca2+ within these cells.
Efect of Na+ and/or K+ in the incubation medium at concentrations which are optimum for the stimulated hydrolysis of ATP (29) resulted in a significant increase in the quantity of Ca2+ taken up by the cells (Table V). A previous observation noted enhanced Ca2f binding following the addition of K+ to isolated renal membranes (19), but Naf had been reported to cause a displacement of bound Ca2f in cardiac sarcoplasmic reticulum (38) and to inhibit the binding of the divalent cation in nerve membranes (18). Effect of Inhibitors en Ca2+ Accumulation-The enhanced Ca2f uptake by Naf and K+ was decreased slightly by ouabain, a cardiac glycoside and inhibitor of cation transport and the Naf + Kf-stimulated Mg2+ATPase (39), but the effect of this steroid was not consistent (Table V and Fig. 10). Valinomycin, a cyclic polypeptide which stimulates respiration and Kf transport in mitochondria (40), at a level of 1 pg per ml, decreased the amount of Ca2+ accumulated by the intact cells (Table V and Fig. 10). At lower concentrations of valinomycin there was no inhibition. The ATP-dependent accumulation of Ca2+ was inhibited completely by oligomycin at concentrations of 1.0 and 0.1 pg per ml (Fig. 10) ; this antibiotic also inhibits ion transport in mitochondria (41) and Naf-activated -4TPase in erythrocyte membranes (42)  but rather result from the phosphorylation of components present in the membrane, with the concomitant binding of Ca2f by these components.
An example of such a system is the phosphorylation of phosphatidylinositol.
A kinase, localized in the plasma membrane (46)) phosphorylates phosphatidylinositol to diphosphatidylinositol, which can subsequently be phosphorylated to triphosphatidylinositol.
The phosphoinositides have a strong affinity for Ca2+ (47). The feasibility of such a Ca2+ accumulation mechanism was examined by incubating cells with ATP for 8, 16, 30, and 60 min and adding Ca2f 2 and 6 min before the termination of these incubations.

Conditions of incubation
It could be anticipated that the longer the period of incubation with ATP prior to the addition of Ca2f, the greater would be the phosphorylation and the greater would be the subsequent cation binding.
This, however, did not occur. In a control series, with Ca2f and ATP incubated together, the maximum accumulation of cation occurred by 16 min of incubation and decreased slightly at 30 and 60 min (Fig. 11). When Ca*+ was added at 2 and 6 min before the termination of incubation, an amount was bound which was proportional to the period the cation was present with the cells and substrate.
The delayed addition of Ca*+ to cells incubated for 16 min resulted in no greater uptake than when the cation was added to cells incubated for 8 min and by 30 min of incubation significantly less Ca2+ was taken up. Effect of Hydrozylamine on Ca %+ A ccumulation,-The inhibit,ion by hydrosylamine of membrane phosphorylation (48, 49) and Cazf uptake (48) in muscle reticulum implicates the formation of an acid-stable acylphosphorylated intermediat'e in cation transport.
The ATP-dependent binding of Ca*+ by chick embryo fibroblasts was also inhibited by hydroxylamine at 0.1 M concentrations (Table  VIII). The level of Ca*+ uptake in the presence of higher concentrations of hydroxylamine was even lower than in unt>reated cells incubated in the absence of ATP.
Accumulation of Ca*+ by Cells Which Have Been Incubated with A TP Prior to Addition of Cation-The accumulation of Ca2f by fibroblasts in the presence of ATP may not involve cation transport functions analogous to that of the muscle reticulum EJect of Trypsin and EDTA on Ca2+ Bindin.g-Studies by Burger and Goldberg (50), Burger (51), and Inbar and Sachs (52), as determined with glycoproteins which bind to specific carbohydrates, established that the cell membrane has carbohydrate sites which are ordinarily not exposed for agglutination but become available when the cells are transformed by chemicals or viruses. This additional binding in the transformed cell is not caused by the synthesis of new carbohydrate; these sit,es are present but masked in the normal cells. Treatment with trypsin Ca2+ accumulation by cells released with EDT-J, trypsin, or by scraping Chick embryo fibroblasts were relezsed from culture dishes by a 10.min incubation at 24" with phosphate-buffered, pH 7.3, EDTA (0.02% in saline) ; by a 3.min incubation at 37" with 0.17, trypsin in isotonic Tris, or by scraping.
The released cells were washed twice in 0.25 M sucrose and incubated for 30 min et 24" inGmMMg 2+. 20 PM Ca2+;5 mM Bicine,pH 8.4;  for va,rying periods of incubation makes these sites accessible to the specific agglutinin. It was possible that the ATP-dependent binding of Cai+ by the fibroblasts resulted from a modification of the cell membrane by the trypsin used to release the cells from the culture dishes. To test this possibility, cells were released with phosphate-buffered EDTA, trypsin, or by scraping, and the Ca2+-binding activities of these preparations were determined. Cells released by EDTA or trypsin were equally as capable of accumulating Ca2+ following the addition of ATP as were cells obtained by scraping (Table  IX).

Binding of Ca 2f by Attached
Cells-Another check on the possibility that the accumulation of Ca2+ resulted from the release of cells was to study this binding with attached cells. Fibroblasts were grown on culture dishes 60 mm in diameter, and 1 to 5 days after plating the Ca2f uptake was determined with and without ATP.
Attached cells accumulat,ed Ca2+ like suspended cells following the addition of ATP (Fig. 12), but this uptake was agedependent.
Cells cultured for 1 to 3 days took up more Cazf than did those cultured for 4 and 5 days. This changing capacity to accumulate Ca'+ Ivas not correlated with cell grzsth, which was linear for 4 days. Fractions-Plasma membrane from cultured chick fibroblasts was isolated within two bands of differing density, the A and B, following flotation equilibrium centrifugation (23). Band A was the purest cell m.embrane preparation, whereas the membranes found in the denser B band were contaminated with intracellular membranes. The membranes in bands A and 13 bound 1.9 nmoles of Ca2+ per mg of protein in the absence of ATP (Table X).
This quantity of bound Ca2+ may be due to a complexing of the cation with sialic acid, which is concentrated in these two fractions.
Additional Caz+ was accumulated by the A and B baud membrane preparations following the addition of ATP.
The C band, containing mitochondria and intracellular membranes, took up about one-fourth the amount of Ca2f accumulated by the A band membranes.
Band D, a composite of two RNA-rich bands and a DNA-containing pellet, had less bound Ca2f following the addition of ATP. The accumulation of Cat+ by fibroblasts required ATP and Mg2+, appeared to be associated with components of the cell membrane, and could be carried out by attached cells as well as by cells released from the culture dish with trypsin, EDTA, or by scraping.
The ATP-dependent Ca2+ uptake was the result of cation transport.
Cells which have been made permeable by heating lose their accumulated Ca 2+ during filtration and washing. The majority of this transported Ca2+ must be in the unbound form, since only small quantities of cation remained after this treatment (Table I). These experiments also eliminate the possibility that damaged cells were the ones which accumulated the Ca2+. The method of filtration under vacuum would in all probability have forced Ca2+ out of "leaky" cells: The permeation studies with trypan blue also indicated that the number of dye-permeable cells did not increase during the incubation period.
The magnitude of Ca2+ accumulation was small, 1 to 25;) when compared with skeletal muscle reticulum (14,15,24,44), but it was similar to the reported values for Ca"+ uptake in some prep arations of cardiac muscle reticulum incubated in the absence of oxalate (38). In the presence of osalate, heart reticulum accumulated about 400 pmoles of Ca2+ per mg of protein (33, 43), whereas the fibroblasts took up 16 to 20 nmoles of Ca2f per mg of protein (Figs. 7 and 8). Membranes derived from blood platelets (20) and those considered to be from the golgi of rat parotid and submaxillary glands (34) transported levels of Ca2+ which are comparable to that of muscle reticulum.
Microsomes from rabbit kidney cortex (19) and crab axons (18) accumulated about the same amount of Ca*f as those from chick fibroblasts.
The requirements for the uptake of Ca2+ by fibroblasts were more specific than had been reported for its accumulation by the reticulum of muscle. Both preparations required Mg2f. Ilowever, in contrast to muscle membranes, Mn2f could not substitute for Mg2+ in carrying out the energy-dependent uptake by fibroblasts. The nucleotide substrate requirement for cation accumulation was also specific. Adenosine triphosphate was the only substrate which would promote Ca2+ transport in fibroblasts even though enzymes which hydrolyze CTP were concentrated in the Ca2f Accumulation by Fibroblasts Vol. 246,No. 22 plasma membrane (23, 29). Osalate, which had a marked effect on Ca2+ accumulation in muscle preparations, produced a smaller accumulation of cations in the chick cells. Both membrane preparations are inhibited by mercurials which react with -SH groups, but the chick fibroblasts were unique in the inhibition of Ca2+ transport produced by oligomycin.
An inhibition by oligomycin of the dTl'-dependent Ca2+ translocation in mitochondria (41) and of the Ca2f accumulation in fibroblasts infers that, in the plasma membrane, the generation of high energy intermediates may be an essential requirement for cation uptake.
The synthesis of phosphorylated intermediates as a step in Ca 2f transport bv the reticulum of muscle is a generally accepted concept (14,15,44,48,49,53). In this regard, the inhibition of Ca2f uptake in chick fibroblasts by hydroxylamine was not unlike the observations reported by Martonosi (48) for sarcoplasmic reticulum and suggests that a similar Ca2f-transport mechanism may be functioning in the cultured cells. However, possibly because of t,he much smaller levels of Ca2f binding, t,he "extra" splitting of AT1 (14,15,44) and a change in the ratio of bound i4C-and 32P-labeled -Vl'P (19) following the addition of cation did not occur. These effects, observed in muscle reticulum (14) and in the membrane preparations from kidney cortex (19), have been considered as evidence for a Ca2f-binding mechanism which requires the phosphorylation of carrier protein.
In this regard, the inability to bind Ca2f in heat-denatured cells supports the premise that cation accumulation requires an energized utilization of ATP rather than the binding of Ca2+ as a chelate with XTP to a membrane site. Experiments in which Cazf was added to t,he cells at various intervals following incubation with RTP also have established that cation accumulation is not the result of randomized membrane phosphorylation and the concomitant Ca2f binding at these sites. Ca2+ must be at the binding sites in the presence of Mg*+ and ATP if the cell is to energetically take it up.
The reason for the decrease in cation binding at 30" and 37" and after periods of incubation longer than 30 min has not been determined.
An energy-requiring Ca2+ extrusion mechanism, like that which is operable in red blood cells (12, 36), does not appear to be the reason, nor can changes in the capacity to bind Ca2f be related to digestion of cell membrane components by residual trypsin (data not presented).
The experiments in which fibroblasts accumulated Ca2f during 30 min of incubation and were subsequently incubated for an additional 30 min in media of different compositions indicated that Ca2f was transported to a maximal level and reached a steady state within the initial incubation (Fig. 9). In the presence of ATP and labeled cation, the net accumulation remained unchanged during the next 30 min. In the absence of XTP and dsCa2+ there was a marked loss of cation.
That an exchange mechanism was operating is suggested by the levels of isotope which remained with the cells when they were incubated again with 'isCa2+. However, it was anticipated that the addition of unlabeled Ca2f to the medium would have led to the ret,entiou of even less b5Ca2+; this was not observed.
If the determinations had been made at earlier periods, it is possible that an increased rate of cation exchange might have been observed.
The capacity of chick fibroblasts to energetically take up Ca2+ from an external medium contrasts with the observations of the passive entrance of this cation and probable energy-dependent extrusion in red blood cells (12, 36), in cultured cells (10,11,54) and in nerve cells (10). It is conceivable that the binding and subsequent uptake of Ca 2+ by these cells and the fibroblasts is associated with sites, localized within special regions of the cell membrane, which are in equilibrium with cations in the internal and external media. Under normal physiological conditions, the Ca2f concentrations in the external medium are much higher than t.he free Ca2+ concentration in the intracellular compartment (10). The energy source, ATP, in the extracellular medium is low. Much higher concentrations of ATP'are found in the intracellular compartment, and in this situation Ca* extrusion occurs to maintain the physiological concentration of cation. Under the conditions of this study, the external Ca2+ concentration of 20 ~L-"I was not greatly different from the internal Ca2+ levels reported for some cells including those of nerve (10). Furthermore, the detachment of the fibroblasts with trypsin and EDTA results in the loss of the majority of the Ca2+ of the cell (54), increasing even further the difference between intracellular and extracellular cation concentrations. The resultant low intracellular Ca2+ level and the high external ATP concentration could initiate the binding of cation to the plasma membrane and its accumulation by the cell.
The presence of Ca2f transport sites with the plasma membrane of fibroblasts could function to maintain critical intracellular levels of this ion, a recently recognized requirement for growth control and cell division which is lost in some virus-transformed cells (55). The maintenance of a spindle shape and motility also requires Ca"+ (56). These cells are ameboid in their movements, they contain microfilaments which have MgZ+-and Cat+-stimulated ATPase activity (57), they can exert tensions of about 2 to 3.4 x lo4 dynes per cm2 (58), and a contractile protein has been isolated from them which has properties similar to those of rabbit muscle actin.
If motility and shape changes are the result of contractile-like activity by cytoplasmic elements, then energized Ca2f binding and uptake by the plasma membrane may represent one facet of the control of those cellular activities.
Aclmowledgments-I wish to express my appreciation for the assistance and comments of Drs. E. Briskey, I. Riegel, and A. Springer in the preparation of this manuscript. The technical assistance of K. Miller and M. Ponosh is gratefully acknowledged.