the journal of biological chemistry val. 256, no. 12. …€¦ · the journal of biological...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 256, No. 12. I.sssue of dune 25, pp 6148-6154, 1981 Printed in U. S. A.

Calcium Transport and Calcium-ATPase Activity in Human Lymphocyte Plasma Membrane Vesicles*

(Received for publication, February 17, 1981, and in revised form, March 24, 1981)

Andrew H. Lichtmant, George B. Segelg, and Marshall A. Lichtman From the Department of Radiation Biology and Biophysics, the Department of Pediatrics, and the Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York I4642

We have studied Ca transport and the Ca-activated Mg-ATPase in plasma membrane vesicles prepared from normal human lymphocytes. Membrane vesicles that were exposed to oxalate as a Ca-trapping agent accumulated Ca in the presence of M g ’ and ATP. ADP, AMP, GTP, UTP, ITP, ‘ITP, or CTP did not substitute for ATP in energizing uptake. The VmaX for Ca uptake was 2.4 pmol of Ca/pg of protein/min, and the K, values for Ca and ATP were 1.0 and 80 ,UM, respectively. One p~ A23187, added initially, completely inhibited net Ca uptake and, if added later, caused the release of Ca accumulated previously. Cyanide, oligomycin, ouabain, or varying Na’ or K’ concentrations had no effect on Ca uptake. A Ca-activated ATPase was present in the same membrane vesicles, which had a V,, of 25 pmol of Pi/pg of protein/min at a free Ca concentration of 4-5 p ~ . This Ca-ATPase had K, values for Ca and ATP of 0.6 and 90 PM, respectively. These kinetic parameters were similar to those observed for uptake of Ca by the vesicles. The Ca-ATPase activity was insensitive to azide, oligomycin, ouabain, or varying Na’ or K’ concentrations. No Ca-activated hydrolysis of GTP or UTP was observed. Both Ca transport and the Ca-ATP- ase activity of ethylene glycol bis(P-aminoethyl ether)- N,N,N’,N’-tetraacetic acid-treated lymphocyte plasma membranes were stimulated 2-fold by a cytoplasmic component (calmodulin) that was purified 500-fold from lymphocyte cytoplasm. Thus, human lymphocyte plasma membranes have both a Ca transport activity and a Ca-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivi- ties to calmodulin.

Intracellular and extracellular Ca activity has been linked closely to the response of lymphocytes to antigens or plant mitogens in oitro by three key observations. First, markedly reduced extracellular Ca prevents lectin stimulation of lymphocytes (1-5). Second, ionophores for Ca added to the exter-

* This work was supported by United States Public Health Service Research Grant CA 12970, by the Ruth Estrin Goldberg Foundation, by the University of Rochester Pediatric Blood Research “Jimmy” Fund, and by a contract with the Environmental Research Develop- ment Agency at the University of Rochester Biomedical and Envi- ronmental Research Project (Report No. UR-3490-1963). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Supported by Grant GM 07356-03 from the United States Public Health Service Medical Scientist Trainee Program.

3 T o whom reprint requests should be addressed at the University of Rochester, Department of Pediatrics, Box 777,601 Elmwood Ave., Rochester, NY 14642.

nal medium can induce cell responses that mimic the response to mitogens (6-9). Third, radiocalcium uptake increases rapidly after exposure of lymphocytes to mitogens ( lO-E?) , a phenomenon which recently has been shown to represent exchange of Ca (13). A rapid extrusion of Ca presumably maintains the low intracellular free Ca in spite of the increase in Ca uptake.

Although Ca transport and a transport-related Ca-ATPase have been described in erythrocytes (14, 15), plasma membrane Ca extrusion mechanisms have not been as well characterized in nucleated ceUs. Two previous studies of lymphocyte ATPase have explored the effects of Ca. Neither study identified an enzyme with the characteristics of a physiologic Ca transport ATPase. One was an ecto-ATPase and the other had a very low affinity for Ca (16, 17).

We have studied highly purified human lymphocyte plasma membrane vesicles in order to better understand how the lymphocyte regulates its intracellular ionic content. The kinetic parameters and the substrate specifcity of Ca transport and Ca-stimulated nucleotide triphosphatase activities of the plasma membranes are described. A lymphocyte cytoplasmic component (calmodulin) has been purified and its effects on Ca-ATPase and Ca transport are presented.’

EXPERIMENTAL PROCEDURES

Preparation of Lymphocyte Plasma Membranes-Lymphocytes were prepared from the mononuclear cell-rich residues from the plateletpheresis of normal human donors (18). This method of lymphocyte procurement was essential for the harvest of sufficient human cells for preparation of plasma membranes for study. The cell suspen- sions used for each membrane preparation had approximately 3 X IO* total cells and were composed of more than 95% lymphocytes by morphology of stained smears and by the size distribution of cells measured with a Coulter electronic particle counter.

The lymphocytes were washed three times in 0.9% NaC1, resuspended in a lysis medium (1 m~ NaHCOs; 0.5 mM CaCL), and disrupted by 25 strokes in a Dounce homogenizer with a tight-fitting pestle. The lysate was centrifuged at 500 X g for 20 min, and the supernatant medium was removed and recentrifuged at 12,800 X g for 20 min. The pellet was resuspended in lysis medium plus 40% sucrose and was layered under a 30% sucrose solution. The gradient was centrifuged at 54,450 x g for 4 h. The plasma membrane fraction located just above the interface of 30-40% sucrose layers was removed and diluted with 40 mM Tris, 110 mM KC1 (Tris-KCI), pH 7.5, at 4 “C. The membrane suspension was sedimented at 45,000 X g for 1 h at 4 “C and resuspended in Tris-KC1. The protein concentration was estimated by the method of Lowry el al. (19) using bovine serum albumin standards and was adjusted to approximately 200 &mL The lymphocyte membranes were stored at -80 “C for up to 1 week prior to analysis.

These membranes showed a 30-fold enrichmeht of the plasma membrane marker, 5‘-nucleotidase, and less than 4% of the succinate

I A preliminary account of the data presented herein has been previously reported: Lichtman, A. H., Segel, G. B., and Lichtman, M. A. (1980) Fed. Proc. 39,2466.

6148

dehydrogenase specific activity found in whole cell homogenates. When examined by transmission electron microscopy, the membranes were vesicular, and nuclei or mitochondria were not identified (20).

Lymphocyte plasma membranes were also prepared so as to mini- mize the presence of bound cytoplasmic activator (calmodulin). In these preparations, MgC12 was substituted for CaCb in the lysis medium, and the lympholysate was treated with 5 mM EGTA' for 30 min at 4 "C before the membranes were separated.

Preparation of RBC Ghosts-Red cell ghosts were used to assay the potency of lymphocyte cytoplasmic activator. The ghosts were prepared by a modification of a previously described method (21). Twenty milliliters of whole blood were obtained from a normal donor by venipuncture. The red blood cells were washed 4 times in 10 volumes of 172 mM Tris-HC1 (pH 7.0 at 4 "C) by centrifugation at 1,500 X g with complete removal of the buffy coat. The cells were then lysed in 100 ml of ice-cold distilled water with constant agitation for 5 min. The membranes were pelleted by centrifugation at 15,000 X g for 30 min and the hemolysate was removed. The pelleted ghosts were washed 3 times in 1 mM EDTA, 10 m~ imidazole-HC1 (pH 7.0 at 23 "C) in order to remove endogenous calmodulin from the membranes. The EDTA buffer was removed by 1 wash in 40 mM imidazole- HC1 (pH 7.0 a t 23 "C). The ghosts were resuspended in Tris-KC1 and stored at -80 "C until use. Ghosts prepared in this way had stable Ca-activated ATPase activity for at least 2 weeks.

ATPase Activity-Lymphocyte plasma membrane and HBC ghost ATPase activities were measured in a medium containing 110 mM KCI, 40 m~ Tris/Cl, pH 7.0 at 37 "C, 20 rm NaCI, 5 mM MgCL, 100 p~ EGTA, and 125 p~ CaCL (5 p~ free Ca). Plasma membranes were added to the reaction mixture and preincubated at 37 "C in a heating block (Bronwill Scientific Co., Rochester, N.Y.) on a shaking platform. In some experiments, the membranes were preincubated in the presence of 10 pg/ml of calmodulin for 30 min at 4 "C before transfer to the heating block. After 10 min of preincubation at 37 "C, the reaction was started by the addition of [y-"P]ATP (0.4 pCi/pmol), to achieve a final concentration of 5 mM ATP. The radiolabeled ATP stock solution was prepared in Tris-KC1 buffer that was adjusted so that the final reaction mixture pH was 7.0. The reaction was stopped after 20 min by transferring each 0.25-ml sample to melting ice and adding 0.4 ml of 10% trichloroacetic acid. The samples were then sedimented at 500 X g for 10 min and 0.2 ml of the supernate was added to 0.4 ml of 2.5% ammonium molybdate in 0.7 N HzSO,. Butyl acetate (0.8 ml) was added and the mixture was mixed thoroughly and sedimented for 10 min at 500 X g. An aliquot (0.4 ml) of the top organic layer containing extracted inorganic phosphate was transferred to 10 ml of Bray's scintillation fluid, and the /3 radioactivity from "P was determined in a Packard Tri-Carb P-spectrophotometer (20).

The ATPase activity is expressed as picomoles of P,/pg of protein/ min. All ATPase values were corrected for P, release measured in the absence of plasma membranes. Basal activity was determined in the presence of 10 p~ ouabain and 100 p~ EGTA without added Mg or Ca. Mg-ATPase activity was measured as the increment in P, liber- ated upon addition of 5 mM Mg. Ca-activated Mg-ATPase was calculated as the increment in P, above the Mg-ATPase activity upon the addition of Ca. Maximal Ca-ATPase activity was observed at a total Ca concentration of 125 p ~ , which provided 5 PM free Ca in this assay.

The (Na,K)-ATPase activity was measured as the ouabain-sensitive portion of the ATPase in the presence of 5 mM Mg, 30 mM Na, 110 mM KC1, and 100 p~ EGTA without added Ca. This was calculated as the difference in P, release in the absence and presence of 10 p~ ouabain. The ouabain-sensitive ATPase is identical with the (Na,K)-activated ATPase under these conditions (20).

CaZrium Uptake-Ca uptake by lymphocyte plasma membrane vesicles was measured as described for the Ca-activated ATPase assay, except that 5 mM ammonium oxalate was added to the assay. Ten micrograms of plasma membrane protein were added, and the mixture was preincubated for 5 min at 37 "C. The reaction was started by the addition of CaC12 to a final Ca concentration of 125 p~ (5 p~ free Ca) with 2 pCi/ml of 45Ca. In some experiments, membranes were preincubated in the presence of 10 pg/ml of calmodulin and 5 p~ free Ca for 30 min at 4 "C. In these studies, the reaction was started by addition of 5 m~ ATP at 37 "C. The reaction mixture (total volume, 250 d) was incubated in a heating block at 37 "C on a shaking

The abbreviations used are: EGTA, ethylene glycol bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid; EDTA, ethylenediaminetet- raacetate; CDTA, transcyclohexane-1,2-diamine-iV,N,N',~-tetraacetic acid.

platform. At appropriate time intervals, 175-pl samples were removed and vacuum-filtered on 0.45-pm pore cellulose acetate filters (Milli- pore) that had been pre-washed with Tris-KC1 at 23 "C. The membrane vesicles trapped on the filter were placed in scintillation vials with 10 ml of Bray's scintillation solution for measurement of the /3 radioactivity. Ca uptake was expressed in picomoles of Ca/pg of protein/min. Uptake values were corrected for radioactivity bound to the filter when an identical reaction mixture without plasma membranes was filtered. This accounted for less than 10% of the total measured radioactivity. ATP-dependent uptake was determined from the difference in radioactivity bound to the filter in the presence and absence of ATP. In control studies, phosphate did not serve as an effective intravesicular trapping agent, but potassium oxalate could substitute for ammonium oxalate.

Determination of Total and Free Calcium Concentrations of Re- action Mixtures-The total Ca concentration of the reaction mixture including the plasma membranes was determined by graphite furnace atomic absorption spectrophotometry. In these measurements, stan- dardization was achieved by successive additions of a standard solution of Ca (prepared from weighed CaCI2. 2H20) to each sample (22). The contaminating Ca in a reaction mixture without added Ca was 7 r 2 (S.E.) p ~ . The quantity of Mg present in the reaction mixture with no added Mg was 7 f 1 p~ by flame atomic absorption spectrophotometry. The free concentrations of Ca and Mg were buffered by EGTA (or CDTA) in all experiments and were calculated with an iterative computer program using pH, the total concentrations of Ca, Mg, EGTA (or CDTA), and ATP, and the appropriate association constants for the interaction of EGTA, CDTA, and ATP with H', Ca", and Mgz+ (23). The true and apparent association constants and the conservation equations for ligands and cations were defined as described by Portzehl et al. (24). The definitions, equations, and algorithms for computing the values of the unknown free cation concentrations are described thoroughly elsewhere (25).

Purification ofLymphoryte Cytoplasmic Activator (Calmodu1in)- Fig. 1 shows the scheme used for the isolation and purification of lymphocyte calmodulin. This procedure is an adaptation of a method

LYMPHOLYSATE

OO°C x 30 MIN

A INBOLUBLE FRACTION FRACTION

SOLUBLE

I ULTRAFILTRATION AMICON PM-10

A FILTRATE RETENTATE

( >10.000 MW)

h DEAE-SEPHACEL COLUMN STEP NaCI QRADIENT CHROMATOQRAPHY

0.3 M NaCI FRACTION

0.6 M NaCI FRACTION

CHROMATOQRAPHY ULTROGEL AcA54 COLUMN

ACTIVE FRACTIONS (APPARENT MW: 30-40,000)

DEAE SEPHACEL COLUMN LINEAR NmCI GRADIENT CHROMATOGRAPHY

ACTIVE FRACTION (0.45 M NaCI)

FIG. 1. Purification of lymphocyte cytoplasmic activator (calmodulin). Lympholysates were obtained by disruption of lymphocytes in hypotonic medium as described in the text. Purification was monitored by assaying each fraction for activation of RBC ghost Ca-ATPase described in the text.

6150 Calcium Transport of Lymphocyte Plasma Membranes

for isolating erythrocyte calmodulin (21). Lympholysates were obtained during the procedure for preparation of EGTA-stripped plasma membranes. The supernate from the 12,000 X g centrifugation, i.e. the cytoplasmic fraction, was heated to 80 "C for 30 min and centrifuged a t 15,000 X g for 30 min. The clarified supernate of the lympholysate was frozen a t -20 "C until material from 20 lymphocyte membrane preparations was obtained over a 3-month period. The lysates were then thawed to 4 "C. pooled, and concentrated 100-fold using an Amicon PM-10 membrane filter (10,000-dalton exclusion).

The concentrated lysate was washed 5 times by filtration with 10 volumes of 0.15 M NaCI, 10 mM imidazole-HCI. pH 6.5, and applied to a DEAE-Sephacel column equilibrated with the same buffer. The column was washed with 3 volumes of 0.3 M NaCI, 10 mM imidazole- HCI, pH 6.5. Bound calmodulin was eluted by changing the NaCl concentration to 0.6 M. The pooled fractions from the 0.6 M NaCl elution were concentrated by PM-10 Amicon filtration, washed with Tris-KC1 buffer, and applied to a Ultrogel AcA-54 column. The calmodulin activity eluted from the column with an apparent molecular weight of 30,000-40,000, this molecular weight estimate likely represents dimerization of the calmodulin molecule. A similarly high molecular weight elution of calmodulin activity from gel filtration chromatography had been described (21).

The active fractions were pooled and concentrated in 0.1 M NaCI, 10 mM imidazole-HCI, pH 6.5. by I'M-10 Amicon filtration. The protein solution was then applied to a DEAE-Sephacel column and eluted by a linear gradient of NaCl in 10 mM imidazole-HCI. The calmodulin activity was found in a sharp elution peak a t 0.45 M NaCI. The active fractions were pooled and washed in Tris-KC1 buffer and concentrated to 100pg/ml by I'M-10 Amicon filtration. Discontinuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 15% acrylamide, 0.1% bisacrylamide by the method of Weber and Osborn (26). The preparation migrated as a single band with an apparent molecular weight of 17,000 (Fig. 2). Calmodulin activity was assayed a t all stages of the purification by measuring stimulation of RBC ghost Ca-ATPase as described above. The purification was calculated by determining the quantity of protein from each step required to half-maximally activate the RBC ghost Ca- ATPase. The final preparation was purified 500-fold from the initial cytoplasmic fraction. The yield was 20-40% of the original quantity of calmodulin activity. The calmodulin was stored at -20 "C up to 4 months before use without loss of activity.

Materials-Tris, oligomycin, and ATP (Tris salt) were purchased from Sigma Chemical Co., St. Louis, Mo. ATP (disodium salt), ADP, AMP, GTP, UTI', ITP, CTI', and T T P were purchased from Boeh- ringer Mannheim Corp., Indianapolis, lnd., and were stored as 50 mM stock solutions in Tris-KC1 buffer, pH 7.0 a t -30 "C, for less than 1 week. [y-'"PIATP, GTP, and UTP, and 45Ca were purchased from Amersham Corp., Arlington Heights, Ill. A23187 was a gift of Dr. Hamill of the Eli Lilly Co.. and a 25 p~ stock solution was prepared

O V A C.A. sa11 mas nmo LISO

I I t i I 4

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lymphocyte cytoplasmic activator. Twenty-five pg of the activator were applied to the gel. The migration proceded from left to right. The absorbance at 550 nm of a Coomassie blue-stained gel is shown. The arrows indicate origin, tracking dye, and position of molecular weight standards on identical gels. OVA, ovalbumin ( M , = 43,000); C. A.. carbonic anhydrase ( M , = 29,000); SBTZ, soybean trypsin inhibitor ( M , = 21,500); MGB, myoglobin ( M , = 16,000); RZBO, ribonuclease ( M , = 13,700); LYSO, lysozyme ( M , = 14,300).

in dimethyl sulfoxide. KC1 and NaCl were of ultrapure grade purchased from Ventron Corp., Danvers, Mass. CDTA was purchased from Eastman Kodak, Rochester, N. Y. ECTA was purchased from J. T. Baker Chemical Co., I'hillipsburg, N. J. DEAE-Sephacel was purchased from I'harmacia Fine Chemicals, Uppsala, Sweden. Ultro- gel AcA54 was purchased from LKB Instruments, Inc., Hockville. Md. All other reagents were purchased from Fisher Scientific CO., l'ittsburg, Pa. All aqueous solutions were prepared with distilled water, deionized by passage through a Barnstead mixed-bed ion exchange resin from Sybron Corp., Rochester, N. Y.

RESULTS

Calcium Uptake by Membrane Vesicles: Time Course, Cofactor Requirements, and Temperature Dependence-The time course of Ca uptake by lymphocyte plasma membrane vesicles is shown in Fig. 3. In the absence of ATP, approximately 1.9 pmol of Ca/pg of protein bound to the plasma membranes within 10 s (the time required between addition of Ca to the reaction mixture and transfer of the filter). No significant additional uptake occurred in the absence of added ATP. No ATP-dependent uptake occurred when Mg2' was omitted from the assay. In the presence of ATP and Mg'+, Ca uptake proceeded in a linear fashion for about 15 min. When 1.0 p~ A23187 was present, no Ca accumulation was observed. The addition of dimethyl sulfoxide without ionophore had no effect on the Ca uptake. The addition of A23187 after 10 min of incubation caused the release of accumulated Ca.

Ca uptake by lymphocyte membrane vesicles specifically required ATP. The Mg salts of ADP, AMP, GTP, CTP, UTP, and TTP did not support Ca uptake by lymphocyte plasma membranes (Table I). Further, the ATP-dependent Ca uptake was temperature-dependent. A marked reduction in the uptake measured at 37 "C occurred a t 4,25, or 45 "C (Table I).

Experiments were performed to determine the response of the plasma membrane ATP-dependent Ca uptake activity to various conditions known to affect Ca transport systems in mitochondria and plasma membranes of other cell types. The presence of 5 mM Na azide, 5 mM Na cyanide, or 10 p~ oligomycin did not significantly affect the Ca uptake in 4 plasma membrane preparations. The elimination of Na or K

0

FIG. 3. Calcium uptake by lymphocyte p lasma membrane vesicles. Ca uptake activity was assayed in a medium containing 110

Mg), 5 mM ammonium oxalate, 100 p~ EGTA, 125 PM Ca (5 p~ free Cab, '"Ca (2 pCi/ml), 5 mM ATP, and 10 pg of membrane protein in a total volume of 250 pl at 37 "C. The membrane vesicles were preincubated in the above medium without 4"CaCln for 5 min and the reaction was started by addition of Ca. At the appropriate times, 175- pI samples were removed and filtered, and "Ca uptake was measured as described in the text. Each poinf represents the mean f S.E. of triplicate sample determinations from 3 membrane preparations. The S.E. was less than 0.5 pmol/pg of protein on all points without bars. The following conditions are shown: 0, optimal medium; A. Mi'+ or oxalate omitted 0, ATP omitted; 0, 1 PM A23187 added from the start; ., A23187 added a t 10 min. The A23187 was dissolved in dimethyl sulfoxide, which had no effect on measured uptake.

mM KCI. 40 mM Tris/Cl, pH 7.0.20 mM NaCI, 5 mM Mg'+ (1 mM free

Calcium Transport of Lymphocyte Plasma Membranes 6151

TABLE I Calcium uptake by lymphocyte plasma membranes: nucleotide

specificities and effect of temperature Calcium uptake was assayed as described in the text and in the

legend to Fig. 3, except that the nucleotide triphosphate or temperature was varied as indicated. All incubations were 15 min. The means 2 S.E. of measurements on 3 membrane preparations are given.

Nucleotide Temperature Calcium uptake “ C pmol Cafpg protein/rnin

ATP 45 0.11 f 0.01 37 1.04 f 0.01 25 0.22 f 0.01

4 0 f 0.01 ADP 37 0.01 f 0.01 AMP 37 0.01 f 0.01 GTP 37 0.06 f 0.02 CTP 37 0.02 f 0.01 UTP 37 0.01 f 0.01 ITP 37 0.05 f 0.01 TTP 37 0.01 f 0.01

3-i

FREE CALCIUM (yM) FIG. 4. Calcium uptake by lymphocyte plasma membrane

vesicles: dependence on free Ca2+ concentration. Uptake was assayed after 15 min of incubation as described in the legend to Fig. 3. The free Ca” concentrations were calculated as described in the text. The ordinate represents ATP-dependent Ca uptake, i.e. the difference in Ca uptake in the presence and absence of ATP. Each point represents the mean -t S.E. of triplicate sample determinations in 3 membrane preparations. The S.E. was less than 0.05 pmol/lg of protein/min on all points without bars.

from the medium or the addition of 10 PM ouabain also had no effect on the Ca uptake activity. In the Na-free medium, Tris-ATP was used, and in the K-free medium, Na was substituted as the major cation.

Calcium Uptake: Kinetic Features-The initial velocity of ATP-dependent Ca uptake by lymphocyte plasma membrane vesicles was measured as a function of the free Ca concentration in the medium (Fig. 4). The Ca uptake reached a maxi- mum at a free Ca concentration of approximately 5 p ~ . A double reciprocal plot of the data was used to calculate a maximal velocity ( VmaX) of 2.6 pmol of Ca/pg of protein/min and a K,,, for free Ca of 1.0 p ~ .

The initial velocity of plasma membrane vesicle Ca uptake was also measured as a function of ATP concentration. In these studies, the incubation times were adjusted so that no more than 10% of the initial ATP was hydrolyzed. The total Ca concentration was adjusted so that free Ca would remain 5 FM at the different ATP concentrations. A biphasic activation of Ca uptake was measured under these conditions (Fig. 5 ) . This pattern of ATP activation could represent a complex

T T

0 . 5

V -1 4 v 00

0 1 1 9 4 B 8 7

ATP CONCENTRATION (mM)

FIG. 5. Calcium uptake by lymphocyte plasma membrane vesicles: dependence on ATP concentration. Uptake was assayed as described in the legend to Fig. 3, except that total ATP concentration was varied as shown. The free Ca concentration was maintained at 5 p ~ . The incubation time was varied from 2 min at low ATP concentrations to 15 min at high ATP concentrations, so that <lo% of the initial ATP present was hydrolyzed. Each point represents the mean f S.E. of triplicate sample determinations in 3 membrane preparations.

kinetic mechanism with 2 saturable components and different affinities for ATP as described for the red cell Ca pump (27, 28). The initial component of the ATP activation curve had a K, for Ca uptake of approximately 80 p ~ . The K, of the lower affinity or second component of the ATP activation curve was 2.3 mM, and the V,,, was 2.2 pmol of Ca/pg of protein/min.

Calcium-ATPase Activity by Membrane Vesicles: Time Course, Cofactor Requirements, a n d Kinetic Parameters- Ouabain-sensitive (Na,K)-Mg-ATPase was assayed to confirm purity and to ensure enzyme activity and integrity of the isolated plasma membranes. The mean ouabain-sensitive ATPase activity was 74 f 5 pmol of PJpg of protein/min in 10 membrane preparations. This represented a greater than 50-fold increase in specific activity over that measured in unfractionated lympholysates.

The total Mg-dependent and Ca-activated Mg-ATPase activities were linear with respect t,o time up to 30 min and protein concentration up to 20 pg/0.25 ml. Maximal activation of the Ca-ATPase was measured at a free Ca concentration of 4-5 PM (Fig. 6). Kinetic analysis revealed a K , for Ca of 0.55 p~ and a V,,, of 25 pmol of PJpg of protein/min.

The Ca-ATPase was assayed at different ATP concentrations. The incubation times were adjusted so that less than 10% of the initial total ATP was hydrolyzed. Total Ca was adjusted so that the free Ca was 4 p~ at all ATP concentrations, and total Mg was kept constant a t 5 m. The ATP concentration required for maximal Ca-ATPase activity was approximately 0.7 m (Fig. 7). The KnZ for ATP was 86 ,UM and the V,,, was 28 pmol of P,/pg of protein/min.

M), a Ca-activated ATPase activity remained that was approximately equal in magnitude to that measured in the presence of 1 mM free Mg. The chelator, CDTA, has a high affinity for Ca and Mg and was employed to assess the Mg requirements of the Ca-ATPase (29). In medium buffered with 100 PM CDTA, either Ca alone (Mg < 10“ M ) or Mg alone (Ca < io” M) in concentrations up to 50 p~ stimulated basal ATPase activity, and these activities were additive when both Ca and Mg were added.

The Ca-ATPase activity was not affected by either changes in Na or K concentrations or by the presence of oxalate (Table 11). A23187, however, caused a 70% stimulation of the ATPase

When Mg was omitted from the medium (free Mg <


phohydrolysis in the presence of either ATP, GTP, or UTP. Effects of Cytoplasmic Activator (Calmodulin) on Lympho-

cyte Membrane Vesicles-In order to further characterize the Ca transport and Ca-activated ATP hydrolysis, we tested the effects of lymphocyte calmodulin on the membrane vesicles.

Calmodulin stimulated the red cell ghost Ca-ATPase 10- fold from 3.2 to 35.5 pmol/pg of protein/min. EGTA treatment of lymphocyte plasma membranes reduced the Ca-ATPase

111). The addition of lymphocyte calmodulin stimulated the EGTA-treated membrane Ca-ATPase. Ca-ATPase increased approximately 2-fold from 15.9 to 29.0 pmol of Pi/pg of pro-

3 * 8 '* l4 '6 ATP-dependent Ca uptake by lymphocyte membranevesicles FREE CALCIUM (uM) from 0.51 to 0.37 pmol/pg of protein/min ( p < 0.05). The

addition of the lymphocyte calmodulin to EGTA-treated

e - from 26.5 to 15.9 pmol/pg of protein/min ( p < 0.01) (Table V Q 5 5-

0 tein/min ( p < 0.01) (Table 111). EGTA treatment reduced

FIG. 6. Calcium-activated ATPase activity of lymphocyte plasma membranes: dependence on free calcium concentration. ATPase activity was-assayed in a medium containing 110 mM KCI, 40 mM Tris-C1, pH 7.0, 20 mM NaCl, 5 mM Mgz', 10 p~ ouabain, 100 p~ EGTA, 5 mM [y3'P]ATP (1 pCi/ml), varying free Ca concentrations from 0 to 14 p~ (calculated as described in the text), and 10 pg of membrane protein in a total volume of 250 pl at 37 "C. The membranes were preincubated for 5 min in the above medium without ATP, and the reaction was started by addition of [y-"*P]ATP. After 20 min, the reaction was stopped, and the "P released was measured as described in the text. The ordinate represents net Ca-activated ATPase, i.e. the difference between ATPase in the presence and absence of Ca. Each point represents the mean f S.E. of triplicate sample determinations in 4 membrane preparations.

0 a . .- a

w v,

a a l-

ATP CONCENTRATION (mM)

FIG. 7. Calcium-activated ATPase activity of lymphocyte plasma membranes: dependence on ATP concentration. ATP- ase activity was assayed as described in the legend to Fig. 6, except that the incubation was varied between 2 and 20 min to ensure 4 0 % ATP hydrolysis. Free Ca" concentration was kept a t 5 PM, and total Mg2+ concentration was kept at 5 mM. The ordinate represents the Ca-activated ATPase. Each point represents the mean k S.E. of triplicate sample determinations in 4 membrane Preparations.

from 19-34 pmol of P,/pg of protein/min, which was shown t,o be unrelated to the dimethyl sulfoxide solvent. Oligomycin and azide did not have an effect on the Ca-ATPase activity.

In order to ascertain whether the hydrolysis of other nucleotide triphosphates by lymphocyte membrane vesicles was stimulated by Ca, assays were performed with [ Y - ~ ~ P I G T P and [y-"'P]UTP under standard conditions, using 4 p~ free Ca to elicit activation. Three membrane preparations studied had no significant Ca-activated GTPase or UTPase activities. These membranes were shown to have a mean Ca-ATPase activity of 38 f 6 (S.E.) pmol/pg of proein/min. There was approximately the same magnitude of Mg-activated phos-

TABLE I1 Lymphocyte plasma membrane Ca-activated ATPase: effects of Nu

and K concentration, oxalate, azide, oligomycin, and A23187 Calcium-activated ATPase activity was assayed as described in the

text and the legend to Fig. 6. The data are the mean f S.E. of experiments on 3 membrane preparations. Each group represents a different set of membrane Preparations. The control in each group was assayed in standard reaction medium without any additions. Tris- ATP was used for Group I experiments. A23187 and oligomycin were dissolved in dimethyl sulfoxide and added to yield a 0.04% (v/v) dimethyl sulfoxide Concentration.

Condition Ca-activated ATPase pmol P,/kgpprotein/min

Group I 0 mM Na; 120 mM K 26 f 1 60 mM Na; 60 mM K 25 f 2 120 mM Na; 0 mM K 24 f 1

Group I1 Control 5 mM NH, oxalate 1 mM Na azide

30 f 2 33 f 1 35 f 2

Group I11 Control 19 f 1 10 p~ oligomycin 20 f 2

0.04% (v/v) dimethyl sulfoxide 23 f 2 1 pM A23187 34 f 1

TABLE I11 Stimulation of calcium uptake and calcium ATPase by lymphocyte

cytoplasmic activator Lymphocyte plasma membranes (both untreated and EGTA-

treated) and RBC ghosts (EDTA-treated) were prepared and Ca- activated Mg-ATPase and ATP-dependent Ca uptake activities were measured as described in the text. Cytoplasmic activator was prepared as shown in Fig. 1 and was used at a concentration of 10 pg/ml. Membranes were preincubated in the absence or presence of the activator for 30 min at 4 "C prior to each assay. The low levels of Ca uptake reflect a loss of activity during this preincubation. The mean f S.E. of measurements on 5 membrane preparations are presented.

Membrane preparation

Lymphocyte plasma membranes EGTA-treated lymphocyte plasma

EDTA-treated RBC ghosts membranes

Lymphocyte plasma membranes EGTA-treated lymphocyte plasma

membranes

__ -Cytoplasmic +Cytoplasmic

activator activator Ca-activated ATPase

pmol P,/lrg protein/min 26.5 k 1.6 25.8 * 3 15.9 f 4.1 29.0 f 4.6

3.2 f 0.6 35.5 f 5.5

ATP-dependent calcium uptake pmol Ca/Gg protein/min

0.51 f 0.18 0.64 f 0.22 0.37 f 0.12 0.62 f 0.16

Calcium Transport of Lymphocyte Plasma Membranes 6153

membranes stimulated Ca uptake from 0.37 to 0.62 pmol/pg of protein/min ( p < 0.01) (Table 111).

DISCUSSION

We have described a lymphocyte plasma membrane ATP- dependent Ca transport system with properties similar to the Ca transport systems described in plasma membranes of erythrocytes (15), macrophages (30), and synaptic plasma membrane vesicles (31, 32). These properties include ATP and Mg dependence, insensitivity to ouabain and Na or K, and a high affinity for Ca.

In order to study the energy transduction system responsi- ble for net Ca transport across the plasma membrane, presumably against an electrochemical gradient, we evaluated the Ca-activated ATPase activity. This ATPase has characteristics similar to the Ca-ATPase in the plasma membranes of erythrocytes (15), adipocytes (29), macrophages (33 ) , and neutrophils (34), all of which are thought to be Ca transport enzymes. The lymphocyte ATPase has a high affinity for Ca and is insensitive to ouabain and changes in Na or K concentration. There are certain similarities between the ATPase and the transport activities which support the hypothesis that they are expressions of the same enzyme. The Ca affinities (0.6 PM for ATPase and 1.0 p~ for transport) are reasonably close in light of the differences in the experimental conditions and probably represent the same Ca-binding site. The ATP activation curves for Ca transport and Ca-ATPase are also similar when measured under similar conditions with ATP affinities of 80 and 90 p ~ , respectively. A further increase in the Ca uptake was seen with increases in the ATP concentration to 4 mM, yet the ATPase activity was not enhanced by ATP concentrations above 1 mM. This discrepancy raises the possibility of a nonenzymatic effect of high ATP concentrations on membrane integrity, preventing Ca egress or enhanced intravesicular Ca sequestration.

The maximal velocities of the Ca uptake and Ca-ATPase activities (2 pmol of Ca/pg of protein/min and 29 pmol of Pi/ pg of protein/min, respectively) yield a very low ratio of Ca transported per ATP hydrolyzed. The 2 assay systems differ, however. The leakiness of the vesicles to Ca, the presence of right-side-out vesicles which may hydrolyze ATP but do not accumulate Ca, and the inefficiency of membrane recovery during fitration could all contribute to a significant underes- timation of Ca translocation.

The Ca transport activity was Mg-dependent. When Mg was omitted from the assay medium, the free Mg concentration was below 1 PM, and no ATP-dependent Ca uptake was observed. In contrast, when Mg was removed from the ATP- ase assay, Ca-activated ATPase activity was not altered. Sim- ilarly, when Ca was removed, Mg alone stimulated the ATP- ase. The precise role of Mg in Ca-activated ATPase activity of erythrocyte membranes is also not clearly understood, since studies of the purified ATPase molecule have yielded results different from those found in intact membranes (35) . Taken together, the transport and ATPase data in lymphocytes suggest that the nonselective Ca- or Mg-activated ATPase activities represent nonspecific divalent cation stimulation of phosphohydrolysis and that both ions are required for the Ca transport ATPase.

A23187 inhibited net Ca uptake and stimulated Ca-ATPase. Both of these effects are consistent with the action of the ionophore in rendering the plasma membrane vesicles perme- able to Ca. With A23187, Ca pumped into the vesicles leaks out rapidly enough to prevent the increase in intravesicular Ca concentration necessary for Ca oxalate precipitation. Stim- ulation of Ca-ATPase by A23187 has been reported in studies of inside-out erythrocyte membrane vesicles (36) and in sar-

coplasmic reticulum (37) , and may be due to a reduction in intravesicular Ca, since intravesicular Ca can inhibit the Ca- ATPase (38).

The cytoplasmic component that we partially purified from lymphocyte cytoplasm is very likely calmodulin. I t is heat- stable and behaves like calmodulin in gel fitration and ion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, this component stimulates EDTA-treated erythrocyte ghost Ca-ATPase. There have been preliminary reports of the presence of calmodulin in lymphoid tissue (39). Our data show that both Ca transport and Ca-ATPase activities of lymphocyte plasma membranes are reduced by EGTA treatment, and both can be stimulated by addition of a calmodulin-like fraction of the same cells from which the membranes were derived. Thus, we have described a lymphocyte plasma membrane ATP-dependent, calmodulin-activated Ca transport system.

The Ca transport system described above may be essential to lymphocyte Ca homeostasis when plasma membrane permeability is altered during early mitogenesis. An increase in Na and K permeability occurs immediately after lymphocytes are exposed to the mitogenic lectin, phytohemagglutinin (40). There is little net change in intracellular K or Na concentration because of the homeostatic response of the plasma membrane Na,K pump (41). Similarly, there is no increase in total lymphocyte Ca after phytohemagglutinin treatment, although "Ca-labeling is increased (13). The Ca transport system characterized here presumably mediates this increased plasma membrane Ca exchange.

Acknowledgments-We thank Geraldine Roberts for providing excellent technical assistance and Beth Rice for carefully preparing the manuscript.

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