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Molecular properties of the red cell calcium pump
Cell Calcium 1: 299-310,1980
MOLECULAR
PROPERTIES
OF THE RED CELL CALCIUM PUMP
11,EFFECTS OF CALMODULIN, PROTEOLYTIC DIGESTION AND DRUGS ON THE CALCIUM-INDUCED MEMBRANE PHOSPHORYLATION BY ATP IN INSIDE-OUT RED CELL MEMBRANE VESICLES ~n~;r~~~pQi2,
B. Sarkadil,
Ilma Szaszl, G. Bot2, and
.
National Instftute of Haematology and Blood Transfusion, 1113 Budapest , and Institute of Mepcal Chemistry, University Medical School, Debrecen , Hungary I + reprint requeststo GG / ABSTRACT
In inside-out hy$n red cell membrane vesicles /IOV/, in th52absence of Mg , the only calcium-induced labelling by g P-ATP occurs in a 140-150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated int?$mediate /EP/ of the calcium pump. In calcium-induced phosphorylation is the presence of Mg accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accels+ates EP formation both in the absence and presence of Mg . Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVS, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of In trypsin-digested IOVs the molecular weight of the %. P-1,abelled EP is shifted to lower values / 110-120 000 I We suggest that trypsin digestion cleaves off a 20-40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule. INTRODUCTION Calcium-dependent formation of a phosphorylated prgtein intermediate of the red cell calcium pump from P-ATP has been shown by several authors lrefs. l-S/. d Investigation of the effects of regulatory proteins and various membrane modifications on the phosphoenzyme
formation may significantly contribute to our better understanding of the molecular properties of the calcium pump enzyme. It is a special advantage in using IOVs that changes in transport properties can be directly compared to the alterations in membrane phosphorylation. In close connection with our preceding paper in this volume /9/ we describe here the effects of calmodulin, trypsin digestion and SH-reagents on the calcium-induced membrane phosphorylation in IOVs. MATERIALS
AND METHODS
The source of the chemicals used, the methods for IOV and calmodulin preparation3yere the same as described in our preceding paper 191. d P-ATP with a specific activity of 600 GBqlmmol was purchased from the Radiochemical Centre, Amersham. Phosphorylation of IOVs was carried out in reaction media containing 130 mM KCl, 17 mM imidazole-HCl, EDTA, CaC12, MgC12 and calmodulin as indicated, and 0.25-O-5 mg IOV protein in a final Volume 8f 0.5 ml. The tubes we'& preincubated for 5 min at 37 C and then cooled to39 C. The reaction was started by the quick addition of d P-ATP to obtain a fin+ concentration of le. The tubes were incubated at 0 C under vigorous stirring and the reaction was stopped by the addition of 4 ml of ice-cold 6% trichloroacetic acid /TCA/, containing 1 mM ATP and 10 mM Pi. The suspension was supplemented with 2 mg of bovine albumine, centrifuged at 10 000 x g for 5 min, and washed three times with 5 ml of the TCA-ATP-P. solution. The washed precipit3te was dissolved in l%isodium dodecyl sulfate /SDS/. P radioactivity was measured in an Intertechnique SL-30 liquid scintillation spectrometer in 2:l mixtures of scintillation liquid /Liquid Scintillator, Nuclear E.L., diluted with toluenej and Triton X-100. Hydroxylamine treatment of the phosphorylated proteins was carried out according to Rega et al. 141. SDS-polyacrylamide gel electrophoresis was performed according to Fairbanks et al. /lOI with a current of 8-10 mA/tube. From three identical gels one was stained for proteins with Coomassie-blue R-250, and3the other two were P radioactivity. cut into 2.5-5 mm slices for measuring Molecular weights were estimated from the relative mobilities of spectrin 2, band 3 and band 5, as references, by using both ghosts and IOV membranes. Trgpsin digestion of the IOV membrane was carried out at 37 C in a medium containing 130 mM KCl, 17 mM imidazole-HCl IpH 7.01, with 0.2 mg trypsinjmg IOV protein for 3 min. Trypsin treatment was stopped by the addition
300
of S-fold excess of soybean trypsin inhibitor, and the IOVs were washed three times in the KCl-imidazole solution. In the control experiments a similar incubation was carried out with the trypsin + trypsin inhibitor mixture. RESULTS In our preceding paper /9/ we showed that the addition of calmodulin or mild trypsin digestion of the IOV membrane significantly stimulate active calcium uptake. Trypsin digestion eliminates calcium-dependent calmodulin binding to the membrane and calmodulin activation of calcium uptake. In the following experiments we investigated the membrane phosphorylation under these conditions. Fig. 1 shows the time course of the cg$cpt;;p -induced membrane phosphorylation in IOVs from x as studied in Ca-EDTA buffers, that is in the absence of free magnesium ions.
EDrA
*co=:
0%
% -A--------_--_AtCALMODULIN A CONTROL
___------_
0
.3’ TK YP.S/N o -‘CALMODULIN
120
60
+ SEC
Fig. 1. Effects of calmodulin and trypsin digestion on the EP-formation of the calcium pump in IgVs. Phosphorylation was carried out at 0 C in media containing 130 mM KCl, 17 my imidazole-IX1 /P&&7.0/, 0.5 mq Tris-EDTA, 0.55 x@4 CaC12 and 1pM d P-ATP. IOV concentration was 0.4 mg/ml, cslmodulin 3 mg/ml. Trypsin pretreatment was rried out as described in the Method29ection. s9 P-incorporation in the absence of Ca was less than 0.05 picomol/mg IOV protein. 301
1
2
3
4
5
6
7
0-Q
CONTROL
e---o
3’ TRYPSIN
6
9
MIGRATION
1
2
3
4
5
6
7
8
!O DISTANCE
11 (Cm)
-
CONTROL
-----
3’ TRYPSIN
9
MIGRATION
10
II
DISlANCf
12 (c rr
Fig. 2a. SDS-polyacrylamide gel electropho52togram of IOV proteins phosphorylated by d P-ATP in the p.resence of Ca-CDTA for 60 seconds. For the conditions of phosphorylation see the legend of Fig. 1. Fig. 2b. Polypeptides of the IOVs separated by SDS--polyacrylamide gel electrophoresis.
302
32 Ca-induced P-incorporation is significantly accelerated by calmodulin although the steady-state level of phosphorylation is only slightly elevated. In contrast to this, trypsin digestion /stimulating active calcium transport similarly to calmodulinl reduces the steady-state value of membrane-labelling. There is no effect of calmodulin on membrane phosphorylation in the trypsin-treated IOVs. As it is shown in Fig. 2, when examining the phosphorylated membrane proteins by SDS-polyacrylamide gel electrophoresis, in IOVs phosphorylated in a Ca-EDTA medium the only significant labelling occurs in a 140-150 000 molecular weight fraction. When phosphory&tion is accelerated by calmodulin, the location of the P-1abelling is unchanged /not shown/, while in trypsin-treated IOVs a significant shift in the molecular weight of the phosphoprotein occurs. The calculated molecular weight of the calcium pump EP in the trypsin-treated IOVs is 110-120 000.
~+CALMODULIN
__e_-------‘-_‘.
OB
* 0
120
60
SEC
Fig. 3. Effects of Mg2+ and Mg2+ + calmodulin on the membrane phosphorylation in IOVs. Curve A shows membrane phosphorylation in a Ca-EDTA buTfer, as described in Fig. 1. The other phosphorylation media contained 5 mM MgC12 and either 50 ,uM CaCl containing media, respectively. concentration = 3 ug/ml.
303
Calmodulin
.-CONTROL D---0
HYOROWLAMINE
co=++hg=+
SDS-PAGE
-.
CONrROL
o----o+tib-DROXFUhVNE
1
2
3
4
5
6
0
7
MIGRANON
9
10 D/STANCE
11 [cm)
Fig. 4a. SD,$PAGE of IOV proteins phosphorylated by d P-ATP in the presence of 50 pM EGTA + . 5 mMMgC1 Fig. 4b. SDQZPAGE 8 f IOV proteins phosphorylated by d P-ATP in the presence of 50 m CaC12+ 5 mM MgCl . For the c2 nditions of phosphorylation and hydroxylamine treatment see the Hethod section.
304
SDS
PAGE
co=*+/ug*+ Ad A.--A3
CONTROL rRYPS,N
~..&.A..&.& I
2
3
4
5
6
7
6
MIGRATION
9
IO
DISTANCE
tl
12
(cm)
Fig. 5a-5b. Effect of trypsin-pretreatment on 32P-incorporation. 5a: SDS-PAGE of IOV proteins phosphorylated in the presence of 50 ,uM EGTA + 5 mY MgCl . 5b: SDS-PAGE of IOV proteins phosphoryfated in the presence of 50 @4 CaCl + 5 W MgC12. For the conditions of phosphorylz ation and trypsin digestion see the -Yethod section.
305
As it is shown in Fig. 3, calcium-induced membrane phosphorylation is rapid in the presence of magnesium ions, and it can be further accelerated by addition of calmodulin. The T f r calcium-induced phosphorylation kc20 8C in a Ca-EDTA buffer and the decreases this value to about 10 seconds. + calmodulin reduces T to less than 4 seconds. The presence of magnesium i ns increases the steady-state lLJ2 level of the phosphoenzyme by about 30%. Fig. 4 shows the SDS-polyacrylamide gel elec-t,2;h;oc;tograms of the IOV membsqne proteins labelled by in the presence of Mg and in the absence /Fig. 4aj or in the presence /Fig. 4b/ sf calcium ions. In both cases considerable amount of P-incorporation into spectrin 2 and band 3 proteins is observed but these phosphate groups are in hydroxylamine-insensitive nds. The only calcium99 P-incorporation is induced, hydroxylamine-sensitive observed again in the 140-150 000 dalton, protein-poor region of the electrophoretogram. As it 32 shown in Fig. 5, trypsin-treatment of the I,OVs decreases P-incorporation and shifts the molecular weight of the calcium-induced phosphoprotein to lower values /llO-120 000 dalton Fig. 5b/. A large portion of spectrin and band 3 is degraded by trypsin treatment of the IOVs /see also Fig. 2b/, and therefore several different phosphorylated polypeptides appear at the lower molecular weight regions ctrophoretogram /Fig. Sa,b/. These fractions P in hydroxylamine-insensitive bonds, while the calcium-induced phosphoprotein is also hydroxylamine-sensitive in the trypsin-digested IOVs. Fig. 6 shows the effect of PCMB-treatment on the calcium-induced EP-formation in IOVzj2in Ca-EDTA buffers. PCMB-pretreatment strongly reduces P-incorporation but a calmodulin stimulation of the EP-formation is present321so in these vesicles. A similar strong reduction of the PVaz';"o;a;;T;s+o;;3f-ved in the .
DISCUSSION In red ~$11 rn2p rane fragments /l-6/ as well as in purified Ca + Mg -ATPase preparations /7-S/ a specific, calcium-induced phosphorylation of a 140-150 000 dalton molecular weight protein was demonstrated. The isolated protein, wh incorporated into lipid vesicles, catalyzed an ATP + Mgsp -dependent accumulation of calcium ions /11-131. Based on these data this protein fraction can be regarded as the molecular basis of the red cell calcium PumP. It has been also demonstrated that the formation of the phosphoprotein /EP/ does not require the presence of magnesium ions /4,5,6/, although t@ process is accelerated by magnesium /4,5/. Mg plays a basic role in the dephosphorylation of the pump protein /4,5,14/.
306
iOTA
.
co*+,
0-c
A~A-Pcma /
A
/ 0
__-_-_---.---
--------_-~+PCMB+CALMOWLIN O+PCMB
60
120
* SEC
Fig. 6. Effect of PCMB-pretreatment on the EP-formation in IOVs. IOVs were preincubated for 5 min with 20 nanomoles PCMB/mg IOV protein and the control IOVs were reactivated by the addition of 1 nY' dithiothreitol. EP formation was measured as described in the legend to Fig. 1. According to the experimental results reported ig2the present paper, membrane phosphorylation in IOVs by E P-ATP in a Ca-EDTA medium, that is in the absence of free magnesium, results in the appearence of a single phosphoprotein with a molecular weight of 140-150 000. Addition of magnesium ions accelerates phosphorylation of this protein, however, due to protein kinase action, several other membrane proteins, such as spectrin 2 and band 3, are also phosphorylated. Calmodulin stimulates EP-formation both in the absence and presence of magnesium and this protein does not affect calcium-independent phosphorylation processes. These findings are in accordance with those by Muallem and Karlish 1151 and Rega and Garrahan 1161, and indicate that the predominant effect of calmodulin in stimulating the calcium pump is to increase the rate of calcium-induced phosphoprotein formation. In the above two reports 115,161 an increased steady-state level of EP was noted in the presence of calmodulin. However, &e to the slow rate of EP formation in the absence of Mg /see Fig. 11, a real steady-state may not have been reached in these experiments 120-30 second incubation periods/, and an
307
underestimation of the calcium-induced EP level possibly also occurred. Rega and Garrahan 1161, based on EP-dephosphorylation measurements, conclude that calmodulin also increases the turnover of the calcium pump cycle. The large amount of hydroxylamine-insensitive, magnesium-dependent membrane phosphorylation in IOVs shows the presence of active, membrane-bound protein kinase enzymes in this system. The actual role of these membrane phosphorylations is still largely unknown /see ref. 17,181. Mild proteolytic digestion of the IOV membrane mimics calmodulin action concerning the kinetics of active calcium transport 191. At the same time this proteolytic treatment decreases the steady-state value of the calcium-induced phosphoprotein formation. We have no ready explanation for this finding, but it emphasises again the multi-step characteristic of the calcium pump cycle and the possibility of one effector acting at more than one site in this cycle. The elimination of calmodulin action by trypsin digestion and a significant decrease in the molecular weight of the calcium-induced phosphoprotein /from 140150 000 to 110-120 OOO/ suggest that a calmodulin-binding regulatory subunit of the pump is digested at the internal membrane surface. According to our preceding paper /9/, probably this subunit determines the "calcium affinity" of the pump. As it is demonstrated in this paper, SH-reagents, inhibiting active calcium transport, also inhibit the formation of the calcium-induced phosphoprotein. As these reagents do not affect the ATP-, calcium- and calmodulin-binding characteristicsof the system /9/, their action is most probably concerned with the inhibition of the intramolecular conformation changes producing the phosphorylated protein intermediate and calcium translocation. ACKNOWLEDGEMENTS This work was supported by the Scientific Research Council, Ministry of Health, Hungary /6-03-0306-01-1/G&/. The authors wish to thank Mrs. II. Sarkadi for the skilful technical assistance. REFERENCES 1. 2.
Katz, S., and Blostein, R. 119731. Calcium-dependent phosphorylation of erythrocyte membranes. Fed. Proc. 32, 287. 2+ Katz, S., and Blostein, R. 119751. Ca -stimulated membrane phosphorylation and ATPase activity of the human erythrocyte. Biochim. Biophys. Acta 389, 314-324.
308
3.
4. 5. 6.
7.
8.
9.
Knauf, P.A., Proverbio, F., and Hoffman, J.F. 119741. Electrophoretic separation of different phosphoproteins associated with Ca-ATPase and Na+K-ATPase in human red cell ghosts. J. Gen. Physiol. 63, 324-336. Rega, A-F., and Garrahan, P.J. /1975/. Calcium ion-dependent phosphorylation of human erythrocyte membranes. J. Membrane Biol. 22, 313-327. Schatzmann, H.J., and Biirgin, H. 119781. Calcium in human red blood cells. Ann.N.Y.Acad.Sci. 307, 125-147. Szdsz, I., Hasitz, M., Sarkadi, B2+ and Gardos, G. 119781. Phosphorylation of the Ca pump intermediate in intact red cells, isolated membranes and inside-out vesicles. Mol. Cell. Biochem. 22, 147-152. Wolf, H.U., Dieckvoss, G., and Lichtner, R. /1937/. Purification and properties of high-affinity Ca -ATPase of human erythrocyte membranes. Acta Biol. Med. Germ. 36, 847-858. Niggli, V., Penniston, g+T., 9qd Carafoli, E. 119791. Purification of the /Ca + Mg /-ATPase from human erythrocyte membranes using a calmodulin affinity column. J. Biol. Chem. 254, 9955-9958. Sarkadi, B., Enyedi, A., and Gbrdos, G. 119801. Molecular properties of the red cell calcium pump. I. Effects of calmodulin, proteolytic digestion and drugs on the kinetics of active calcium transport in inside-out red cell membrane vesicles. Cell Calcium
10. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. 119711. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606-2617. 11. Haaker, H., and Racker, 5+ 119791. Purification and reconstitution of the Ca -ATPase from plasma membranes of pig erythrocytes. J. Biol. Chem. 254, 6598-6602. 12. Carafoli, E., Niggli, V., and Penniston, J2T. /12$0/. Purification and reconstitution of the /Ca + .Mg /-ATPase of the erythrocyte membrane. Proc. New York Acad. Sci., in the press. 13. Gietzen, K Seiler, S., Fleischer, S., and Wolf, H.U. dl~$Jl. Ca2' transport by reconstituted high-affinity -ATPase of human erythrocytes. In: Function and Molecular Aspects of Biomembrane Transport leds. E. auagliariello, et al./; pp 519-522, Elsevier, Amsterdam 14. Garrahan, P.J., and Rega, A2F. 119781. Activation of partial reas$ions of the Ca -ATPase from human red cells by Mg and ATP. Biochem. Biophys. Acta, 513, 59-65. 15. Muallem, S., and Karlish, S.J.D. /1980/. Regulatory raction between calmodulin and ATP on the red cell ;ir? Pump- Biochim. Biophys. Acta, 597, 631-636. 16. Rega, A-F., and Garrahan, P.J. /1980/. Ef&cts of calmodulin on the phosphoenzyme of the Ca -ATPase of human red cell membranes. Biochim. Biophys. Acta, 596, 487-489.
17. Greenquist, A.C., and Shohet, S.B. /1975/. Phosphorylation and dephosphorylation in the erythrocyte membrane. In: Erythrocyte Structure and Function /eds. G.J. Brewer 1, pp 515-534, Alan R. Liss, New York. 18. Sarkadi, B. 119801. Active calcium transport in human red cells. Biochim. Biophys. Acta, in the press.
310
MOLECULAR
PROPERTIES
OF THE RED CELL CALCIUM PUMP
11,EFFECTS OF CALMODULIN, PROTEOLYTIC DIGESTION AND DRUGS ON THE CALCIUM-INDUCED MEMBRANE PHOSPHORYLATION BY ATP IN INSIDE-OUT RED CELL MEMBRANE VESICLES ~n~;r~~~pQi2,
B. Sarkadil,
Ilma Szaszl, G. Bot2, and
.
National Instftute of Haematology and Blood Transfusion, 1113 Budapest , and Institute of Mepcal Chemistry, University Medical School, Debrecen , Hungary I + reprint requeststo GG / ABSTRACT
In inside-out hy$n red cell membrane vesicles /IOV/, in th52absence of Mg , the only calcium-induced labelling by g P-ATP occurs in a 140-150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated int?$mediate /EP/ of the calcium pump. In calcium-induced phosphorylation is the presence of Mg accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accels+ates EP formation both in the absence and presence of Mg . Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVS, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of In trypsin-digested IOVs the molecular weight of the %. P-1,abelled EP is shifted to lower values / 110-120 000 I We suggest that trypsin digestion cleaves off a 20-40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule. INTRODUCTION Calcium-dependent formation of a phosphorylated prgtein intermediate of the red cell calcium pump from P-ATP has been shown by several authors lrefs. l-S/. d Investigation of the effects of regulatory proteins and various membrane modifications on the phosphoenzyme
formation may significantly contribute to our better understanding of the molecular properties of the calcium pump enzyme. It is a special advantage in using IOVs that changes in transport properties can be directly compared to the alterations in membrane phosphorylation. In close connection with our preceding paper in this volume /9/ we describe here the effects of calmodulin, trypsin digestion and SH-reagents on the calcium-induced membrane phosphorylation in IOVs. MATERIALS
AND METHODS
The source of the chemicals used, the methods for IOV and calmodulin preparation3yere the same as described in our preceding paper 191. d P-ATP with a specific activity of 600 GBqlmmol was purchased from the Radiochemical Centre, Amersham. Phosphorylation of IOVs was carried out in reaction media containing 130 mM KCl, 17 mM imidazole-HCl, EDTA, CaC12, MgC12 and calmodulin as indicated, and 0.25-O-5 mg IOV protein in a final Volume 8f 0.5 ml. The tubes we'& preincubated for 5 min at 37 C and then cooled to39 C. The reaction was started by the quick addition of d P-ATP to obtain a fin+ concentration of le. The tubes were incubated at 0 C under vigorous stirring and the reaction was stopped by the addition of 4 ml of ice-cold 6% trichloroacetic acid /TCA/, containing 1 mM ATP and 10 mM Pi. The suspension was supplemented with 2 mg of bovine albumine, centrifuged at 10 000 x g for 5 min, and washed three times with 5 ml of the TCA-ATP-P. solution. The washed precipit3te was dissolved in l%isodium dodecyl sulfate /SDS/. P radioactivity was measured in an Intertechnique SL-30 liquid scintillation spectrometer in 2:l mixtures of scintillation liquid /Liquid Scintillator, Nuclear E.L., diluted with toluenej and Triton X-100. Hydroxylamine treatment of the phosphorylated proteins was carried out according to Rega et al. 141. SDS-polyacrylamide gel electrophoresis was performed according to Fairbanks et al. /lOI with a current of 8-10 mA/tube. From three identical gels one was stained for proteins with Coomassie-blue R-250, and3the other two were P radioactivity. cut into 2.5-5 mm slices for measuring Molecular weights were estimated from the relative mobilities of spectrin 2, band 3 and band 5, as references, by using both ghosts and IOV membranes. Trgpsin digestion of the IOV membrane was carried out at 37 C in a medium containing 130 mM KCl, 17 mM imidazole-HCl IpH 7.01, with 0.2 mg trypsinjmg IOV protein for 3 min. Trypsin treatment was stopped by the addition
300
of S-fold excess of soybean trypsin inhibitor, and the IOVs were washed three times in the KCl-imidazole solution. In the control experiments a similar incubation was carried out with the trypsin + trypsin inhibitor mixture. RESULTS In our preceding paper /9/ we showed that the addition of calmodulin or mild trypsin digestion of the IOV membrane significantly stimulate active calcium uptake. Trypsin digestion eliminates calcium-dependent calmodulin binding to the membrane and calmodulin activation of calcium uptake. In the following experiments we investigated the membrane phosphorylation under these conditions. Fig. 1 shows the time course of the cg$cpt;;p -induced membrane phosphorylation in IOVs from x as studied in Ca-EDTA buffers, that is in the absence of free magnesium ions.
EDrA
*co=:
0%
% -A--------_--_AtCALMODULIN A CONTROL
___------_
0
.3’ TK YP.S/N o -‘CALMODULIN
120
60
+ SEC
Fig. 1. Effects of calmodulin and trypsin digestion on the EP-formation of the calcium pump in IgVs. Phosphorylation was carried out at 0 C in media containing 130 mM KCl, 17 my imidazole-IX1 /P&&7.0/, 0.5 mq Tris-EDTA, 0.55 x@4 CaC12 and 1pM d P-ATP. IOV concentration was 0.4 mg/ml, cslmodulin 3 mg/ml. Trypsin pretreatment was rried out as described in the Method29ection. s9 P-incorporation in the absence of Ca was less than 0.05 picomol/mg IOV protein. 301
1
2
3
4
5
6
7
0-Q
CONTROL
e---o
3’ TRYPSIN
6
9
MIGRATION
1
2
3
4
5
6
7
8
!O DISTANCE
11 (Cm)
-
CONTROL
-----
3’ TRYPSIN
9
MIGRATION
10
II
DISlANCf
12 (c rr
Fig. 2a. SDS-polyacrylamide gel electropho52togram of IOV proteins phosphorylated by d P-ATP in the p.resence of Ca-CDTA for 60 seconds. For the conditions of phosphorylation see the legend of Fig. 1. Fig. 2b. Polypeptides of the IOVs separated by SDS--polyacrylamide gel electrophoresis.
302
32 Ca-induced P-incorporation is significantly accelerated by calmodulin although the steady-state level of phosphorylation is only slightly elevated. In contrast to this, trypsin digestion /stimulating active calcium transport similarly to calmodulinl reduces the steady-state value of membrane-labelling. There is no effect of calmodulin on membrane phosphorylation in the trypsin-treated IOVs. As it is shown in Fig. 2, when examining the phosphorylated membrane proteins by SDS-polyacrylamide gel electrophoresis, in IOVs phosphorylated in a Ca-EDTA medium the only significant labelling occurs in a 140-150 000 molecular weight fraction. When phosphory&tion is accelerated by calmodulin, the location of the P-1abelling is unchanged /not shown/, while in trypsin-treated IOVs a significant shift in the molecular weight of the phosphoprotein occurs. The calculated molecular weight of the calcium pump EP in the trypsin-treated IOVs is 110-120 000.
~+CALMODULIN
__e_-------‘-_‘.
OB
* 0
120
60
SEC
Fig. 3. Effects of Mg2+ and Mg2+ + calmodulin on the membrane phosphorylation in IOVs. Curve A shows membrane phosphorylation in a Ca-EDTA buTfer, as described in Fig. 1. The other phosphorylation media contained 5 mM MgC12 and either 50 ,uM CaCl containing media, respectively. concentration = 3 ug/ml.
303
Calmodulin
.-CONTROL D---0
HYOROWLAMINE
co=++hg=+
SDS-PAGE
-.
CONrROL
o----o+tib-DROXFUhVNE
1
2
3
4
5
6
0
7
MIGRANON
9
10 D/STANCE
11 [cm)
Fig. 4a. SD,$PAGE of IOV proteins phosphorylated by d P-ATP in the presence of 50 pM EGTA + . 5 mMMgC1 Fig. 4b. SDQZPAGE 8 f IOV proteins phosphorylated by d P-ATP in the presence of 50 m CaC12+ 5 mM MgCl . For the c2 nditions of phosphorylation and hydroxylamine treatment see the Hethod section.
304
SDS
PAGE
co=*+/ug*+ Ad A.--A3
CONTROL rRYPS,N
~..&.A..&.& I
2
3
4
5
6
7
6
MIGRATION
9
IO
DISTANCE
tl
12
(cm)
Fig. 5a-5b. Effect of trypsin-pretreatment on 32P-incorporation. 5a: SDS-PAGE of IOV proteins phosphorylated in the presence of 50 ,uM EGTA + 5 mY MgCl . 5b: SDS-PAGE of IOV proteins phosphoryfated in the presence of 50 @4 CaCl + 5 W MgC12. For the conditions of phosphorylz ation and trypsin digestion see the -Yethod section.
305
As it is shown in Fig. 3, calcium-induced membrane phosphorylation is rapid in the presence of magnesium ions, and it can be further accelerated by addition of calmodulin. The T f r calcium-induced phosphorylation kc20 8C in a Ca-EDTA buffer and the decreases this value to about 10 seconds. + calmodulin reduces T to less than 4 seconds. The presence of magnesium i ns increases the steady-state lLJ2 level of the phosphoenzyme by about 30%. Fig. 4 shows the SDS-polyacrylamide gel elec-t,2;h;oc;tograms of the IOV membsqne proteins labelled by in the presence of Mg and in the absence /Fig. 4aj or in the presence /Fig. 4b/ sf calcium ions. In both cases considerable amount of P-incorporation into spectrin 2 and band 3 proteins is observed but these phosphate groups are in hydroxylamine-insensitive nds. The only calcium99 P-incorporation is induced, hydroxylamine-sensitive observed again in the 140-150 000 dalton, protein-poor region of the electrophoretogram. As it 32 shown in Fig. 5, trypsin-treatment of the I,OVs decreases P-incorporation and shifts the molecular weight of the calcium-induced phosphoprotein to lower values /llO-120 000 dalton Fig. 5b/. A large portion of spectrin and band 3 is degraded by trypsin treatment of the IOVs /see also Fig. 2b/, and therefore several different phosphorylated polypeptides appear at the lower molecular weight regions ctrophoretogram /Fig. Sa,b/. These fractions P in hydroxylamine-insensitive bonds, while the calcium-induced phosphoprotein is also hydroxylamine-sensitive in the trypsin-digested IOVs. Fig. 6 shows the effect of PCMB-treatment on the calcium-induced EP-formation in IOVzj2in Ca-EDTA buffers. PCMB-pretreatment strongly reduces P-incorporation but a calmodulin stimulation of the EP-formation is present321so in these vesicles. A similar strong reduction of the PVaz';"o;a;;T;s+o;;3f-ved in the .
DISCUSSION In red ~$11 rn2p rane fragments /l-6/ as well as in purified Ca + Mg -ATPase preparations /7-S/ a specific, calcium-induced phosphorylation of a 140-150 000 dalton molecular weight protein was demonstrated. The isolated protein, wh incorporated into lipid vesicles, catalyzed an ATP + Mgsp -dependent accumulation of calcium ions /11-131. Based on these data this protein fraction can be regarded as the molecular basis of the red cell calcium PumP. It has been also demonstrated that the formation of the phosphoprotein /EP/ does not require the presence of magnesium ions /4,5,6/, although t@ process is accelerated by magnesium /4,5/. Mg plays a basic role in the dephosphorylation of the pump protein /4,5,14/.
306
iOTA
.
co*+,
0-c
A~A-Pcma /
A
/ 0
__-_-_---.---
--------_-~+PCMB+CALMOWLIN O+PCMB
60
120
* SEC
Fig. 6. Effect of PCMB-pretreatment on the EP-formation in IOVs. IOVs were preincubated for 5 min with 20 nanomoles PCMB/mg IOV protein and the control IOVs were reactivated by the addition of 1 nY' dithiothreitol. EP formation was measured as described in the legend to Fig. 1. According to the experimental results reported ig2the present paper, membrane phosphorylation in IOVs by E P-ATP in a Ca-EDTA medium, that is in the absence of free magnesium, results in the appearence of a single phosphoprotein with a molecular weight of 140-150 000. Addition of magnesium ions accelerates phosphorylation of this protein, however, due to protein kinase action, several other membrane proteins, such as spectrin 2 and band 3, are also phosphorylated. Calmodulin stimulates EP-formation both in the absence and presence of magnesium and this protein does not affect calcium-independent phosphorylation processes. These findings are in accordance with those by Muallem and Karlish 1151 and Rega and Garrahan 1161, and indicate that the predominant effect of calmodulin in stimulating the calcium pump is to increase the rate of calcium-induced phosphoprotein formation. In the above two reports 115,161 an increased steady-state level of EP was noted in the presence of calmodulin. However, &e to the slow rate of EP formation in the absence of Mg /see Fig. 11, a real steady-state may not have been reached in these experiments 120-30 second incubation periods/, and an
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underestimation of the calcium-induced EP level possibly also occurred. Rega and Garrahan 1161, based on EP-dephosphorylation measurements, conclude that calmodulin also increases the turnover of the calcium pump cycle. The large amount of hydroxylamine-insensitive, magnesium-dependent membrane phosphorylation in IOVs shows the presence of active, membrane-bound protein kinase enzymes in this system. The actual role of these membrane phosphorylations is still largely unknown /see ref. 17,181. Mild proteolytic digestion of the IOV membrane mimics calmodulin action concerning the kinetics of active calcium transport 191. At the same time this proteolytic treatment decreases the steady-state value of the calcium-induced phosphoprotein formation. We have no ready explanation for this finding, but it emphasises again the multi-step characteristic of the calcium pump cycle and the possibility of one effector acting at more than one site in this cycle. The elimination of calmodulin action by trypsin digestion and a significant decrease in the molecular weight of the calcium-induced phosphoprotein /from 140150 000 to 110-120 OOO/ suggest that a calmodulin-binding regulatory subunit of the pump is digested at the internal membrane surface. According to our preceding paper /9/, probably this subunit determines the "calcium affinity" of the pump. As it is demonstrated in this paper, SH-reagents, inhibiting active calcium transport, also inhibit the formation of the calcium-induced phosphoprotein. As these reagents do not affect the ATP-, calcium- and calmodulin-binding characteristicsof the system /9/, their action is most probably concerned with the inhibition of the intramolecular conformation changes producing the phosphorylated protein intermediate and calcium translocation. ACKNOWLEDGEMENTS This work was supported by the Scientific Research Council, Ministry of Health, Hungary /6-03-0306-01-1/G&/. The authors wish to thank Mrs. II. Sarkadi for the skilful technical assistance. REFERENCES 1. 2.
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10. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. 119711. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606-2617. 11. Haaker, H., and Racker, 5+ 119791. Purification and reconstitution of the Ca -ATPase from plasma membranes of pig erythrocytes. J. Biol. Chem. 254, 6598-6602. 12. Carafoli, E., Niggli, V., and Penniston, J2T. /12$0/. Purification and reconstitution of the /Ca + .Mg /-ATPase of the erythrocyte membrane. Proc. New York Acad. Sci., in the press. 13. Gietzen, K Seiler, S., Fleischer, S., and Wolf, H.U. dl~$Jl. Ca2' transport by reconstituted high-affinity -ATPase of human erythrocytes. In: Function and Molecular Aspects of Biomembrane Transport leds. E. auagliariello, et al./; pp 519-522, Elsevier, Amsterdam 14. Garrahan, P.J., and Rega, A2F. 119781. Activation of partial reas$ions of the Ca -ATPase from human red cells by Mg and ATP. Biochem. Biophys. Acta, 513, 59-65. 15. Muallem, S., and Karlish, S.J.D. /1980/. Regulatory raction between calmodulin and ATP on the red cell ;ir? Pump- Biochim. Biophys. Acta, 597, 631-636. 16. Rega, A-F., and Garrahan, P.J. /1980/. Ef&cts of calmodulin on the phosphoenzyme of the Ca -ATPase of human red cell membranes. Biochim. Biophys. Acta, 596, 487-489.
17. Greenquist, A.C., and Shohet, S.B. /1975/. Phosphorylation and dephosphorylation in the erythrocyte membrane. In: Erythrocyte Structure and Function /eds. G.J. Brewer 1, pp 515-534, Alan R. Liss, New York. 18. Sarkadi, B. 119801. Active calcium transport in human red cells. Biochim. Biophys. Acta, in the press.
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