Phosphorylation of the Red Blood Cell Membrane during the Active Transport of Ca ++ YOUNG
NAM C H A a n d K W A N G S O O LEE
From the Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203
ABSTRACT The phosphorylation of red blood cell membrane fragments (RBCMF) during Ca ++ transport was investigated. When red cell membrane fragments are incubated with [y-a2P]ATP under the experimental condition which minimizes the phosphorylation of Na+-K+-ATPase, RBCMF are labeled in the presence of Mg ++ without Ca ++. When Ca ++ is added, the labeling decreases due to dephosphorylation of RBCMF. The initial reaction of phosphorylation is reversed in the presence of excess ADP. The treatment of RBCMF with n-ethylmaleimide (NEM) does not interfere with the initial phosphorylation reaction, but blocks the dephosphorylation in the presence of Ca ++. These data suggest that the enzymatic sequence of the Ca ++ transport mechanism may be very similar to that of the Na + transport mechanism. I N T R O D U C T I O N
It has b e e n shown previously that the red b l o o d cell m e m b r a n e has an active t r a n s p o r t m e c h a n i s m for o u t w a r d extrusion o f Ca ++ ( S c h a t z m a n n a n d Vincenzi, 1969; Lee a n d Shin, 1969), a n d A T P is the specific e n e r g y source o f the Ca ++ t r a n s p o r t system (Vincenzi a n d S c h a t z m a n n , 1967; Cha et al., 1971). Previously, it was f o u n d that w h e n r e d blood cells are f r a g m e n t e d , red blood cell m e m b r a n e f r a g m e n t s (RBCMF) were f o u n d to take u p Ca ++ a n d this Ca ++ u p t a k e is a c c o m p a n i e d by A T P hydrolysis by Mg++-activated A T P a s e (Lee a n d Shin, 1969; Cha et al. 1971). It has b e e n s u g g e s t e d that this Ca ++ u p t a k e is d u e to the p r e s e n c e o f inside o u t vesicles in the p r e s e n t system a l t h o u g h not all o f the vesicles are inside out (Steck et al., 1970; W e i n e r a n d Lee, 1972). R B C M F were f o u n d to have both Na+-K+-ATPase a n d Ca++-ATPase which are intimately associated with the s o d i u m a n d calcium t r a n s p o r t m e c h a n i s m s , respectively ( S c h a t z m a n n , 1966; Skou, 1965; Lee a n d Shin, 1969). N u m e r o u s studies on the s o d i u m t r a n s p o r t m e c h a n i s m in the r e d blood cell m e m b r a n e indicate the i n v o l v e m e n t o f p h o s p h o r y l a t e d i n t e r m e d i a t e s d u r i n g the e n z y m a t i c process (Albers, 1967; Albers et al., 1968; Post et al., 1969). T h e r e f o r e , an a t t e m p t has b e e n m a d e to investigate the possibility that the similar p h o s p h o rylation o f R B C M F m a y also be involved with the calcium t r a n s p o r t m e c h a n i s m . It was f o u n d that the calcium t r a n s p o r t m e c h a n i s m a p p e a r s to s h a r e similarities with the s o d i u m t r a n s p o r t m e c h a n i s m with r e g a r d to p h o s p h o r y l a t i o n m e c h a nisms involved with the enzymatic action. THE JOURNAL
OF G E N E R A L P H Y S I O L O G Y
" VOLUME
67,
1976 " pages
251-261
251
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THE JOURNAL
OF GENERAL PHYSIOLOGY
' VOLUME
67 " 1976
METHODS
Preparation of RBCMF RBCMF were prepared according to the procedure used previously in this laboratory (Cha et al., 1971).
Measurement of ATPase Activities T h e Ca++-ATPase activity (in the presence of Mg ++) was measured by incubating RBCMF in the standard medium for l0 rain at 37°C. T h e standard m e d i u m for Ca++-ATPase contained: 120 mM KCI; 30 mM histidine-imidazole buffer, pH 7.0; 3 mM MgClz; 2 mM ATP; and 0.5 mM CaCl2. T h e Na+-K+-ATPase was measured by incubating RBCMF for 10 min at 37°C in the medium containing: 100 mM NaC1; l0 mM KCI; 30 mM histidineimidazole buffer, pH 7.0; 3 mM MgCl2; and 2 mM ATP.
Measurement of A TP-Dependent Ca ++ Uptake by RBCMF T h e Ca ++ uptake of RBCMF was measured according to the method of Cha et al. (1971) employing 4"~Ca. In this method, the radioactivity of RBCMF after incubation of RBCMF with 4"~Ca in the presence of A T P was measured.
Measurements of Phosphorylation of RBCMF In phosphorylation experiments, RBCMF (1 mg protein/ml reaction mixture) were incubated at 37°C in a standard medium for phosphorylation which contained 120 mM KCI, 30 mM histidine-imidazole buffer (pH 7.0), 0.1 mM EGTA (ethylene glycol bis[/3aminoethylether]N,N'-tetraacetic acid), 2 p,M radioactive A T P (1 /~Ci/M), and 3 mM MgCI2. Before the actual incubation, RBCMF were equilibrated at 37°C for 3 min in the standard medium without MgC12 and the reaction was started by a d d i n g MgCl~ into the mixture after the equilibration. T h e absence of Na + from the reaction mixture in the presence of KC1 was essential to exclude any possibility that the phosphorylation might result from Na+-dependent phosphorylation of Na+-K+-ATPase present in RBCMF. After various periods of incubation the reaction was stopped by adding 5 ml of ice-cold 5% trichloroacetic acid (TCA) (with 0.01 mM A T P and 0.1 mM Pi) to the reaction mixture and placing it in an ice bath immediately. When Ca++-dependent dephosphorylation was desired, Ca ++ (0.2 raM) was introduced at an appropriate time after the reaction had been started, and the incubation was continued until the reaction was stopped by adding TCA. T h e mixture was then centrifuged at 2,000 g for 10 rain at 4°C. T h e precipitate was washed twice with 5 ml of the same TCA solution, and the final precipitate was suspended in 2 ml of 0.1 N NaOH. A half milliliter of the suspension was applied to a planchet t)r put into a scintillation vial with 10-ml Bray's solution (Bray, 1960), and counted either in a thin-window Nuclear Chicago gas flow counter or in a Packard scintillation counter (Packard I n s t r u m e n t Co., Inc., Downers Grove, Ill.), respectively. RESULTS
ATPase Activities and ATP-Dependent Ca ++ Uptake T h e N a + - K + - a c t i v a t e d a n d Ca++-activated A T P a s e (in the p r e s e n c e o f Mg ++) o f R B C M F were 0.45 a n d 1.68 /~mol P i / m g p r o t e i n / h , r e s p e c t i v e l y . T h e Ca ++ uptake of R B C M F in the presence a n d absence of A T P after 1 h of i n c u b a t i o n were 45.2 a n d 6.1 n m o l / m g p r o t e i n , r e s p e c t i v e l y . T h i s i n d i c a t e s t h a t t h e p r e s e n t R B C M F has a n active Ca ++ t r a n s p o r t m e c h a n i s m .
CHA ANO LEE Phosphorylationof Erythrocyte Membraneduring Ca++Active Transport
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Binding of [w32P]ATP and [y32P]ATP to R B C M F
Radioactive A T P , labeled at oL or y position was incubated in the standard m e d i u m for p h o s p h o r y l a t i o n for 30 and 60 rain with RBCMF, and the radioactivity taken u p by R B C M F d u r i n g the incubation period was m e a s u r e d . Results are shown in Table I. TABLE
I
BINDING OF [o~-32P]ATP AND [y-32P]ATP T O RBCMF Time incubation Treatment 30 min [a-azP]ATP
[~,-s2P]ATP
60 min
558
670
5962
6169
n2P-labeling is expressed in terms o f counts per minute per 0.25 m g protein o f RBCMF.
As can be seen in this table, the radioactivity o f RBCMF after T C A t r e a t m e n t was significant with [T-3zP]ATP, but not with [ot-32P]ATP. This indicates that RBCMF is p h o s p h o r y l a t e d with [T-32P]ATP only, and not by [~-3zP]ATP. In all the following e x p e r i m e n t s , only [T-32P]ATP was e m p l o y e d for all phosphorylation experiments. It should be noted that the phosphorylation o f RBCMF by [y3zP]ATP occurs in the m e d i u m which contains 120 mM KC1 and no NaCI, the condition which minimizes the possibility o f p h o s p h o r y l a t i o n o f Na+-K+-ATPase by A T P . Influence of M g ++ and Ca ++ on Phosphorylation
Radioactive labeling o f R B C M F was m e a s u r e d after incubation o f RBCMF in mixtures containing the basic c o m p o n e n t s (BC) o f 120 mM KCI, 30 mM histidine-imidazole b u f f e r (pH 7.0), 0.1 mM E G T A , 2/zM radioactive A T P (1 txCi/M) and the following variations: BC only, (no Mg ++, no Ca ++ in Fig. 1), BC + 3 mM MgC12 (Mg ++ in Fig. 1), BC + 0.5 mM CaC12 (Ca ++ but no Mg ++ in Fig. 1), BC + 3 m M MgCl2 + 0.5 m M CaCl2 (Ca ++ at zero time in Fig. 1). Also, in some e x p e r i m e n t s with BC + 3 mM MgCI2 (Mg ++ in Fig. 1) 0.5 mM CaClz was a d d e d 5 rain after the incubation started. Results are shown in Fig. 1. In the presence o f Mg ++ alone (Mg ++ in Fig. 1), RBCMF were very rapidly p h o s p h o r y l a t e d in the first few seconds, and then c o n t i n u e d at a diminished rate d u r i n g the r e m a i n d e r of the 10-rain incubation period. Without Mg ++ in media (no Mg ++, no Ca ++, and Ca ++ but no Mg ++, in Fig. 1), no significant labeling o f R B C M F o c c u r r e d . Although it is not shown in Fig. 1, the addition o f KCI u p to 5 rain after the incubation in Mg ++ solution did not have any effect on the labeling o f RBCMF. W h e n both Ca ++ and Mg ++ were present f r o m the start o f the reaction (Ca ++ at zero time in Fig. 1), R B C M F were p h o s p h o r y l a t e d slightly within the first few seconds and progressed slowly d u r i n g the rest o f the incubation time. T h e s e results indicate that the presence o f Mg ++ and Ca ++ are r e q u i r e d for phosphorylation and d e p h o s p h o r y l a t i o n o f RBCMF, respectively. T h u s , the greatest
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labeling o f RBCMF occurs when media contained Mg ++ only, a n d the labeling decreases when Ca ++ is also present in media.
Influence of Addition of ADP on Labeling during Incubation Experiments were p e r f o r m e d where 0.2 mM ADP was a d d e d 5 min after R B C M F were incubated in the standard m e d i u m for p h o s p h o r y l a t i o n . It should be m e n t i o n e d that the s t a n d a r d m e d i u m is the same as the m e d i u m noted 7-
6-5.
~
o
Mg++
C~4"
Ca++at5th rain
x
E3O. (,.) 2-
| ~
_~-÷
l "
01
Ca++otZerot~me CO+ +
f
~
x
butnoMg+ +
(-)(-;
rain incubotion
FIGURE 1. Labeling of RBCMF after incubation under varii/us experimental conditions. Experimental conditions: All media contained 120 mM KC1, 30 mM histidine-imidazole buffer (pH 7.0), 0.1 inM FGTA, and 2 /xM radioactive ATP. In addition: ( - ) (-), none; Mg++, 3 mM MgC12; Ca ++ but no Mg++, 0.5 mM CaCI2; Ca ++ at zero time, 3 mM MgCI~, and 0.5 mM CaCI2; Ca ++ at 5th min, 3 nlM MgCI2 from the beginning, and 0.2 mM CaCI,, at 5 rain. "Mg ++'' in Fig. 1. As shown in Fig. 2, the addition o f ADP caused an initial rapid fall o f radioactivity o f RBCMF, which was followed by an increase in labeling at the same rate as that o f the control with Mg ++ alone. I n Fig. 2, the effect o f addition o f nonradioactive A T P (0.2 mM) d u r i n g the incubation is also shown. T h e labeling was diluted by formation of nonradioactive p h o s p h a t e intermediaries, when cold A T P was a d d e d . Since Ca ++ is absent in the m e d i u m , the effect o f A D P in decreasing the labeling may be d u e to the shift of reaction E + A T P ,~ A D P + E - P to the left, a l t h o u g h o t h e r u n k n o w n mechanisms maybe responsible for this p h e n o m e n o n .
Influence of EDTA on the Response of R B CMF to the Addition of ADP or Ca ++ Five minutes after R B C M F were incubated in the standard m e d i u m for phosphorylation, E D T A (ethylene diamine tetraacetate) in a c o n c e n t r a t i o n o f 5 mM was a d d e d to the reaction mixture. Results are shown in Fig. 3. As can be seen in this figure, the addition o f E D T A is followed by the cessation o f f u r t h e r labeling o f RBCMF. This probably is due to the chelation of Mg ++ by E D T A , which
CHA AND LEE Phosphorylationof Erythrocyte Membraneduring Ca++Active Transport
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results in the lack o f Mg ++ necessary for the p h o s p h o r y l a t i o n reaction. W h e n 0.2 mM A D P is a d d e d immediately after E D T A , no effect o f A D P is observed. This s u p p o r t s the suggestion m a d e previously that the effect o f A D P is d u e to the reversal o f the initial p h o s p h o r y l a t i o n reaction, which requires Mg ++. T h e addition o f Ca ++ after E D T A still causes the decrease in labeling o f R B C M F probably because the d e p h o s p h o r y l a t i o n requires the presence o f Ca ++ but not Mg ++ .
S 9
CONTROL ADP
70 0 x
6-
5"
j
E
o. u
4-
32-
/
ATP 0.2mM
min i n c u b a t i o n
FIGURE 2. Effects of ADP and ATP on labeling of RBCMF. Experimental conditions: Incubation medium contained 120 mM KCI, 30 mM histidine-imidazole buffer (pH 7.0) 0.1 mM EGTA, 2 p.M radioactive ATP, and 3 mM MgCI2. ADP (0.2 raM) or ATP (0.2 mM) was added 5 rain after incubation was started.
Influence of A D P and Ca ++ on Labeling of NEM-Treated R B C M F R B C M F were treated with 0.2 mM NEM at 37°C for 20 rain b e f o r e the incubation. T h e NEM-treated R B C M F were t h e n incubated with [y-~2P]ATP in the s t a n d a r d m e d i u m for p h o s p h o r y l a t i o n and either A D P or Ca ++ was a d d e d 5 min after incubation. T h e radioactivity o f R B C M F d u r i n g various stages o f experiments is shown in Fig. 4. As can be seen in this figure, the addition o f Ca ++ does not influence the labeling o f N E M - t r e a t e d RBCMF. T h e addition o f ADP, however, still causes a fall in labeling o f N E M - t r e a t e d R B C M F similar to that observed with n o n t r e a t e d RBCMF. This suggests that N E M t r e a t m e n t blocks d e p h o s p h o r y l a t i o n in the presence o f Ca ++, but does not block the effect of ADP, which acts on the initial p h o s p h o r y l a t i o n step.
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Addition of 45Ca to the Previously Phosphorylated RBCMF Five minutes after R B C M F were incubated in a m e d i u m c o n t a i n i n g 20 g M radioactive A T P in the p r e s e n c e o f Mg ++ alone, 45Ca (1/~Ci/mM) was i n t r o d u c e d into the reaction m i x t u r e . T h e 10-times g r e a t e r concentration o f A T P above the A T P concentration used in the p h o s p h o r y l a t i o n e x p e r i m e n t s was used h e r e , because a significant a m o u n t o f 45Ca u p t a k e was n e e d e d in this e x p e r i m e n t . As can be seen in Fig. 5, the level o f p h o s p h o r y l a t i o n d e c r e a s e d to a certain level a n d r e m a i n e d so, while the 45Ca was continuously b e i n g t a k e n up by the R B C M F . T h u s , it a p p e a r s that a steady level o f p h o s p h o r y l a t i o n is m a i n t a i n e d d u r i n g the active Ca ++ u p t a k e in R B C M F , a l t h o u g h f u r t h e r investigation is r e q u i r e d to ascertain this aspect.
~
7, 0 0
o x
E o
6"
5" 4-
/
CONTROL
EDTAADP aflerEDTA
after EOTA
rain incubation
FIGURE 3. Effect of EDTA and labeling of RBCMF. Experimental conditions: Incubation medium contained 120 mM KCI, 30 mM histidine-imidazole buffer (pH 7.0), 0.1 mM EGTA, 2 txM radioactive ATP, and 3 mM MgC12. Seven minutes after the reaction after additions were made. EDTA; EDTA (5 mM). ADP after EDTA; EDTA (5 raM) then ADP (0.2 mM). Ca ++ after EDTA; EDTA (5 raM) then CaCl~ (0.5 mM). DISCUSSION
It has b e e n established t h a t Na+-K+-activated A T P a s e a n d Ca++-activated A T P ase are intimately associated with o u t w a r d extrusion o f Na + a n d Ca 2+, respectively (Skou, 1957 a n d 1965; H o f f m a n , 1961; S c h a t z m a n n , 1966; Albers, 1967; Post et al., 1969; S c h a t z m a n n and Vincenzi, 1969; Lee and Shin, 1969). Since the p r e s e n t R B C M F has b o t h o f the above two A T P a s e activities, an a t t e m p t has b e e n m a d e to investigate the similarity o f these two active t r a n s p o r t systems in m o l e c u l a r mechanisms. T h e sequence o f enzymatic steps involved in Na+-K +A T P a s e is intensively studied by m a n y investigators, whereas very little is known
CI~A AND LEE Phosphorylation of Erythrocyte Membrane during Ca ++Active Transport
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with r e g a r d to the e n z y m a t i c sequence o f the Ca ++ t r a n s p o r t system o f Ca ++A T P a s e in RBCMF. With Na+-K+-ATPase, evidence suggests that the following sequential reaction takes place ( B a d e r et al., 1966; Albers et al., 1968; Post et al., 1969): ATP
+ El,
Pi
N a + M g ++ ' ~ EI-P
+ ADP Mg ++ blocked by N E M
+ E2 •
' '
E2-P
+
H~O
I n the p r e s e n t study the possibility that the Ca ++ t r a n s p o r t m e c h a n i s m o f R B C M F may go t h r o u g h a similar sequence has been e x p l o r e d . T h e radioactivities o f R B C M F i n c u b a t e d in the s t a n d a r d m e d i u m for phosp h o r y l a t i o n with either [a-a2p]ATP or [T-a2p]ATP are shown in T a b l e I. T h e data indicate that the t e r m i n a l p h o s p h a t e o f A T P f o r m s an acid stable c o m p l e x with R B C M F u n d e r e x p e r i m e n t a l conditions.
6"
5"
0 o
4-
x E
3-
9, Q.
2-
/ I
t
2
5
7
I0
min incubotion
FmURE 4. Effects of ADP and Ca ++ on NEM-treated RBCMF. Experimental conditions: Same as in Fig. 2. ADP (0.2 raM) and CaCI2 (0.5 raM) were added 5 rain after incubation. T h e participation o f p h o s p h o r y l a t e d i n t e r m e d i a t e s in Na + t r a n s p o r t has b e e n investigated by n u m e r o u s investigators, a n d it is k n o w n that labeling o f the Na +activated m e m b r a n e A T P a s e occurs d u r i n g the t r a n s p o r t o f Na + (Avruch a n d Fairbanks, 1972; Post et al., 1969; Blostein, 1968; K n a u f et al., 1974 a, b). A l t h o u g h the p r e s e n t R B C M F has both Na+-K+-ATPase a n d Ca++-ATPase, the e x p e r i m e n t a l conditions m a k e it unlikely that this labeling o f R B C M F is d u e to the p h o s p h o r y l a t i o n o f Na+-K+-ATPase. T h i s incubation m e d i u m for phosp h o r y l a t i o n contains no Na + which is n e e d e d f o r p h o s p h o r y l a t i o n o f Na+-K +A T P a s e . O n the o t h e r h a n d , the m e d i u m has K + (120 m M ) which mediates d e p h o s p h o r y l a t i o n o f Na+-K+-ATPase. F u r t h e r m o r e , the addition o f K + to the labeled R B C M F did not have a n y effect, whereas the addition o f Ca ++ d e c r e a s e d
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the labeling (Fig. 1). T h e s e data indicate that the labeling o f R B C M F u n d e r the p r e s e n t e x p e r i m e n t a l condition is d u e to the p h o s p h o r y l a t i o n o f Ca++-ATPase of RBCMF. Data in Fig. 1 show that the p h o s p h o r y l a t i o n o f R B C M F p r o c e e d s t h r o u g h o u t the e x p e r i m e n t in the p r e s e n c e o f Mg ++ only. T h u s the initial p h o s p h o r y l a t i o n step a p p e a r s to require Mg ++, but not Ca ++. T h e addition o f Ca ++ to labeled R B C M F is followed by a r a p i d fall o f radioactivity o f RBCMF. T h e r a p i d fall of
~
O
00 3 14
_--_2
o
1
0
5
10
15
30
rain i n c u b a t i o n Double labeling of RBCMF with :~P and 4:'Ca. O .
. . . O :~2p-labeling, FIGURE 5. c.... c a2p-labeling after addition of 4 5 C a + + , x . . . . X 45Ca++-labeling. Experimental conditions: Incubation medium contained: 120 mM KCI, 30 mM histidineimidazole buffer (pH 7.0), 0.1 mM EGTA, 20 ~M radioactive ATP, and 2 mM MgC12. At 5 rain, *"Ca (1 >Ci/mM) was added.
radioactivity o f R B C M F m a y be d u e either to the inhibition o f p h o s p h o r y l a t i o n or acceleration of d e p h o s p h o r y l a t i o n . T h e inhibition of p h o s p h o r y l a t i o n probably d u e to c o m p e t i t i o n o f Ca ++ with Mg ++ a p p e a r s unlikely because in NEMt r e a t e d RBCMF, the addition of Ca ++ has no effect whatsoever on the p h o s p h o rylation as indicated by the continuous labeling o f R B C M F (Fig. 4). T h u s the fall o f labeling after the addition o f Ca ++ p r o b a b l y is d u e to d e p h o s p h o r y l a t i o n o f p h o s p h o r y l a t e d i n t e r m e d i a t e s . Since, as discussed above, the Ca ++ t r a n s p o r t A T P a s e and not Na+-K+-ATPase is p h o s p h o r y l a t e d u n d e r the e x p e r i m e n t a l conditions, it a p p e a r s that the fall o f labeling a f t e r the addition o f Ca ++ is d u e to d e p h o s p h o r y l a t i o n o f Ca ++ t r a n s p o r t A T P a s e in the p r e s e n c e o f Ca ++. In this connection, results o b t a i n e d in the recent work by K n a u f et al. (1974 b) should be m e n t i o n e d . T h e s e w o r k e r s m e a s u r e d p h o s p h o r y l a t i o n of r e d b l o o d cell m e m b r a n e in m e d i a containing Mg ++ a n d Na + (or Na+-K +) at 0°C, a n d f o u n d that the p r e s e n c e o f Ca ++ increased p h o s p h o r y l a t i o n . T w o possibilities were suggested. O n e possibility was that the Ca ++ p h o s p h o p r o t e i n in their study m a y be d i f f e r e n t f r o m that of Ca ++ t r a n s p o r t A T P a s e , since conditions o f their study ( t e m p e r a -
CHA AND LEE
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ture, A T P concentrations, etc.) were d i f f e r e n t f r o m those o f previous works on the Ca ++ t r a n s p o r t A T P a s e . A n o t h e r possibility suggested was that the Ca ++ p h o s p h o p r o t e i n characterized in their work may r e p r e s e n t an i n t e r m e d i a t e in the Mg++-Ca++-ATPase involved with Ca ++ t r a n s p o r t . I f this is the case, then the d i f f e r e n t results obtained in their and the p r e s e n t study may be reconciled by assuming that Ca ++ is r e q u i r e d for the step f r o m E1-P to Ez-P. Since the Ca ++ t r a n s p o r t ATPase is very sensitive to t e m p e r a t u r e and no d e p h o s p h o r y l a t i o n occurs at 0°C (Cha et al., 1971), the presence o f Ca ++ would increase phosphorylation by f o r m i n g E2-P without d e p h o s p h o r y l a t i o n o f this i n t e r m e d i a t e at 0°C in the study o f K n a u f et al. (1974 b). As discussed by the a u t h o r s in their study, a n o t h e r cause o f increased phosphorylation by Ca ++ was the inhibition o f d e p h o s p h o r y l a t i o n o f Na+-K+-ATPase by Ca ++. In the p r e s e n t study, on the o t h e r h a n d , this condition o f increasing p h o s p h o r y l a t i o n by Ca ++ does not exist, since no p h o s p h o r y l a t i o n o f Na+-K+-ATPase presumably occurs in the present e x p e r i m e n t a l conditions. T h u s the present Ca ++ transport A T P a s e seems to require Mg ++ and Ca ++ for the initial p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n , respectively. A n o t h e r aspect to be considered with the p h o s p h o r y l a t e d intermediates o f t h e C a ++ t r a n s p o r t system in the R B C M F in this study is to c o m p a r e the present system with that o f sarcoplasmic reticulum. Previously, the participation o f p h o s p h o r y l a t e d intermediates in the Ca ++ t r a n s p o r t system in sarcoplasmic reticulum o f skeletal muscle has been r e p o r t e d (Hasselbach and Makinose, 1962; Y a m o m o t o and T o n o m u r a , 1967; Martonosi, 1967, 1969). T h e r e are m a n y differences between p h o s p h o r y l a t i o n o f the Ca ++ transport system in sarcoplasmic reticulum and in RBCMF. In sarcoplasmic reticulum, Ca ++ increases p h o s p h o r y l a t i o n and Mg ++ decreases the level o f p h o s p h o r y l a t i o n , whereas in R B C M F it is the opposite, namely, Mg ++ and Ca ++ are r e q u i r e d for phosphorylation and d e p h o s p h o r y l a t i o n , respectively. T h u s , Martonosi (1969) f o u n d that p h o s p h o r y l a t e d intermediates of sarcoplasmic reticulum a p p e a r in the presence o f Ca ++ only and almost no p h o s p h o r y l a t e d intermediates with Mg ++ alone, whereas the opposite is true with the R B C M F in this study. F u r t h e r m o r e , the concentrations o f A T P a n d p H o f media for the optimal p h o s p h o r y l a t i o n o f sarcoplasmic reticulum are quite d i f f e r e n t f r o m those o f RBCMF. It is interesting to note that when Ca ++ is present at zero time along with Mg ++, labeling o f R B C M F occurs very slowly, since the steady state o f p h o s p h o rylation and d e p h o s p h o r y l a t i o n is r e a c h e d f r o m the beginning o f the experiment. Also it is n o t e d that the considerable initial p h o s p h o r y l a t i o n is f o u n d in the absence o f Ca ++ and Mg ++. This may be d u e to the presence o f e n d o g e n o u s Mg ++ and Ca ++ in RBCMF. T h e effect o f ADP on the p h o s p h o r y l a t e d RBCMF is shown in Fig. 2. T h e addition o f ADP initially causes a rapid fall o f labeling, then the relabeling o f R B C M F proceeds at the same rate as the control. Since this m e d i u m does not contain Ca ++, d e p h o s p h o r y l a t i o n would not occur. T h e m e c h a n i s m o f this effect o f ADP is not known at p r e s e n t . H o w e v e r , the following possibility is suggested. This effect o f ADP in decreasing the labeling (Fig. 2) may be d u e to the shift o f the initial p h o s p h o r y l a t i n g step: E + A T P ~-- E-P + ADP to left. This is s u p p o r t e d by the finding that ADP decreases the labeling o f RBCMF only
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THE JOURNAL OF GENERALPHYSIOLOGY• VOLUME 67 • 1976
t e m p o r a r i l y (Fig. 2), a n d t h e A D P e f f e c t is n o t o b s e r v e d w h e n M g ++ is n o t p r e s e n t (Fig. 3, a f t e r a d d i t i o n o f E D T A ) . F u r t h e r m o r e , t h e e f f e c t o f A D P is o b s e r v e d in t h e m e d i u m w h i c h d o e s n o t c o n t a i n C a ++ . H o w e v e r , t h e a d d i t i o n o f Ca ++ a f t e r E D T A still b r i n g s a b o u t t h e d e c r e a s e in l a b e l i n g o f R B C M F (Fig. 3). T h i s a g a i n i n d i c a t e s t h a t t h e d e p h o s p h o r y l a t i o n o f Ca ++ A T P a s e r e q u i r e s t h e p r e s e n c e o f C a ++, b u t n o t M g ++. I n N E M - t r e a t e d R B C M F , t h e a d d i t i o n o f C a ++ d i d n o t h a v e a n y e f f e c t o n t h e labeling of RBCMF. This suggests that NEM blocks somewhere along the e n z y m a t i c p r o c e s s e s w h i c h l e a d to t h e d e p h o s p h o r y l a t i o n o f C a + + - A T P a s e . T h i s suggests that NEM blocks the reaction some~vhere after the phosphorylation and b e f o r e t h e d e p h o s p h o r y l a t i o n . T h e e f f e c t o f A D P in d e c r e a s i n g t h e l a b e l i n g is still o b s e r v e d in N E M - t r e a t e d R B C M F , a n d this i n d i c a t e s t h a t N E M d o e s n o t i n t e r f e r e with t h e initial p h o s p h o r y l a t i n g s t e p . T h u s , t h e e n z y m a t i c s e q u e n c e o f t h e c a l c i u m t r a n s p o r t m e c h a n i s m (Ca ++ A T P a s e ) m a y b e p r e s e n t e d in t h e following scheme: Mg ++ A T P + E.~ "E-P + ADP Ca++
NEM-sensitive E +P:
T h u s , t h e e n z y m a t i c s e q u e n c e o f t h e Ca ++ t r a n s p o r t s y s t e m is v e r y s i m i l a r to t h a t o f t h e N a + t r a n s p o r t m e c h a n i s m in t h e r e d b l o o d cell m e m b r a n e . This work was supported by Grants HE-2875 and HL 16706 from National Heart and Lung Institute, National Institute of Health, United States Public Health Service. Dr. Lee is a Career Scientist of the Health Research Council, City of New York. This work is in partial fulfillment of the Ph.D. degree in pharmacology by Dr. Cha at the State University of New York, Downstate Medical Center. Received for publication 10 February 1975.
REFERENCES AImERS, R. W. 1967. Biochemical aspect of active transport. Annu. Rev. Biochem. 36:727756. ALBEr~S, R. W., G . J . KovaL, and G . J . SIEGEL. 1968. Studies on the interaction of ouabain and other cardioactive steroids with sodium-potassium activated adenosinetriphosphatase. Mol. Pharmacol. 4:324-336. AVRUCH, J., and G. FAIRBANKS. 1972. Demonstration of a p h o s p h o p e p t i d e intermediate in the Mg++-dependent, Na +- and K+-stimulated adenosine triphosphatase reaction of the erythrocyte m e m b r a n e . Proc. Natl. Acad. Sci. U. S. A. 69:1216-1220. BADER, H., A. K. SEN, and R. L. POST. 1966. Isolation a n d characterizations of a phosphorylated intermediate in the (Na + + K +) system-dependent ATPase. Biochim. Biophys. Acta. 118:106-115. BLOSTEIN, R. 1968. Relations between erythrocyte m e m b r a n e phosphorylation and adenosine triphosphate hydrolysis. J. Biol. Chem. 243:1957-1965. BuAv, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. h279-285.
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CHA, Y. N., B. C. SHIN, and K. S. LEE. 1971. Active uptake o f Ca ++ and Ca++-activating Mg++-ATPase in red cell m e m b r a n e f r a g m e n t s . J . Gen. Physiol. 57:202-215. HASSELBACH, W., and M. MAKINOSE. 1962. A T P and active transport. Biochem. Biophys. Res. Commun. 7:132-136. HOF~MAN, J. H. 1961. Molecular mechanism o f active cation transport. In Biophysics o f Physiological.and Pharmacological Actions. A.M. Shanes, editor. Washington AAAs Publication, No. 69.3-17. KNAUF, P. A., F. PROVERBIO, and J. F. HO~FMAN. 1974 a. Chemical characterization a n d Pronase susceptibility o f the Na: K pump-associated p h o s p h o p r o t e i n of h u m a n r e d blood cells. J. Gen. Physiol. 63:305-323. KNAUV, P. A., F. PROVERBIO, a n d J. F. HOFFMAN. 1974 b. Electrophoretic separation o f different p h o s p h o p r o t e i n s associated with Ca-ATPase and Na +, K-ATPase in h u m a n red cell ghosts. J. Gen. Physiol. 63:324-336. LEE, K. S., and B. C. SHIN. 1969. Studies on the active t r a n s p o r t o f Ca ++ in h u m a n red ceils. J. Gen. Physiol. 54:713-729. MARTONOSI, A. 1967. T h e role o f phospholipids in the ATPase activity o f skeletal muscle microsomes. Biochem. Biophys. Res. Commun. 29:753-757. MARTONOSI, A. 1969. Sarcoplasmic reticulum VII properties o f a p h o s p h o p r o t e i n intermediate implicated in calcium transport. J. Biol. Chem. 244:613-620. POST, R. L., S. KUME, T. TOBIN, B. ORCUTT, and A. K. SEN. 1969. Flexibility o f an active center in sodium-plus-potassium adenosine t r i p h o s p h a t a s e . J . Gen. Physiol. 54:306-326. SCHATZMAN, H . J . 1966. A T P d e p e n d e n t Ca+-extrusion from h u m a n red cells. Experientia (Basel). 22:364-365. SCHATZMANN, H. J., and F. F. VINCENZI. 1969. Calcium m o v e m e n t across the m e m b r a n e of h u m a n r e d cells. J. Physiol. (Lond.). 201-369-395. SKOU, J. C. 1957. T h e influence o f some cations on an adenosine triphosphatase from p e r i p h e r a l nerves. Biochim. Biophys. Acta. 23:394-401. S s o v , J. C. 1965. Enzymatic basis for active t r a n s p o r t o f Na + and K + across cell membrane. Physiol. Rev. 45:596-617. STECK, T . L., R. S. WEXNSTEIr%J. H. STRAt'S, and D. F. H. WALtaCH. 1970. Inside-out r e d cell m e m b r a n e vesicles; Preparatio~ and purification. Science (Wash. D. C.). 168:255-257. VINCENZl, F. F., a n d H . J . SCHATZMAN. 1967. Some properties of Ca ++ activated A T P a s e in h u m a n r e d cell m e m b r a n e . Helv. Physiol. Pharmacol. Acta. 25:CR 233-234. WEINER, M. L., and K. S. LEE. 1972. Active calcium ion uptake by inside-out a n d r i g h t side-out vesicles o f red blood cell m e m b r a n e s . J. Gen. Physiol. 59:462-475. YAMAMOTO, T . , and Y. TONOMURA. 1967. Reaction mechanism of the Ca++-dependent ATPase o f sarcoplasmic reticulum from skeletal muscle. I. Kinetic studies. J. Biochem. 62:558-575.
NAM C H A a n d K W A N G S O O LEE
From the Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203
ABSTRACT The phosphorylation of red blood cell membrane fragments (RBCMF) during Ca ++ transport was investigated. When red cell membrane fragments are incubated with [y-a2P]ATP under the experimental condition which minimizes the phosphorylation of Na+-K+-ATPase, RBCMF are labeled in the presence of Mg ++ without Ca ++. When Ca ++ is added, the labeling decreases due to dephosphorylation of RBCMF. The initial reaction of phosphorylation is reversed in the presence of excess ADP. The treatment of RBCMF with n-ethylmaleimide (NEM) does not interfere with the initial phosphorylation reaction, but blocks the dephosphorylation in the presence of Ca ++. These data suggest that the enzymatic sequence of the Ca ++ transport mechanism may be very similar to that of the Na + transport mechanism. I N T R O D U C T I O N
It has b e e n shown previously that the red b l o o d cell m e m b r a n e has an active t r a n s p o r t m e c h a n i s m for o u t w a r d extrusion o f Ca ++ ( S c h a t z m a n n a n d Vincenzi, 1969; Lee a n d Shin, 1969), a n d A T P is the specific e n e r g y source o f the Ca ++ t r a n s p o r t system (Vincenzi a n d S c h a t z m a n n , 1967; Cha et al., 1971). Previously, it was f o u n d that w h e n r e d blood cells are f r a g m e n t e d , red blood cell m e m b r a n e f r a g m e n t s (RBCMF) were f o u n d to take u p Ca ++ a n d this Ca ++ u p t a k e is a c c o m p a n i e d by A T P hydrolysis by Mg++-activated A T P a s e (Lee a n d Shin, 1969; Cha et al. 1971). It has b e e n s u g g e s t e d that this Ca ++ u p t a k e is d u e to the p r e s e n c e o f inside o u t vesicles in the p r e s e n t system a l t h o u g h not all o f the vesicles are inside out (Steck et al., 1970; W e i n e r a n d Lee, 1972). R B C M F were f o u n d to have both Na+-K+-ATPase a n d Ca++-ATPase which are intimately associated with the s o d i u m a n d calcium t r a n s p o r t m e c h a n i s m s , respectively ( S c h a t z m a n n , 1966; Skou, 1965; Lee a n d Shin, 1969). N u m e r o u s studies on the s o d i u m t r a n s p o r t m e c h a n i s m in the r e d blood cell m e m b r a n e indicate the i n v o l v e m e n t o f p h o s p h o r y l a t e d i n t e r m e d i a t e s d u r i n g the e n z y m a t i c process (Albers, 1967; Albers et al., 1968; Post et al., 1969). T h e r e f o r e , an a t t e m p t has b e e n m a d e to investigate the possibility that the similar p h o s p h o rylation o f R B C M F m a y also be involved with the calcium t r a n s p o r t m e c h a n i s m . It was f o u n d that the calcium t r a n s p o r t m e c h a n i s m a p p e a r s to s h a r e similarities with the s o d i u m t r a n s p o r t m e c h a n i s m with r e g a r d to p h o s p h o r y l a t i o n m e c h a nisms involved with the enzymatic action. THE JOURNAL
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METHODS
Preparation of RBCMF RBCMF were prepared according to the procedure used previously in this laboratory (Cha et al., 1971).
Measurement of ATPase Activities T h e Ca++-ATPase activity (in the presence of Mg ++) was measured by incubating RBCMF in the standard medium for l0 rain at 37°C. T h e standard m e d i u m for Ca++-ATPase contained: 120 mM KCI; 30 mM histidine-imidazole buffer, pH 7.0; 3 mM MgClz; 2 mM ATP; and 0.5 mM CaCl2. T h e Na+-K+-ATPase was measured by incubating RBCMF for 10 min at 37°C in the medium containing: 100 mM NaC1; l0 mM KCI; 30 mM histidineimidazole buffer, pH 7.0; 3 mM MgCl2; and 2 mM ATP.
Measurement of A TP-Dependent Ca ++ Uptake by RBCMF T h e Ca ++ uptake of RBCMF was measured according to the method of Cha et al. (1971) employing 4"~Ca. In this method, the radioactivity of RBCMF after incubation of RBCMF with 4"~Ca in the presence of A T P was measured.
Measurements of Phosphorylation of RBCMF In phosphorylation experiments, RBCMF (1 mg protein/ml reaction mixture) were incubated at 37°C in a standard medium for phosphorylation which contained 120 mM KCI, 30 mM histidine-imidazole buffer (pH 7.0), 0.1 mM EGTA (ethylene glycol bis[/3aminoethylether]N,N'-tetraacetic acid), 2 p,M radioactive A T P (1 /~Ci/M), and 3 mM MgCI2. Before the actual incubation, RBCMF were equilibrated at 37°C for 3 min in the standard medium without MgC12 and the reaction was started by a d d i n g MgCl~ into the mixture after the equilibration. T h e absence of Na + from the reaction mixture in the presence of KC1 was essential to exclude any possibility that the phosphorylation might result from Na+-dependent phosphorylation of Na+-K+-ATPase present in RBCMF. After various periods of incubation the reaction was stopped by adding 5 ml of ice-cold 5% trichloroacetic acid (TCA) (with 0.01 mM A T P and 0.1 mM Pi) to the reaction mixture and placing it in an ice bath immediately. When Ca++-dependent dephosphorylation was desired, Ca ++ (0.2 raM) was introduced at an appropriate time after the reaction had been started, and the incubation was continued until the reaction was stopped by adding TCA. T h e mixture was then centrifuged at 2,000 g for 10 rain at 4°C. T h e precipitate was washed twice with 5 ml of the same TCA solution, and the final precipitate was suspended in 2 ml of 0.1 N NaOH. A half milliliter of the suspension was applied to a planchet t)r put into a scintillation vial with 10-ml Bray's solution (Bray, 1960), and counted either in a thin-window Nuclear Chicago gas flow counter or in a Packard scintillation counter (Packard I n s t r u m e n t Co., Inc., Downers Grove, Ill.), respectively. RESULTS
ATPase Activities and ATP-Dependent Ca ++ Uptake T h e N a + - K + - a c t i v a t e d a n d Ca++-activated A T P a s e (in the p r e s e n c e o f Mg ++) o f R B C M F were 0.45 a n d 1.68 /~mol P i / m g p r o t e i n / h , r e s p e c t i v e l y . T h e Ca ++ uptake of R B C M F in the presence a n d absence of A T P after 1 h of i n c u b a t i o n were 45.2 a n d 6.1 n m o l / m g p r o t e i n , r e s p e c t i v e l y . T h i s i n d i c a t e s t h a t t h e p r e s e n t R B C M F has a n active Ca ++ t r a n s p o r t m e c h a n i s m .
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Binding of [w32P]ATP and [y32P]ATP to R B C M F
Radioactive A T P , labeled at oL or y position was incubated in the standard m e d i u m for p h o s p h o r y l a t i o n for 30 and 60 rain with RBCMF, and the radioactivity taken u p by R B C M F d u r i n g the incubation period was m e a s u r e d . Results are shown in Table I. TABLE
I
BINDING OF [o~-32P]ATP AND [y-32P]ATP T O RBCMF Time incubation Treatment 30 min [a-azP]ATP
[~,-s2P]ATP
60 min
558
670
5962
6169
n2P-labeling is expressed in terms o f counts per minute per 0.25 m g protein o f RBCMF.
As can be seen in this table, the radioactivity o f RBCMF after T C A t r e a t m e n t was significant with [T-3zP]ATP, but not with [ot-32P]ATP. This indicates that RBCMF is p h o s p h o r y l a t e d with [T-32P]ATP only, and not by [~-3zP]ATP. In all the following e x p e r i m e n t s , only [T-32P]ATP was e m p l o y e d for all phosphorylation experiments. It should be noted that the phosphorylation o f RBCMF by [y3zP]ATP occurs in the m e d i u m which contains 120 mM KC1 and no NaCI, the condition which minimizes the possibility o f p h o s p h o r y l a t i o n o f Na+-K+-ATPase by A T P . Influence of M g ++ and Ca ++ on Phosphorylation
Radioactive labeling o f R B C M F was m e a s u r e d after incubation o f RBCMF in mixtures containing the basic c o m p o n e n t s (BC) o f 120 mM KCI, 30 mM histidine-imidazole b u f f e r (pH 7.0), 0.1 mM E G T A , 2/zM radioactive A T P (1 txCi/M) and the following variations: BC only, (no Mg ++, no Ca ++ in Fig. 1), BC + 3 mM MgC12 (Mg ++ in Fig. 1), BC + 0.5 mM CaC12 (Ca ++ but no Mg ++ in Fig. 1), BC + 3 m M MgCl2 + 0.5 m M CaCl2 (Ca ++ at zero time in Fig. 1). Also, in some e x p e r i m e n t s with BC + 3 mM MgCI2 (Mg ++ in Fig. 1) 0.5 mM CaClz was a d d e d 5 rain after the incubation started. Results are shown in Fig. 1. In the presence o f Mg ++ alone (Mg ++ in Fig. 1), RBCMF were very rapidly p h o s p h o r y l a t e d in the first few seconds, and then c o n t i n u e d at a diminished rate d u r i n g the r e m a i n d e r of the 10-rain incubation period. Without Mg ++ in media (no Mg ++, no Ca ++, and Ca ++ but no Mg ++, in Fig. 1), no significant labeling o f R B C M F o c c u r r e d . Although it is not shown in Fig. 1, the addition o f KCI u p to 5 rain after the incubation in Mg ++ solution did not have any effect on the labeling o f RBCMF. W h e n both Ca ++ and Mg ++ were present f r o m the start o f the reaction (Ca ++ at zero time in Fig. 1), R B C M F were p h o s p h o r y l a t e d slightly within the first few seconds and progressed slowly d u r i n g the rest o f the incubation time. T h e s e results indicate that the presence o f Mg ++ and Ca ++ are r e q u i r e d for phosphorylation and d e p h o s p h o r y l a t i o n o f RBCMF, respectively. T h u s , the greatest
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labeling o f RBCMF occurs when media contained Mg ++ only, a n d the labeling decreases when Ca ++ is also present in media.
Influence of Addition of ADP on Labeling during Incubation Experiments were p e r f o r m e d where 0.2 mM ADP was a d d e d 5 min after R B C M F were incubated in the standard m e d i u m for p h o s p h o r y l a t i o n . It should be m e n t i o n e d that the s t a n d a r d m e d i u m is the same as the m e d i u m noted 7-
6-5.
~
o
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C~4"
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E3O. (,.) 2-
| ~
_~-÷
l "
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~
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FIGURE 1. Labeling of RBCMF after incubation under varii/us experimental conditions. Experimental conditions: All media contained 120 mM KC1, 30 mM histidine-imidazole buffer (pH 7.0), 0.1 inM FGTA, and 2 /xM radioactive ATP. In addition: ( - ) (-), none; Mg++, 3 mM MgC12; Ca ++ but no Mg++, 0.5 mM CaCI2; Ca ++ at zero time, 3 mM MgCI~, and 0.5 mM CaCI2; Ca ++ at 5th min, 3 nlM MgCI2 from the beginning, and 0.2 mM CaCI,, at 5 rain. "Mg ++'' in Fig. 1. As shown in Fig. 2, the addition o f ADP caused an initial rapid fall o f radioactivity o f RBCMF, which was followed by an increase in labeling at the same rate as that o f the control with Mg ++ alone. I n Fig. 2, the effect o f addition o f nonradioactive A T P (0.2 mM) d u r i n g the incubation is also shown. T h e labeling was diluted by formation of nonradioactive p h o s p h a t e intermediaries, when cold A T P was a d d e d . Since Ca ++ is absent in the m e d i u m , the effect o f A D P in decreasing the labeling may be d u e to the shift of reaction E + A T P ,~ A D P + E - P to the left, a l t h o u g h o t h e r u n k n o w n mechanisms maybe responsible for this p h e n o m e n o n .
Influence of EDTA on the Response of R B CMF to the Addition of ADP or Ca ++ Five minutes after R B C M F were incubated in the standard m e d i u m for phosphorylation, E D T A (ethylene diamine tetraacetate) in a c o n c e n t r a t i o n o f 5 mM was a d d e d to the reaction mixture. Results are shown in Fig. 3. As can be seen in this figure, the addition o f E D T A is followed by the cessation o f f u r t h e r labeling o f RBCMF. This probably is due to the chelation of Mg ++ by E D T A , which
CHA AND LEE Phosphorylationof Erythrocyte Membraneduring Ca++Active Transport
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results in the lack o f Mg ++ necessary for the p h o s p h o r y l a t i o n reaction. W h e n 0.2 mM A D P is a d d e d immediately after E D T A , no effect o f A D P is observed. This s u p p o r t s the suggestion m a d e previously that the effect o f A D P is d u e to the reversal o f the initial p h o s p h o r y l a t i o n reaction, which requires Mg ++. T h e addition o f Ca ++ after E D T A still causes the decrease in labeling o f R B C M F probably because the d e p h o s p h o r y l a t i o n requires the presence o f Ca ++ but not Mg ++ .
S 9
CONTROL ADP
70 0 x
6-
5"
j
E
o. u
4-
32-
/
ATP 0.2mM
min i n c u b a t i o n
FIGURE 2. Effects of ADP and ATP on labeling of RBCMF. Experimental conditions: Incubation medium contained 120 mM KCI, 30 mM histidine-imidazole buffer (pH 7.0) 0.1 mM EGTA, 2 p.M radioactive ATP, and 3 mM MgCI2. ADP (0.2 raM) or ATP (0.2 mM) was added 5 rain after incubation was started.
Influence of A D P and Ca ++ on Labeling of NEM-Treated R B C M F R B C M F were treated with 0.2 mM NEM at 37°C for 20 rain b e f o r e the incubation. T h e NEM-treated R B C M F were t h e n incubated with [y-~2P]ATP in the s t a n d a r d m e d i u m for p h o s p h o r y l a t i o n and either A D P or Ca ++ was a d d e d 5 min after incubation. T h e radioactivity o f R B C M F d u r i n g various stages o f experiments is shown in Fig. 4. As can be seen in this figure, the addition o f Ca ++ does not influence the labeling o f N E M - t r e a t e d RBCMF. T h e addition o f ADP, however, still causes a fall in labeling o f N E M - t r e a t e d R B C M F similar to that observed with n o n t r e a t e d RBCMF. This suggests that N E M t r e a t m e n t blocks d e p h o s p h o r y l a t i o n in the presence o f Ca ++, but does not block the effect of ADP, which acts on the initial p h o s p h o r y l a t i o n step.
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Addition of 45Ca to the Previously Phosphorylated RBCMF Five minutes after R B C M F were incubated in a m e d i u m c o n t a i n i n g 20 g M radioactive A T P in the p r e s e n c e o f Mg ++ alone, 45Ca (1/~Ci/mM) was i n t r o d u c e d into the reaction m i x t u r e . T h e 10-times g r e a t e r concentration o f A T P above the A T P concentration used in the p h o s p h o r y l a t i o n e x p e r i m e n t s was used h e r e , because a significant a m o u n t o f 45Ca u p t a k e was n e e d e d in this e x p e r i m e n t . As can be seen in Fig. 5, the level o f p h o s p h o r y l a t i o n d e c r e a s e d to a certain level a n d r e m a i n e d so, while the 45Ca was continuously b e i n g t a k e n up by the R B C M F . T h u s , it a p p e a r s that a steady level o f p h o s p h o r y l a t i o n is m a i n t a i n e d d u r i n g the active Ca ++ u p t a k e in R B C M F , a l t h o u g h f u r t h e r investigation is r e q u i r e d to ascertain this aspect.
~
7, 0 0
o x
E o
6"
5" 4-
/
CONTROL
EDTAADP aflerEDTA
after EOTA
rain incubation
FIGURE 3. Effect of EDTA and labeling of RBCMF. Experimental conditions: Incubation medium contained 120 mM KCI, 30 mM histidine-imidazole buffer (pH 7.0), 0.1 mM EGTA, 2 txM radioactive ATP, and 3 mM MgC12. Seven minutes after the reaction after additions were made. EDTA; EDTA (5 mM). ADP after EDTA; EDTA (5 raM) then ADP (0.2 mM). Ca ++ after EDTA; EDTA (5 raM) then CaCl~ (0.5 mM). DISCUSSION
It has b e e n established t h a t Na+-K+-activated A T P a s e a n d Ca++-activated A T P ase are intimately associated with o u t w a r d extrusion o f Na + a n d Ca 2+, respectively (Skou, 1957 a n d 1965; H o f f m a n , 1961; S c h a t z m a n n , 1966; Albers, 1967; Post et al., 1969; S c h a t z m a n n and Vincenzi, 1969; Lee and Shin, 1969). Since the p r e s e n t R B C M F has b o t h o f the above two A T P a s e activities, an a t t e m p t has b e e n m a d e to investigate the similarity o f these two active t r a n s p o r t systems in m o l e c u l a r mechanisms. T h e sequence o f enzymatic steps involved in Na+-K +A T P a s e is intensively studied by m a n y investigators, whereas very little is known
CI~A AND LEE Phosphorylation of Erythrocyte Membrane during Ca ++Active Transport
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with r e g a r d to the e n z y m a t i c sequence o f the Ca ++ t r a n s p o r t system o f Ca ++A T P a s e in RBCMF. With Na+-K+-ATPase, evidence suggests that the following sequential reaction takes place ( B a d e r et al., 1966; Albers et al., 1968; Post et al., 1969): ATP
+ El,
Pi
N a + M g ++ ' ~ EI-P
+ ADP Mg ++ blocked by N E M
+ E2 •
' '
E2-P
+
H~O
I n the p r e s e n t study the possibility that the Ca ++ t r a n s p o r t m e c h a n i s m o f R B C M F may go t h r o u g h a similar sequence has been e x p l o r e d . T h e radioactivities o f R B C M F i n c u b a t e d in the s t a n d a r d m e d i u m for phosp h o r y l a t i o n with either [a-a2p]ATP or [T-a2p]ATP are shown in T a b l e I. T h e data indicate that the t e r m i n a l p h o s p h a t e o f A T P f o r m s an acid stable c o m p l e x with R B C M F u n d e r e x p e r i m e n t a l conditions.
6"
5"
0 o
4-
x E
3-
9, Q.
2-
/ I
t
2
5
7
I0
min incubotion
FmURE 4. Effects of ADP and Ca ++ on NEM-treated RBCMF. Experimental conditions: Same as in Fig. 2. ADP (0.2 raM) and CaCI2 (0.5 raM) were added 5 rain after incubation. T h e participation o f p h o s p h o r y l a t e d i n t e r m e d i a t e s in Na + t r a n s p o r t has b e e n investigated by n u m e r o u s investigators, a n d it is k n o w n that labeling o f the Na +activated m e m b r a n e A T P a s e occurs d u r i n g the t r a n s p o r t o f Na + (Avruch a n d Fairbanks, 1972; Post et al., 1969; Blostein, 1968; K n a u f et al., 1974 a, b). A l t h o u g h the p r e s e n t R B C M F has both Na+-K+-ATPase a n d Ca++-ATPase, the e x p e r i m e n t a l conditions m a k e it unlikely that this labeling o f R B C M F is d u e to the p h o s p h o r y l a t i o n o f Na+-K+-ATPase. T h i s incubation m e d i u m for phosp h o r y l a t i o n contains no Na + which is n e e d e d f o r p h o s p h o r y l a t i o n o f Na+-K +A T P a s e . O n the o t h e r h a n d , the m e d i u m has K + (120 m M ) which mediates d e p h o s p h o r y l a t i o n o f Na+-K+-ATPase. F u r t h e r m o r e , the addition o f K + to the labeled R B C M F did not have a n y effect, whereas the addition o f Ca ++ d e c r e a s e d
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the labeling (Fig. 1). T h e s e data indicate that the labeling o f R B C M F u n d e r the p r e s e n t e x p e r i m e n t a l condition is d u e to the p h o s p h o r y l a t i o n o f Ca++-ATPase of RBCMF. Data in Fig. 1 show that the p h o s p h o r y l a t i o n o f R B C M F p r o c e e d s t h r o u g h o u t the e x p e r i m e n t in the p r e s e n c e o f Mg ++ only. T h u s the initial p h o s p h o r y l a t i o n step a p p e a r s to require Mg ++, but not Ca ++. T h e addition o f Ca ++ to labeled R B C M F is followed by a r a p i d fall o f radioactivity o f RBCMF. T h e r a p i d fall of
~
O
00 3 14
_--_2
o
1
0
5
10
15
30
rain i n c u b a t i o n Double labeling of RBCMF with :~P and 4:'Ca. O .
. . . O :~2p-labeling, FIGURE 5. c.... c a2p-labeling after addition of 4 5 C a + + , x . . . . X 45Ca++-labeling. Experimental conditions: Incubation medium contained: 120 mM KCI, 30 mM histidineimidazole buffer (pH 7.0), 0.1 mM EGTA, 20 ~M radioactive ATP, and 2 mM MgC12. At 5 rain, *"Ca (1 >Ci/mM) was added.
radioactivity o f R B C M F m a y be d u e either to the inhibition o f p h o s p h o r y l a t i o n or acceleration of d e p h o s p h o r y l a t i o n . T h e inhibition of p h o s p h o r y l a t i o n probably d u e to c o m p e t i t i o n o f Ca ++ with Mg ++ a p p e a r s unlikely because in NEMt r e a t e d RBCMF, the addition of Ca ++ has no effect whatsoever on the p h o s p h o rylation as indicated by the continuous labeling o f R B C M F (Fig. 4). T h u s the fall o f labeling after the addition o f Ca ++ p r o b a b l y is d u e to d e p h o s p h o r y l a t i o n o f p h o s p h o r y l a t e d i n t e r m e d i a t e s . Since, as discussed above, the Ca ++ t r a n s p o r t A T P a s e and not Na+-K+-ATPase is p h o s p h o r y l a t e d u n d e r the e x p e r i m e n t a l conditions, it a p p e a r s that the fall o f labeling a f t e r the addition o f Ca ++ is d u e to d e p h o s p h o r y l a t i o n o f Ca ++ t r a n s p o r t A T P a s e in the p r e s e n c e o f Ca ++. In this connection, results o b t a i n e d in the recent work by K n a u f et al. (1974 b) should be m e n t i o n e d . T h e s e w o r k e r s m e a s u r e d p h o s p h o r y l a t i o n of r e d b l o o d cell m e m b r a n e in m e d i a containing Mg ++ a n d Na + (or Na+-K +) at 0°C, a n d f o u n d that the p r e s e n c e o f Ca ++ increased p h o s p h o r y l a t i o n . T w o possibilities were suggested. O n e possibility was that the Ca ++ p h o s p h o p r o t e i n in their study m a y be d i f f e r e n t f r o m that of Ca ++ t r a n s p o r t A T P a s e , since conditions o f their study ( t e m p e r a -
CHA AND LEE
Phosphorylation of Erythrocyte Membrane during Ca ++Active Transport
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ture, A T P concentrations, etc.) were d i f f e r e n t f r o m those o f previous works on the Ca ++ t r a n s p o r t A T P a s e . A n o t h e r possibility suggested was that the Ca ++ p h o s p h o p r o t e i n characterized in their work may r e p r e s e n t an i n t e r m e d i a t e in the Mg++-Ca++-ATPase involved with Ca ++ t r a n s p o r t . I f this is the case, then the d i f f e r e n t results obtained in their and the p r e s e n t study may be reconciled by assuming that Ca ++ is r e q u i r e d for the step f r o m E1-P to Ez-P. Since the Ca ++ t r a n s p o r t ATPase is very sensitive to t e m p e r a t u r e and no d e p h o s p h o r y l a t i o n occurs at 0°C (Cha et al., 1971), the presence o f Ca ++ would increase phosphorylation by f o r m i n g E2-P without d e p h o s p h o r y l a t i o n o f this i n t e r m e d i a t e at 0°C in the study o f K n a u f et al. (1974 b). As discussed by the a u t h o r s in their study, a n o t h e r cause o f increased phosphorylation by Ca ++ was the inhibition o f d e p h o s p h o r y l a t i o n o f Na+-K+-ATPase by Ca ++. In the p r e s e n t study, on the o t h e r h a n d , this condition o f increasing p h o s p h o r y l a t i o n by Ca ++ does not exist, since no p h o s p h o r y l a t i o n o f Na+-K+-ATPase presumably occurs in the present e x p e r i m e n t a l conditions. T h u s the present Ca ++ transport A T P a s e seems to require Mg ++ and Ca ++ for the initial p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n , respectively. A n o t h e r aspect to be considered with the p h o s p h o r y l a t e d intermediates o f t h e C a ++ t r a n s p o r t system in the R B C M F in this study is to c o m p a r e the present system with that o f sarcoplasmic reticulum. Previously, the participation o f p h o s p h o r y l a t e d intermediates in the Ca ++ t r a n s p o r t system in sarcoplasmic reticulum o f skeletal muscle has been r e p o r t e d (Hasselbach and Makinose, 1962; Y a m o m o t o and T o n o m u r a , 1967; Martonosi, 1967, 1969). T h e r e are m a n y differences between p h o s p h o r y l a t i o n o f the Ca ++ transport system in sarcoplasmic reticulum and in RBCMF. In sarcoplasmic reticulum, Ca ++ increases p h o s p h o r y l a t i o n and Mg ++ decreases the level o f p h o s p h o r y l a t i o n , whereas in R B C M F it is the opposite, namely, Mg ++ and Ca ++ are r e q u i r e d for phosphorylation and d e p h o s p h o r y l a t i o n , respectively. T h u s , Martonosi (1969) f o u n d that p h o s p h o r y l a t e d intermediates of sarcoplasmic reticulum a p p e a r in the presence o f Ca ++ only and almost no p h o s p h o r y l a t e d intermediates with Mg ++ alone, whereas the opposite is true with the R B C M F in this study. F u r t h e r m o r e , the concentrations o f A T P a n d p H o f media for the optimal p h o s p h o r y l a t i o n o f sarcoplasmic reticulum are quite d i f f e r e n t f r o m those o f RBCMF. It is interesting to note that when Ca ++ is present at zero time along with Mg ++, labeling o f R B C M F occurs very slowly, since the steady state o f p h o s p h o rylation and d e p h o s p h o r y l a t i o n is r e a c h e d f r o m the beginning o f the experiment. Also it is n o t e d that the considerable initial p h o s p h o r y l a t i o n is f o u n d in the absence o f Ca ++ and Mg ++. This may be d u e to the presence o f e n d o g e n o u s Mg ++ and Ca ++ in RBCMF. T h e effect o f ADP on the p h o s p h o r y l a t e d RBCMF is shown in Fig. 2. T h e addition o f ADP initially causes a rapid fall o f labeling, then the relabeling o f R B C M F proceeds at the same rate as the control. Since this m e d i u m does not contain Ca ++, d e p h o s p h o r y l a t i o n would not occur. T h e m e c h a n i s m o f this effect o f ADP is not known at p r e s e n t . H o w e v e r , the following possibility is suggested. This effect o f ADP in decreasing the labeling (Fig. 2) may be d u e to the shift o f the initial p h o s p h o r y l a t i n g step: E + A T P ~-- E-P + ADP to left. This is s u p p o r t e d by the finding that ADP decreases the labeling o f RBCMF only
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THE JOURNAL OF GENERALPHYSIOLOGY• VOLUME 67 • 1976
t e m p o r a r i l y (Fig. 2), a n d t h e A D P e f f e c t is n o t o b s e r v e d w h e n M g ++ is n o t p r e s e n t (Fig. 3, a f t e r a d d i t i o n o f E D T A ) . F u r t h e r m o r e , t h e e f f e c t o f A D P is o b s e r v e d in t h e m e d i u m w h i c h d o e s n o t c o n t a i n C a ++ . H o w e v e r , t h e a d d i t i o n o f Ca ++ a f t e r E D T A still b r i n g s a b o u t t h e d e c r e a s e in l a b e l i n g o f R B C M F (Fig. 3). T h i s a g a i n i n d i c a t e s t h a t t h e d e p h o s p h o r y l a t i o n o f Ca ++ A T P a s e r e q u i r e s t h e p r e s e n c e o f C a ++, b u t n o t M g ++. I n N E M - t r e a t e d R B C M F , t h e a d d i t i o n o f C a ++ d i d n o t h a v e a n y e f f e c t o n t h e labeling of RBCMF. This suggests that NEM blocks somewhere along the e n z y m a t i c p r o c e s s e s w h i c h l e a d to t h e d e p h o s p h o r y l a t i o n o f C a + + - A T P a s e . T h i s suggests that NEM blocks the reaction some~vhere after the phosphorylation and b e f o r e t h e d e p h o s p h o r y l a t i o n . T h e e f f e c t o f A D P in d e c r e a s i n g t h e l a b e l i n g is still o b s e r v e d in N E M - t r e a t e d R B C M F , a n d this i n d i c a t e s t h a t N E M d o e s n o t i n t e r f e r e with t h e initial p h o s p h o r y l a t i n g s t e p . T h u s , t h e e n z y m a t i c s e q u e n c e o f t h e c a l c i u m t r a n s p o r t m e c h a n i s m (Ca ++ A T P a s e ) m a y b e p r e s e n t e d in t h e following scheme: Mg ++ A T P + E.~ "E-P + ADP Ca++
NEM-sensitive E +P:
T h u s , t h e e n z y m a t i c s e q u e n c e o f t h e Ca ++ t r a n s p o r t s y s t e m is v e r y s i m i l a r to t h a t o f t h e N a + t r a n s p o r t m e c h a n i s m in t h e r e d b l o o d cell m e m b r a n e . This work was supported by Grants HE-2875 and HL 16706 from National Heart and Lung Institute, National Institute of Health, United States Public Health Service. Dr. Lee is a Career Scientist of the Health Research Council, City of New York. This work is in partial fulfillment of the Ph.D. degree in pharmacology by Dr. Cha at the State University of New York, Downstate Medical Center. Received for publication 10 February 1975.
REFERENCES AImERS, R. W. 1967. Biochemical aspect of active transport. Annu. Rev. Biochem. 36:727756. ALBEr~S, R. W., G . J . KovaL, and G . J . SIEGEL. 1968. Studies on the interaction of ouabain and other cardioactive steroids with sodium-potassium activated adenosinetriphosphatase. Mol. Pharmacol. 4:324-336. AVRUCH, J., and G. FAIRBANKS. 1972. Demonstration of a p h o s p h o p e p t i d e intermediate in the Mg++-dependent, Na +- and K+-stimulated adenosine triphosphatase reaction of the erythrocyte m e m b r a n e . Proc. Natl. Acad. Sci. U. S. A. 69:1216-1220. BADER, H., A. K. SEN, and R. L. POST. 1966. Isolation a n d characterizations of a phosphorylated intermediate in the (Na + + K +) system-dependent ATPase. Biochim. Biophys. Acta. 118:106-115. BLOSTEIN, R. 1968. Relations between erythrocyte m e m b r a n e phosphorylation and adenosine triphosphate hydrolysis. J. Biol. Chem. 243:1957-1965. BuAv, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. h279-285.
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CHA, Y. N., B. C. SHIN, and K. S. LEE. 1971. Active uptake o f Ca ++ and Ca++-activating Mg++-ATPase in red cell m e m b r a n e f r a g m e n t s . J . Gen. Physiol. 57:202-215. HASSELBACH, W., and M. MAKINOSE. 1962. A T P and active transport. Biochem. Biophys. Res. Commun. 7:132-136. HOF~MAN, J. H. 1961. Molecular mechanism o f active cation transport. In Biophysics o f Physiological.and Pharmacological Actions. A.M. Shanes, editor. Washington AAAs Publication, No. 69.3-17. KNAUF, P. A., F. PROVERBIO, and J. F. HO~FMAN. 1974 a. Chemical characterization a n d Pronase susceptibility o f the Na: K pump-associated p h o s p h o p r o t e i n of h u m a n r e d blood cells. J. Gen. Physiol. 63:305-323. KNAUV, P. A., F. PROVERBIO, a n d J. F. HOFFMAN. 1974 b. Electrophoretic separation o f different p h o s p h o p r o t e i n s associated with Ca-ATPase and Na +, K-ATPase in h u m a n red cell ghosts. J. Gen. Physiol. 63:324-336. LEE, K. S., and B. C. SHIN. 1969. Studies on the active t r a n s p o r t o f Ca ++ in h u m a n red ceils. J. Gen. Physiol. 54:713-729. MARTONOSI, A. 1967. T h e role o f phospholipids in the ATPase activity o f skeletal muscle microsomes. Biochem. Biophys. Res. Commun. 29:753-757. MARTONOSI, A. 1969. Sarcoplasmic reticulum VII properties o f a p h o s p h o p r o t e i n intermediate implicated in calcium transport. J. Biol. Chem. 244:613-620. POST, R. L., S. KUME, T. TOBIN, B. ORCUTT, and A. K. SEN. 1969. Flexibility o f an active center in sodium-plus-potassium adenosine t r i p h o s p h a t a s e . J . Gen. Physiol. 54:306-326. SCHATZMAN, H . J . 1966. A T P d e p e n d e n t Ca+-extrusion from h u m a n red cells. Experientia (Basel). 22:364-365. SCHATZMANN, H. J., and F. F. VINCENZI. 1969. Calcium m o v e m e n t across the m e m b r a n e of h u m a n r e d cells. J. Physiol. (Lond.). 201-369-395. SKOU, J. C. 1957. T h e influence o f some cations on an adenosine triphosphatase from p e r i p h e r a l nerves. Biochim. Biophys. Acta. 23:394-401. S s o v , J. C. 1965. Enzymatic basis for active t r a n s p o r t o f Na + and K + across cell membrane. Physiol. Rev. 45:596-617. STECK, T . L., R. S. WEXNSTEIr%J. H. STRAt'S, and D. F. H. WALtaCH. 1970. Inside-out r e d cell m e m b r a n e vesicles; Preparatio~ and purification. Science (Wash. D. C.). 168:255-257. VINCENZl, F. F., a n d H . J . SCHATZMAN. 1967. Some properties of Ca ++ activated A T P a s e in h u m a n r e d cell m e m b r a n e . Helv. Physiol. Pharmacol. Acta. 25:CR 233-234. WEINER, M. L., and K. S. LEE. 1972. Active calcium ion uptake by inside-out a n d r i g h t side-out vesicles o f red blood cell m e m b r a n e s . J. Gen. Physiol. 59:462-475. YAMAMOTO, T . , and Y. TONOMURA. 1967. Reaction mechanism of the Ca++-dependent ATPase o f sarcoplasmic reticulum from skeletal muscle. I. Kinetic studies. J. Biochem. 62:558-575.
