83
Biochimica et Biophysica Acta, 507 (1978) 83--93 © Elsevier/North-Holland Biomedical Press
BBA 77917
IS T H E R E A PLASMA MEMBRANE-LOCATED ANION-SENSITIVE ATPase? III. IDENTITY OF THE E R Y T H R O C Y T E ENZYME WITH ( C a 2+ + Mg 2+)-ATPase
J.M.M. VAN AMELSVOORT, P.M.K.B. VAN HOOF, J.J.H.H.M. DE PONT and S.L. BONTING
Department of Biochemistry, University of Ni]megen, Ni]megen (The Netherlands) (Received July 12th, 1977)
Summary The characteristics of the anion-sensitive Mg:+-ATPase activity of the rabbit erythrocyte have been studied in a lyophilized ghost preparation. The enzyme appears to be different from the anion-sensitive Mg2+-ATPase activity of other tissues in many parameters, such as optimal pH, effects of various anions, oligomycin sensitivity and effects of Triton X-100. The enzyme is insensitive towards inhibition by irreversibly b o u n d 4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). This excludes a relationship between the enzyme and the " b a n d 3 " protein, which is thought to be involved in the anion exchange over the erythrocyte membrane. From the effects of ethyleneglycol-bis-(fi-aminoethylether)-N,N'-tetraacetic acid (EGTA), CaCl~, chlorpromazine and ruthenium red it is concluded that the enzyme activity does n o t represent a separate entity b u t is part of the (Ca 2÷ + Mg2+)-ATPase system of the erythrocyte membrane. A reported stimulatory effect of carbonic anhydrase is attributed to a contamination of the carbonic anhydrase preparation by calcium and/or (Ca 2÷ + Mg2÷)-ATPase activator protein.
Introduction Previous studies by others [1] and ourselves [2--4] have cast serious d o u b t on the presence of an anion-sensitive Mg~+-ATPase in plasma membranes of various tissues like gastric mucosa [1,2], trout gill [2], kidney [3] and pancreas [4] and thus on its alleged role in anion transport. In these tissues the enzyme Abbreviations: H2DIDS, 4,4r-diisothiocyano-dihydrostilbene-2,2r-disulfonic acid; EGTA, ethylene-
glycol-bis-(fl-aminoethylether)-N,N~-tetraacetic acid.
84 seems to be located in the m i t o c h o n d r i a rather than in the plasma membrane. A particulate anion-sensitive Mg2*-ATPase activity has, however, also been reported in rabbit e r y t h r o c y t e s [5], which are devoid of mitochondria. Hence, this activity must be located in the plasma membrane. It differs in one respect from the comparable activity in other tissues: it is not inhibited by thiocyahate. In view of the obvious importance of the question whether this e n z y m e activity can be involved in anion transport, we have decided to make a thorough study of its characteristics. Methods and Materials
Preparation of erythrocyte ghosts. E r y t h r o c y t e ghosts are prepared according to the m e t h o d of Duncan [5] with slight modifications. Blood from New Zealand white rabbits, freshly obtained by heart puncture, is immediately mixed with 0.1 volume 3.8% sodium citrate, is cooled to 0°C, is filtered over four layers of surgical gauze, and is centrifuged for 15 min at 1400 × g. After removal of plasma and " b u f f y c o a t " by aspiration, the e r y t h r o c y t e s are washed three times with isotonic (310 mosM) sodium phosphate buffer at pH 8.0 [6], and h e m o l y z e d in 20 mM imidazole • HC1 (pH 7.5). The membranes are centrifuged for 25 min at 30 000 ×g, are washed once with 4 mM MgC12, 1 mM EDTA, 20 mM imidazole • HC1 (pH 7.5), two or three times with 2 mM MgC12, 20 mM i m i d a z o l e . HC1 (pH 7.5) and finally in double-distilled water. The membranes are resuspended in double-distilled water, lyophilized, and stored in closed tubes at --18°C. For each assay a sample is resuspended in doubledistilled water to a protein c o n c e n t r a t i o n of approx. 5 mg/ml. Assay of anion-sensitive ATPase. For reasons to be explained in Results, media different from those used previously [2] are chosen. Each of the media contains one major anion: HCO~, Cl-, SCN-, acetate, azide or SO~-. After addition of 20 ~l e n z y m e preparation to 300 pl medium, the incubation medium has the following final composition: 30 mM imidazole, 2 mM MgC12, 2 mM Na2ATP (neutralized with imidazole before addition), 10 -4 M ouabain and 50 mM NaHCO3, NaC1, NaSCN, NaN3 or acetate or 27.5 mM Na2SO3. The pH of the medium is adjusted to 7.0 with the corresponding acid, e x c e p t in the case of SCN- or azide where acetic acid is used. After incubation at 37~C the reaction is stopped by addition of 1 ml ice-cold 8.6% trichloroacetic acid. Blanks are run at 0°C. The liberated phosphate is determined as previously described [ 2]. When oligomycin in ethanolic solution is applied, controls containing the same a m o u n t of ethanol (~2%, v/v) are included. Preincubation for 15 min at O C and 5 rain at 37°C in the absence of ATP precedes the incubation in this case. The effect of T r i t on X-100 on the e n z y m e activity is tested by treating the lyophilized preparation, resuspended in 30 mM imidazole • HC1 (pH 7.0) with various Tr ito n X-100 concentrations for 60 min at 0°C after which aliquots are taken to measure ATPase activity. Protein determination. Protein concentrations are estimated by the m e t h o d o f L o w r y et al. [7]. Bovine serum albumin serves as standard. Materials. Na2ATP, Na3CTP, Na3GTP, Na3ITP, Na3UTP, Na2AMP, ADP and
85 carbonic anhydrase (bovine erythrocyte) are obtained from Boehringer (Mannheim, G.F.R.), ruthenium red (Ru(NH3)4(OH)C1-2H20) from Fluka AG (Buchs SG, Switzerland), chlorpromazine from Specia (Amstelveen, The Netherlands), Triton X-100 from The British Drug Houses Ltd. (Poole, U.K.) and acetazolamide from Sigma (St. Louis, Mo., U.S.A.). Oligomycin (Sigma, St. Louis, Mo., U.S.A.) consists of 15% oligomycin A and 85% oligomycin B (average molecular weight, 400). Dichlorphenamide is a gift LaBaz B.V. (Maassluis, The Netherlands), and the ionophore A-23187 from Eli Lilly and Company (Indianapolis, U.S.A.). H2DIDS (4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid) is synthesized by Prof. Dr. H. Fasold and is a gift from Prof. Dr. H. Passow (Max-PlanckInstitut fiir Biophysik, Frankfurt a/M, G.F.R.). All other materials are from E. Merck (Darmstadt, G.F.R.) and are of analytical grade. Results
Effect of HCO~ With the Tris-buffered media used in our previous studies [2] no stimulation of the erythrocyte Mg2*-ATPase in the HCO~ medium compared to the C1medium can be detected between pH 7 and 9. Since Duncan [5] reported such stimulation in a medium buffered with imidazole, we have replaced Tris by 30 mM imidazole. In this medium the activity in the HCO~ medium is higher than that in the C1- medium. In Fig. 1 the pH dependence of the Mg2÷-ATPase activity in the C1- and HCO~ media is presented. The activity in the C1- medium decreases almost linearly with decreasing pH, whereas the activity in HCO~ medium remains fairly constant down to pH 7, and then begins to decrease. Further experiments have been carried out at pH 7.0.
Effect of other anions The effects of various anions are shown in Table I and compared with their effects on the ATPase activity in gastric mucosa microsomal fraction [2]. Only HCO~ raises the enzyme activity above that in C1- medium. The inhibition of the enzyme activity by SCN- is rather small in contrast to the effects of this ion in gastric mucosa [2], which is in agreement with the findings of Duncan [5]. Sulfite, which strongly stimulates the anion-sensitive Mg2÷-ATPase activity from other tissues [2,8--10], slightly inhibits the erythrocyte enzyme. Azide, which strongly inhibits the enzyme activity from gastric mucosa [2,11,12], is ineffective here.
Substrate specificity The substrate specificity of the enzyme activity is shown in Table II. Only GTP and ITP can replace ATP to a certain extent. The high activity with ADP and especially AMP in Cl- medium may be due to the presence of a separate 5'-nucleotidase activity in the erythrocyte membrane. The latter activity is inhibited by EDTA [13], and so here the HCO~ may complex a divalent cation required for the 5'-nucleotidase activity.
86
2,0 o L
E
1.5 G., E
1.0
0.5
:
HC£:~
~nedlu
i
: I
medium
i
i
i
i
6,0
6.5
7.0
7.5
m
$,
~G pH
Fig. 1. E f f e c t o f p H o n the a c t i v i t y o f a n i o n - s e n s i t i v e Mg2+-ATPase o f rabbit e r y t h r o c y t e g h o s t s in H C O ~ m e d i u m ( o ) a n d C1- m e d i u m ( e ) . T h e p H v a l u e s o f the m e d i a a r e a d j u s t e d w i t h the c o r r e s p o n d i n g acid, e x c e p t f o r v a l u e s b e l o w p H 6 , 7 w h e r e H C I is u s e d in b o t h cases. M e a n s o f t h r e e e x p e r i m e n t s w i t h s t a n d a r d errors.
Effect of oligomycin The effect of oligomycin on the enzyme activity is shown in Fig. 2. The inhibition is weak with a P/s0 below 5.6 (P/s0 is the negative logarithm of the molar inhibitor concentration at half-maximal inhibition). For the anion-sensitive Mg2÷-ATPase activity from rabbit gastric mucosa, rabbit kidney and rat pancreas, we have found P/s0 values of 7 or above [2--4]. This indicates that the erythrocyte enzyme is much less sensitive towards inhibition by oligomytin. TABLE
I
E F F E C T S OF V A R I O U S A N I O N S G H O S T S C O M P A R E D TO T H E I R FROM RABBIT GASTRIC MUCOSA
ON ANION-SENSITIVE Mg2+-ATPase FROM ERYTHROCYTE EFFECTS ON MICROSOMAL ANION-SENSITIVE Mg-ATPase
Average relative activities are presented set at 1.00.
Major a n i o n
Relative activity Erythrocyte ghosts
SO~ SCNAzide Acetate ClHCO3
with S.E. for four experiments,
0.79 0.85 0.95 0.96
± 0.06 ± 0.04 -+ 0 . 0 4 -+ 0 . 0 2
~1.00
1.27 ± 0.09
* T a k e n f r o m r e f . 2.
Gastric m u c o s a 2.59 0.28 0.10 1.38 ~1.00 1.54
± ± ± ±
0.16 0.02 0.01 0.06
± 0.04
*
w i t h the a c t i v i t y in C1- m e d i u m
87 r
> 100-
// /f
c - / ~
50
OLIGOMYCIN ERYTHROCYTE GHOSTS
0
r //
-log [ohgomycln] Fig. 2. E f f e c t o f o l i g o m y c i n o n t h e r e l a t i v e M g 2 + - A T P a s e a c t i v i t y in H C O ~ m e d i u m . activity without added oligomycin (~100%) are shown for two experiments.
Mean ratios over the
Effect of Triton X-IO0 The effect of the non-ionic detergent Triton X-100 on the enzyme activity is shown in Fig. 3. The enzyme activity in both media appears to be relatively unaffected by preincubation with up to 1 mg/ml Triton X-100, but decreases sharply at higher concentrations. At a Triton X-100/protein ratio of 3 the activity is almost abolished. Approx. 25% of the membrane protein but no anionT A B L E II S U B S T R A T E S P E C I F I C I T Y OF A N I O N - S E N S I T I V E Mg2+-ATPase FR OM E R YT HR OC YT E R e l a t i v e a c t i v i t i e s ( a c t i v i t y f o r A T P set at 1 . 0 0 ) a r e p r e s e n t e d Substrate
ATP AMP ADP GTP CTP UTP ITP
HCO 3 ~1.00 0.02 0.20 0.40 0.17 0.22 0.39
± 0.01 ± 0.10 ± 0.02 -+ 0 . 0 4 + 0.03 -+ 0 . 0 1
Cl------1.00 0.83 0.37 0.39 0.17 0.18 0.35
± ± ± ± ± ±
0.17 0.16 0.04 0.07 0.02 0.04
with S.E. for three experiments.
GHOSTS
88
!i •
T
T
2.0
~o h
-,
\,
h ~.s
l
:2
"
~
' ,,
trltonx
lil I
"00
3j
protein
1,C'
'i
0.5
0
0
"~ ~0 -4
10 2
1 T ~, TON
102 X ~ 100
', m g , r T ,
I
Fig. 3. E f f e c t of p r e i n c u b a t i o n w i t h T r i t o n X - 1 0 0 o n the Mg2+-ATPase a c t i v i t y in HCO~ m e d i u m (l::) a n d C1- m e d i u m (o). Means o f five e x p e r i m e n t s w i t h s t a n d a r d errors.
sensitive Mg2÷-ATPase activity is solubilized at this Triton X-100/protein ratio (not shown). This is in contrast to the findings for the anion-sensitive Mg 2÷ATPase activity from Necturus oxyntic cells [14] and dog [8] or rabbit gastric mucosa (our unpublished results), where a Triton X-100/protein ratio of 3 gives optimal solubilization of the enzyme activity.
Effect of H2DIDS Next, we have tested the effect of H2DIDS, which is a well-known inhibitor of the anion transport over the erythrocyte membrane [15,16]. Treatment of intact cells with 12.5 uM H2DIDS for 25 min at 37°C results in approx. 80% inhibition of sulphate equilibrium exchange [15], but does not affect the anion-sensitive Mg2÷-ATPase activity of erythrocyte ghosts prepared from these cells (Table III). In both the HCOj medium and the C1- medium no or even a slight stimulatory effect of H2DIDS is observed. Also the apparent stimulation of the enzyme activity in both media by CaC12 (see below) is unaffected by H2DIDS. This suggests that no inter-relationship exists between the enzyme activity and the " b a n d 3" protein, which is t h o u g h t to be responsible for the anion exchange over the erythrocyte membrane [ 16].
Effect of ouabain Another possibility, which must be considered, is that the enzyme activity would n o t represent a separate entity but reflect a side-effect of the (Na ÷ + K*)ATPase or (Ca 2÷ + Mg2+)ATPase activities in the erythrocyte membrane which
89 TABLE III EFFECT OF IRREVERSIBLY BOUND RABBIT ERYTHROCYTE GHOSTS
H 2 D I D S ON A N I ON-SE NSIT IVE Mg2+-ATPase AC T IVIT Y IN
R e s u l t s are e x p r e s s e d in p m o l A T P - h -j • m g I p r o t e i n ( m e a n s o f t w o e x p e r i m e n t s ) . CaC12 w a s t e s t e d at a c o n c e n t r a t i o n o f 1 0 0 p M . H 2 D I D S w a s t e s t e d b y r e a c t i n g t h e i n t a c t cells at 10% h e m a t o e r i t in i s o t o n i c p h o s p h a t e b u f f e r at p H 7.4 f o r 25 m i n at 37~C w i t h o r w i t h o u t 12.5 tiM H 2 D I D S . A f t e r this, t h e cells w e r e w a s h e d a n d a l y o p h i l i z e d g h o s t p r e p a r a t i o n w a s p r e p a r e d as d e s c r i b e d in M e t h o d s a n d M a t e r i a l s . Medium
--CaCl2 +CaC12
--H 2 DIDS
+H 2 DIDS
HCO~
CV
HCO~
Cl-
1.47 2.02
1.24 1.51
1.50 2.11
.1.32 1.57
are t h o u g h t to represent separate and distinct enzymes [ 17]. When the ouabain concentration was raised from 10-4 to 10 -3 M in the assay media, no effect was seen on the anion-sensitive Mg2÷-ATPase activity. This result, in addition to the absence of K + in the assay media, excludes a relationship between the enzyme activity and (Na ÷ + K+)ATPase activity. Relation with (Ca 2+ + Mg2÷)ATPase The enzyme could, however, very well be related to the (Ca2+ + Mg2÷)ATPase activity of the erythrocyte membrane (Fig. 4). With increasing concentrations of CaC12 the enzyme activity increases in both the HCO3 medium and the C1- medium, whereas the ratio of these activities remains fairly constant. The lowered activity in the presence of EGTA and the small increase between 1 and 10 pM Ca 2÷ may indicate the presence of some Ca 2+ in assay medium or membrane preparation. The slight decrease in the ratio of the activities in the HCO~ and C1- media at higher Ca 2÷ concentrations may be due to precipitation or complexation of calcium in the HCO~ medium. There thus appears to be a strict coupling between the ATPase activities in both media at increasing calcium concentration. The activity is inhibited by ruthenium red and even stronger by chlorpromazine (Table IV), which are both t h o u g h t to be rather specific inhibitors of erythrocyte (Ca2+ + Mg2÷)ATPase activity [18]. Addition of 100/~M EGTA, a calcium-chelating agent, also lowers the activity. The HCO~-stimulated part of the ATPase activity is inhibited 38, 94 and 73% by ruthenium red, chlorpromazine and EGTA, respectively. When 100 pM CaC12 is added to the media, the enzyme activity increases in both media and is inhibited again by ruthenium red and chlorpromazine. Relation with carbonic anhydrase Since carbonic anhydrase was reported to stimulate the anion-sensitive Mg2÷ATPase activity of renal brush border membranes [19], we have tested its effects on the erythrocyte activity. The erythrocyte enzyme appears to be stimulated somewhat by the addition of carbonic anhydrase (Table V). However, the stimulation is not prevented by acetazolamide or dichlorphenamide, which are both strong inhibitors of carbonic anhydrase activity [20]. Furthermore, the effect is exerted also by denatured (boiled) carbonic anhydrase and it
90
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200
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/"
......
15,3
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--
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T
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i
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CaCl
2
F i g . 4 . E f f e c t o f CaCI 2 o n t h e M g 2 + - A T P a s e a c t i v i t y i n H C O ~ m e d i u m ( o ) a n d in C I - m e d i u m ( e ) a n d o n t h e r a t i o o f A T P a s e a c t i v i t y in b o t h m e d i a ( n ) . E G T A is t e s t e d at a c o n c e n t r a t i o n of 1 0 0 #M. M e a n r a t i o s o v e r t h e a c t i v i t y i n t h e a b s e n c e o f a d d e d CaC12 ( ~ 1 0 0 % ) , w i t h s t a n d a r d e r r o r s , are s h o w n f o r five e x p e r i ments.
is abolished by addition of EGTA. Bovine serum albumin has no effect on the anion-sensitive Mg2+-ATPase activity (not shown), which suggests that the stimulation is not due to a general "protein" effect. Zinc, a cofactor of carbonic anhydrase, is ineffective and at higher concentrations even inhibitory. TABLE
IV
EFFECTS GHOSTS
OF VARIOUS
AGENTS
ON ANION-SENSITIVE
ATPase
ACTIVITIES
IN ERYTHROCYTE
C a C I 2 , r u t h e n i u m r e d , c h l o r p r o m a z i n e a n d E G T A are t e s t e d at 1 0 - 4 M. S p e c i f i c a c t i v i t y is e x p r e s s e d in p m o l A T P • h -1 • m g -1 p r o t e i n w i t h t h e s t a n d a r d e r r o r ; n is t h e n u m b e r o f e x p e r i m e n t s . CaC12 added
Agent
Specific activity HCO3 medium
Clmedium
Difference HCO 3 -- Cl-
Ratio HCO3/C1
-----
Control Ruthenium red Chlorpromazine EGTA
1.07 0.90 0.58 0.81
_+ 0 . 0 6 ± 0.06 + 0.02 ± 0.06
0 . 8 1 -+ 0 . 0 4 0 . 7 5 _+ 0 . 0 4 0.56 ± 0.02 0.75 t 0.06
0.26 ± 0.04 0.16 ± 0.03 0.02 ± 0.02 0 . 0 7 -+ 0 . 0 2
1.32 1.21 1.03 1.09
± ± ± ±
0.04 0.03 0.04 0.03
6 6 6 5
+ + +
Control Ruthenium red Chlorpromazine
1.73 ± 0.08 1.39 + 0.07 1.08 + 0.03
1.24 ± 0.06 1.23 ± 0.10 0 . 9 8 +_ 0 . 0 5
0.49 ± 0.03 0 . 1 6 *~ 0 . 0 4 0.10 ± 0.03
1.39 ± 0.02 1.14 ± 0.04 1 . 1 9 _+ 0 . 0 9
6 6 6
91 TABLE V EFFECTS OF A CARBONIC ANHYDRASE ACTIVITIES IN ERYTHROCYTE GHOSTS
PREPARATION
ON
ANION-SENSITIVE
Mg2+-ATPase
S p e c i f i c a c t i v i t i e s are e x p r e s s e d i n p m o l A T P • h - 1 . r a g - 1 p r o t e i n w i t h t h e s t a n d a r d e r r o r f o r f i v e e x p e r i m e n t s i n b o t h cases. A c e t a z o l a m i d e a n d E G T A w e r e t e s t e d a t a c o n c e n t r a t i o n o f 1 0 - 3 a n d 1 0 - 4 M, respectively. Agent
Specific activity
Difference
Ratio
HCO3 -- Cl-
HCO3/CI-
ncoi
el-
medium
medium
Control Carbonic anhydrase, 200 units/ml Carbonic anhydrase + acetazolamide * Acetazolamide
1.57 2.99 3.35 1.60
+ 0.03 _+ 0 . 2 4 ± 0.29 + 0.07
1.20 1.93 2.13 1.24
~ 0.03 _+ 0 . 0 6 ± 0.14 + 0.05
0.37 1.06 1.23 0.35
± ± ± ±
0.01 0.19 0.18 0.05
1.31 1.54 1.57 1.29
± ± ± ±
0.02 0.08 0.06 0.05
Control Carbonic anhydrase, 100 units/ml Carbonic anhydrase denaturated Carbonic anhydrase + EGTA
1.41 2.10 1.99 1.02
± 0.12 _+ 0 . 1 6 ~ 0.15 ± 0.06
1.08 1.41 1.34 0.91
_+ 0 . 0 9 ± 0.06 ± 0.09 ± 0.04
0.34 0.69 0.66 0.11
± 0.04 _+ 0 . 1 3 ± 0.08 ± 0.04
1.31 1.48 1.49 1.12
* ± ± ±
0.02 0.09 0.05 0.05
* D i c h l o r p h e n a m i d e , a n o t h e r i n h i b i t o r o f c a r b o n i c a n h y d r a s e (n = 2) o r h i g h e r c o n c e n t r a t i o n s a c e t a z o l a m i d e (5 m M ) do n o t a b o l i s h t h e c a r b o n i c a n h y d r a s e effect either.
of
Hence, the stimulatory effect might be due to contamination of the commercial carbonic anhydrase preparation with either calcium or an activator of erythrocyte (Ca2++ Mg2÷)ATPase [21,22]. The first possibility, a stimulatory effect of calcium, cannot fully explain the carbonic anhydrase effect, since the activity ratio also increases (Table V) which is in conflict with the negligible effect of CaC12 on this ratio (Fig. 4). The second possibility, stimulation by an activator in the carbonic anhydrase preparation, is not ruled out by the persistence of the effect upon boiling the preparation, since the activator protein seems to be relatively heat stable [21]. On the other hand, simultaneous addition of carbonic anhydrase and EGTA abolishes the stimulation by carbonic anhydrase (Table V). Thus both calcium and the activator protein could, either together or separately, be responsible for the observed effect.
Effect of ionophore The question arises, whether the anion-sensitivity of the (Ca2++ Mg2+)ATPase activity of the erythrocyte membrane is a direct effect of HCO~ on the enzyme, or whether HCO~ acts by increasing the Ca 2÷ permeability of membrane vesicles. Although vesicle formation is rather improbable in the lyophilized preparation, we have tested the effect of the calcium ionophore A-23187. At 2 • 10 -s M this substance is w i t h o u t effect on the enzyme activity in both the HCO5 medium and the C1- medium, with or w i t h o u t added 100 gM CaC12. This makes a direct effect of HCO~ on the (Ca 2÷ + Mg2÷)ATPase more likely. Discussion T h e p u r p o s e o f our s t u d y was to d e t e r m i n e w h e t h e r the e r y t h r o c y t e anionsensitive Mg2÷-ATPase activity can be a p l a s m a m e m b r a n e - l o c a t e d a n i o n trans-
92 port system. In our previous studies of various tissues we could only detect a mitochondrial localization of the enzyme in all tissues studied, viz. gastric mucosa [2], gill [2], kidney [3] and pancreas [4], thus making a role of the enzyme in anion transport across the plasma membrane in these tissues highly unlikely. However, since erythrocytes do not contain mitochondria, the reported HCO~-stimulated Mg2÷-ATPase activity in this cell [5] could still play a role in such a process. The absence of HCO~ stimulation in the presence of Tris buffer and the pH behaviour emphasize that this enzyme activity differs quite markedly from that in gastric mucosa [2] and kidney [19]. The erythrocyte activity does not only differ from the others by being relatively insensitive towards inhibition by SCN-, as previously reported by Duncan [5] and recently by Izutsu et al. [23], but also by the slight effects of sulfite and azide. Its substrate specificity resembles that of the gastric mucosal anion-sensitive Mg2÷-ATPase [2], except for the high AMPase activity in the C1- medium. It has a much lower sensitivity towards oligomycin compared to the enzyme activity from other tissues [2--4]. Triton X-100 strongly inhibits the erythrocyte activity at the concentration, which optimally solubilizes the gastric mucosal enzyme [8,14]. It is obvious that the erythrocyte enzyme differs in many ways from other anion-sensitive Mg2÷-ATPase activities. The ineffectiveness of H2DIDS appears to exclude a relationship between the anion-exchange protein in the erythrocyte membrane and the enzyme activity. Ouabain, a known inhibitor of (Na÷+ K~}ATPase activity, is also without effect, indicating that the activity does not represent an anion sensitivity of the (Na ÷ + K÷)ATPase system. The inhibition of the enzyme activity by chlorpromazine, ruthenium red and EGTA and its stimulation by Ca 2÷ strongly suggest that it is a part of the (Ca 2÷ + Mg2÷)ATPase system of the erythrocyte membrane. Increasing concentrations of CaC12 result in a parallel increase of the enzyme activity in both the HCO~ medium and the C1- medium, indicating a direct relationship between these two activities. Finally, the activity increase evoked by carbonic anhydrase, which was thought to form a part of the anion transport system, can be attributed to a contamination of the carbonic anhydrase preparation. Boiled carbonic anhydrase also stimulates the enzyme activity, whereas acetazolamide and dichlorphenamide do n o t abolish the carbonic anhydrase effect. EGTA also abolishes the effect, whereas Zn 2÷, a cofactor of carbonic anhydrase, is ineffective. The ineffectiveness of the Ca 2÷ ionophore A-23187 suggests that the HCO~ stimulation is n o t caused by an increase in calcium permeability of membrane vesicles, b u t is rather a direct effect of HCO~ on the (Ca 2÷ + Mg2÷)ATPase activity, such as reported by Ahlers and GSnther [24] for (Ca 2÷ + Mg2÷)ATPase from Escherichia coli in the presence of C1-. We must therefore conclude that the HCOS-stimulated Mg2÷-ATPase of the rabbit erythrocyte membrane does n o t only greatly differ in its properties from the anion-sensitive Mg2÷-ATPase found in other tissues, but that it actually forms part of the (Ca2÷ + Mg2÷)ATPase activity in the erythrocyte membrane and is n o t a separate enzyme. A role of this activity in anion transport across this membrane appears very improbable. In other tissues with anion-sensitive Mg2÷-ATPase activity, the enzyme does not seem to be located in the plasma
93
membrane, but rather in the mitochondria, which also precludes a role of the enzyme in anion transport. Acknowledgements
The skilful technical assistance of Mrs. Carolien van Dreumel-Hermens is gratefully acknowledged. We thank Professor Dr. H. Passow (Max-PlanckInstitut ffir Biophysik, Frankfurt a/M, G.F.R.) for the gift of H2DIDS, which was synthesized by Professor Dr. H. Fasold. This work was supported in part by the Netherlands Organisation for Basic Research (Z.W.O.) through the Netherlands Foundation for Biophysics. References 1 S o u m a r m o n , A., L e w i n , M., Cheret, A.M. a n d Bonffls, S. ( 1 9 7 4 ) Biochim. B i o p h y s . A c t a 3 3 9 , 4 0 3 - 414 2 V a n A m e l s v o o r t , J.M.M., de P o n t , J . J . H . H . M . a n d Bonting, S.L. ( 1 9 7 7 ) Biochim. Biophys. A c t a 4 6 6 , 283--301 3 Van A m e l s v o o r t , J.M.M., de P o n t , J . J . H . H . M . , Stols, A . L . H . a n d Bonting, S.L. ( 1 9 7 7 ) Biochim. Biophys. Acta 471, 78--91 4 V a n A m e l s v o o r t , J.M.M., De P o n t , J . J . H . H . M . a n d Bonting, S.L. ( 1 9 7 7 ) 1 1 t h FEBS Meet. C o p e n hagen, A b s t r . A - 4 - 1 5 - 6 0 3 - 3 5 D u n c a n , C.J. ( 1 9 7 5 ) Life Sei. 1 6 , 9 5 5 - - 9 6 6 6 S c h a r f f , O. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 4 3 , 2 0 6 - - 2 1 8 7 L o w r y , O.H., R o s e b r o u g h , N.J., F a r r , A.L. a n d R a n d a l l , R.J. ( 1 9 5 1 ) J. Biol. C h e m . 193, 2 6 5 - - 2 7 5 8 B l u m , A.L., S h a h , G., Pierre, T. St., H e l a n d e r , H . F . , Sung, C.P., Wiebelhaus, V.D. a n d Sachs, G. ( 1 9 7 1 ) Biochim. B i o p h y s . A c t a 2 4 9 , 1 0 1 - - 1 1 3 9 S i m o n , B. a n d T h o m a s , L. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 8 8 , 4 3 4 - - 4 4 2 10 S i m o n , B., K i n n e , R. a n d K n a u f , H. ( 1 9 7 2 ) Pfliigers A r c h . 3 3 7 , 1 7 7 - - 1 8 4 11 K a s b e k a r , D.K., D u r b i n , R.P. a n d L i n d l e y , D. ( 1 9 6 5 ) Biochim. Biophys. A c t a 1 0 5 , 4 7 2 - - 4 8 2 12 Sachs, G., Wiebelhaus, V.D., B l u m , A.L. a n d H i r s c h o w i t z , B.I. ( 1 9 7 2 ) in Gastric S e c r e t i o n (Sachs, G., Heinz, E. a n d Ullrich, K.J., eds.), pp. 3 2 1 - - 3 4 3 , A c a d e m i c Press, L o n d o n 13 D r u m m o n d , G.I. a n d Y a m a m o t o , M. ( 1 9 7 1 ) in The E n z y m e s ( B o y e r , P.D., ed.), Vol. IV, pp. 3 3 7 - 3 5 4 , A c a d e m i c press, L o n d o n 14 Wiebelhaus, V.D., S u n g , C.P., H e l a n d e r , H.F., S h a h , G., Blum, A.L. a n d Sachs, G. ( 1 9 7 1 ) Biochim. Biophys. Acta 241, 49--56 15 L e p k e , S., F a s o l d , H., Pring, M. a n d Passow, H. ( 1 9 7 6 ) J. M e m b r a n e Biol. 29, 1 4 7 - - 1 7 7 16 R o t h s t e i n , A., C a b a n t c h i k , Z.I. a n d K n a u f , P. ( 1 9 7 6 ) Fed. Proc. 35, 3 - - 1 0 17 S c h a t z m a n n , H.J. ( 1 9 7 5 ) in Calcium T r a n s p o r t in C o n t r a c t i o n a n d S e c r e t i o n (Carafoli, E., Clementi, F., D r a b i k o w s k i , W. a n d M a r g r e t h , A., eds.), p p . 4 5 - - 4 9 , N o r t h - H o l l . Publ. Co., A m s t e r d a m 18 B o n t i n g , S.L. a n d de P o n t , J . J . H . H . M . ( 1 9 7 7 ) in M a m m a l i a n Cell M e m b r a n e s ( J a m i e s o n , G.A. a n d R o b i n s o n , D.M., eds.), Vol. 4, pp. 1 4 5 - - 1 8 3 , B u t t e r w o r t h , L o n d o n 19 Liang, C.T. a n d S a c k t o r , B. ( 1 9 7 6 ) A r c h . B i o c h e m . B i o p h y s . 1 7 6 , 2 8 5 - - 2 9 7 20 Maren, T h . H . , ParceU, A.L. a n d Malik, M.N. ( 1 9 6 0 ) J. P h a r m a c o l . Exp. Ther. 1 3 0 , 3 8 9 - - 4 0 0 21 L u t h r a , M.G., H i l d e n b r a n d t , G.R. a n d H a n a h a n , D.J. ( 1 9 7 6 ) Biochim. B i o p h y s . A c t a 4 1 9 , 1 6 4 - - 1 7 9 22 B o n d , G.H. a n d C l o u g h , D.L. ( 1 9 7 3 ) Biochim. B i o p h y s . A c t a 3 2 3 , 5 9 2 - - 5 9 9 23 Izutsu, K.T., M a d d e n , P.R., Watson, E.L. a n d Siegel, I.A. ( 1 9 7 7 ) Pfltigers A r c h . 3 6 9 , 1 1 9 - - 1 2 4 24 Ahlers, J. a n d G i i n t h e r , Th. ( 1 9 7 5 ) A r c h . B i o c h e m . Biophys. 1 7 1 , 1 6 3 - - 1 6 9
Biochimica et Biophysica Acta, 507 (1978) 83--93 © Elsevier/North-Holland Biomedical Press
BBA 77917
IS T H E R E A PLASMA MEMBRANE-LOCATED ANION-SENSITIVE ATPase? III. IDENTITY OF THE E R Y T H R O C Y T E ENZYME WITH ( C a 2+ + Mg 2+)-ATPase
J.M.M. VAN AMELSVOORT, P.M.K.B. VAN HOOF, J.J.H.H.M. DE PONT and S.L. BONTING
Department of Biochemistry, University of Ni]megen, Ni]megen (The Netherlands) (Received July 12th, 1977)
Summary The characteristics of the anion-sensitive Mg:+-ATPase activity of the rabbit erythrocyte have been studied in a lyophilized ghost preparation. The enzyme appears to be different from the anion-sensitive Mg2+-ATPase activity of other tissues in many parameters, such as optimal pH, effects of various anions, oligomycin sensitivity and effects of Triton X-100. The enzyme is insensitive towards inhibition by irreversibly b o u n d 4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). This excludes a relationship between the enzyme and the " b a n d 3 " protein, which is thought to be involved in the anion exchange over the erythrocyte membrane. From the effects of ethyleneglycol-bis-(fi-aminoethylether)-N,N'-tetraacetic acid (EGTA), CaCl~, chlorpromazine and ruthenium red it is concluded that the enzyme activity does n o t represent a separate entity b u t is part of the (Ca 2÷ + Mg2+)-ATPase system of the erythrocyte membrane. A reported stimulatory effect of carbonic anhydrase is attributed to a contamination of the carbonic anhydrase preparation by calcium and/or (Ca 2÷ + Mg2÷)-ATPase activator protein.
Introduction Previous studies by others [1] and ourselves [2--4] have cast serious d o u b t on the presence of an anion-sensitive Mg~+-ATPase in plasma membranes of various tissues like gastric mucosa [1,2], trout gill [2], kidney [3] and pancreas [4] and thus on its alleged role in anion transport. In these tissues the enzyme Abbreviations: H2DIDS, 4,4r-diisothiocyano-dihydrostilbene-2,2r-disulfonic acid; EGTA, ethylene-
glycol-bis-(fl-aminoethylether)-N,N~-tetraacetic acid.
84 seems to be located in the m i t o c h o n d r i a rather than in the plasma membrane. A particulate anion-sensitive Mg2*-ATPase activity has, however, also been reported in rabbit e r y t h r o c y t e s [5], which are devoid of mitochondria. Hence, this activity must be located in the plasma membrane. It differs in one respect from the comparable activity in other tissues: it is not inhibited by thiocyahate. In view of the obvious importance of the question whether this e n z y m e activity can be involved in anion transport, we have decided to make a thorough study of its characteristics. Methods and Materials
Preparation of erythrocyte ghosts. E r y t h r o c y t e ghosts are prepared according to the m e t h o d of Duncan [5] with slight modifications. Blood from New Zealand white rabbits, freshly obtained by heart puncture, is immediately mixed with 0.1 volume 3.8% sodium citrate, is cooled to 0°C, is filtered over four layers of surgical gauze, and is centrifuged for 15 min at 1400 × g. After removal of plasma and " b u f f y c o a t " by aspiration, the e r y t h r o c y t e s are washed three times with isotonic (310 mosM) sodium phosphate buffer at pH 8.0 [6], and h e m o l y z e d in 20 mM imidazole • HC1 (pH 7.5). The membranes are centrifuged for 25 min at 30 000 ×g, are washed once with 4 mM MgC12, 1 mM EDTA, 20 mM imidazole • HC1 (pH 7.5), two or three times with 2 mM MgC12, 20 mM i m i d a z o l e . HC1 (pH 7.5) and finally in double-distilled water. The membranes are resuspended in double-distilled water, lyophilized, and stored in closed tubes at --18°C. For each assay a sample is resuspended in doubledistilled water to a protein c o n c e n t r a t i o n of approx. 5 mg/ml. Assay of anion-sensitive ATPase. For reasons to be explained in Results, media different from those used previously [2] are chosen. Each of the media contains one major anion: HCO~, Cl-, SCN-, acetate, azide or SO~-. After addition of 20 ~l e n z y m e preparation to 300 pl medium, the incubation medium has the following final composition: 30 mM imidazole, 2 mM MgC12, 2 mM Na2ATP (neutralized with imidazole before addition), 10 -4 M ouabain and 50 mM NaHCO3, NaC1, NaSCN, NaN3 or acetate or 27.5 mM Na2SO3. The pH of the medium is adjusted to 7.0 with the corresponding acid, e x c e p t in the case of SCN- or azide where acetic acid is used. After incubation at 37~C the reaction is stopped by addition of 1 ml ice-cold 8.6% trichloroacetic acid. Blanks are run at 0°C. The liberated phosphate is determined as previously described [ 2]. When oligomycin in ethanolic solution is applied, controls containing the same a m o u n t of ethanol (~2%, v/v) are included. Preincubation for 15 min at O C and 5 rain at 37°C in the absence of ATP precedes the incubation in this case. The effect of T r i t on X-100 on the e n z y m e activity is tested by treating the lyophilized preparation, resuspended in 30 mM imidazole • HC1 (pH 7.0) with various Tr ito n X-100 concentrations for 60 min at 0°C after which aliquots are taken to measure ATPase activity. Protein determination. Protein concentrations are estimated by the m e t h o d o f L o w r y et al. [7]. Bovine serum albumin serves as standard. Materials. Na2ATP, Na3CTP, Na3GTP, Na3ITP, Na3UTP, Na2AMP, ADP and
85 carbonic anhydrase (bovine erythrocyte) are obtained from Boehringer (Mannheim, G.F.R.), ruthenium red (Ru(NH3)4(OH)C1-2H20) from Fluka AG (Buchs SG, Switzerland), chlorpromazine from Specia (Amstelveen, The Netherlands), Triton X-100 from The British Drug Houses Ltd. (Poole, U.K.) and acetazolamide from Sigma (St. Louis, Mo., U.S.A.). Oligomycin (Sigma, St. Louis, Mo., U.S.A.) consists of 15% oligomycin A and 85% oligomycin B (average molecular weight, 400). Dichlorphenamide is a gift LaBaz B.V. (Maassluis, The Netherlands), and the ionophore A-23187 from Eli Lilly and Company (Indianapolis, U.S.A.). H2DIDS (4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid) is synthesized by Prof. Dr. H. Fasold and is a gift from Prof. Dr. H. Passow (Max-PlanckInstitut fiir Biophysik, Frankfurt a/M, G.F.R.). All other materials are from E. Merck (Darmstadt, G.F.R.) and are of analytical grade. Results
Effect of HCO~ With the Tris-buffered media used in our previous studies [2] no stimulation of the erythrocyte Mg2*-ATPase in the HCO~ medium compared to the C1medium can be detected between pH 7 and 9. Since Duncan [5] reported such stimulation in a medium buffered with imidazole, we have replaced Tris by 30 mM imidazole. In this medium the activity in the HCO~ medium is higher than that in the C1- medium. In Fig. 1 the pH dependence of the Mg2÷-ATPase activity in the C1- and HCO~ media is presented. The activity in the C1- medium decreases almost linearly with decreasing pH, whereas the activity in HCO~ medium remains fairly constant down to pH 7, and then begins to decrease. Further experiments have been carried out at pH 7.0.
Effect of other anions The effects of various anions are shown in Table I and compared with their effects on the ATPase activity in gastric mucosa microsomal fraction [2]. Only HCO~ raises the enzyme activity above that in C1- medium. The inhibition of the enzyme activity by SCN- is rather small in contrast to the effects of this ion in gastric mucosa [2], which is in agreement with the findings of Duncan [5]. Sulfite, which strongly stimulates the anion-sensitive Mg2÷-ATPase activity from other tissues [2,8--10], slightly inhibits the erythrocyte enzyme. Azide, which strongly inhibits the enzyme activity from gastric mucosa [2,11,12], is ineffective here.
Substrate specificity The substrate specificity of the enzyme activity is shown in Table II. Only GTP and ITP can replace ATP to a certain extent. The high activity with ADP and especially AMP in Cl- medium may be due to the presence of a separate 5'-nucleotidase activity in the erythrocyte membrane. The latter activity is inhibited by EDTA [13], and so here the HCO~ may complex a divalent cation required for the 5'-nucleotidase activity.
86
2,0 o L
E
1.5 G., E
1.0
0.5
:
HC£:~
~nedlu
i
: I
medium
i
i
i
i
6,0
6.5
7.0
7.5
m
$,
~G pH
Fig. 1. E f f e c t o f p H o n the a c t i v i t y o f a n i o n - s e n s i t i v e Mg2+-ATPase o f rabbit e r y t h r o c y t e g h o s t s in H C O ~ m e d i u m ( o ) a n d C1- m e d i u m ( e ) . T h e p H v a l u e s o f the m e d i a a r e a d j u s t e d w i t h the c o r r e s p o n d i n g acid, e x c e p t f o r v a l u e s b e l o w p H 6 , 7 w h e r e H C I is u s e d in b o t h cases. M e a n s o f t h r e e e x p e r i m e n t s w i t h s t a n d a r d errors.
Effect of oligomycin The effect of oligomycin on the enzyme activity is shown in Fig. 2. The inhibition is weak with a P/s0 below 5.6 (P/s0 is the negative logarithm of the molar inhibitor concentration at half-maximal inhibition). For the anion-sensitive Mg2÷-ATPase activity from rabbit gastric mucosa, rabbit kidney and rat pancreas, we have found P/s0 values of 7 or above [2--4]. This indicates that the erythrocyte enzyme is much less sensitive towards inhibition by oligomytin. TABLE
I
E F F E C T S OF V A R I O U S A N I O N S G H O S T S C O M P A R E D TO T H E I R FROM RABBIT GASTRIC MUCOSA
ON ANION-SENSITIVE Mg2+-ATPase FROM ERYTHROCYTE EFFECTS ON MICROSOMAL ANION-SENSITIVE Mg-ATPase
Average relative activities are presented set at 1.00.
Major a n i o n
Relative activity Erythrocyte ghosts
SO~ SCNAzide Acetate ClHCO3
with S.E. for four experiments,
0.79 0.85 0.95 0.96
± 0.06 ± 0.04 -+ 0 . 0 4 -+ 0 . 0 2
~1.00
1.27 ± 0.09
* T a k e n f r o m r e f . 2.
Gastric m u c o s a 2.59 0.28 0.10 1.38 ~1.00 1.54
± ± ± ±
0.16 0.02 0.01 0.06
± 0.04
*
w i t h the a c t i v i t y in C1- m e d i u m
87 r
> 100-
// /f
c - / ~
50
OLIGOMYCIN ERYTHROCYTE GHOSTS
0
r //
-log [ohgomycln] Fig. 2. E f f e c t o f o l i g o m y c i n o n t h e r e l a t i v e M g 2 + - A T P a s e a c t i v i t y in H C O ~ m e d i u m . activity without added oligomycin (~100%) are shown for two experiments.
Mean ratios over the
Effect of Triton X-IO0 The effect of the non-ionic detergent Triton X-100 on the enzyme activity is shown in Fig. 3. The enzyme activity in both media appears to be relatively unaffected by preincubation with up to 1 mg/ml Triton X-100, but decreases sharply at higher concentrations. At a Triton X-100/protein ratio of 3 the activity is almost abolished. Approx. 25% of the membrane protein but no anionT A B L E II S U B S T R A T E S P E C I F I C I T Y OF A N I O N - S E N S I T I V E Mg2+-ATPase FR OM E R YT HR OC YT E R e l a t i v e a c t i v i t i e s ( a c t i v i t y f o r A T P set at 1 . 0 0 ) a r e p r e s e n t e d Substrate
ATP AMP ADP GTP CTP UTP ITP
HCO 3 ~1.00 0.02 0.20 0.40 0.17 0.22 0.39
± 0.01 ± 0.10 ± 0.02 -+ 0 . 0 4 + 0.03 -+ 0 . 0 1
Cl------1.00 0.83 0.37 0.39 0.17 0.18 0.35
± ± ± ± ± ±
0.17 0.16 0.04 0.07 0.02 0.04
with S.E. for three experiments.
GHOSTS
88
!i •
T
T
2.0
~o h
-,
\,
h ~.s
l
:2
"
~
' ,,
trltonx
lil I
"00
3j
protein
1,C'
'i
0.5
0
0
"~ ~0 -4
10 2
1 T ~, TON
102 X ~ 100
', m g , r T ,
I
Fig. 3. E f f e c t of p r e i n c u b a t i o n w i t h T r i t o n X - 1 0 0 o n the Mg2+-ATPase a c t i v i t y in HCO~ m e d i u m (l::) a n d C1- m e d i u m (o). Means o f five e x p e r i m e n t s w i t h s t a n d a r d errors.
sensitive Mg2÷-ATPase activity is solubilized at this Triton X-100/protein ratio (not shown). This is in contrast to the findings for the anion-sensitive Mg 2÷ATPase activity from Necturus oxyntic cells [14] and dog [8] or rabbit gastric mucosa (our unpublished results), where a Triton X-100/protein ratio of 3 gives optimal solubilization of the enzyme activity.
Effect of H2DIDS Next, we have tested the effect of H2DIDS, which is a well-known inhibitor of the anion transport over the erythrocyte membrane [15,16]. Treatment of intact cells with 12.5 uM H2DIDS for 25 min at 37°C results in approx. 80% inhibition of sulphate equilibrium exchange [15], but does not affect the anion-sensitive Mg2÷-ATPase activity of erythrocyte ghosts prepared from these cells (Table III). In both the HCOj medium and the C1- medium no or even a slight stimulatory effect of H2DIDS is observed. Also the apparent stimulation of the enzyme activity in both media by CaC12 (see below) is unaffected by H2DIDS. This suggests that no inter-relationship exists between the enzyme activity and the " b a n d 3" protein, which is t h o u g h t to be responsible for the anion exchange over the erythrocyte membrane [ 16].
Effect of ouabain Another possibility, which must be considered, is that the enzyme activity would n o t represent a separate entity but reflect a side-effect of the (Na ÷ + K*)ATPase or (Ca 2÷ + Mg2+)ATPase activities in the erythrocyte membrane which
89 TABLE III EFFECT OF IRREVERSIBLY BOUND RABBIT ERYTHROCYTE GHOSTS
H 2 D I D S ON A N I ON-SE NSIT IVE Mg2+-ATPase AC T IVIT Y IN
R e s u l t s are e x p r e s s e d in p m o l A T P - h -j • m g I p r o t e i n ( m e a n s o f t w o e x p e r i m e n t s ) . CaC12 w a s t e s t e d at a c o n c e n t r a t i o n o f 1 0 0 p M . H 2 D I D S w a s t e s t e d b y r e a c t i n g t h e i n t a c t cells at 10% h e m a t o e r i t in i s o t o n i c p h o s p h a t e b u f f e r at p H 7.4 f o r 25 m i n at 37~C w i t h o r w i t h o u t 12.5 tiM H 2 D I D S . A f t e r this, t h e cells w e r e w a s h e d a n d a l y o p h i l i z e d g h o s t p r e p a r a t i o n w a s p r e p a r e d as d e s c r i b e d in M e t h o d s a n d M a t e r i a l s . Medium
--CaCl2 +CaC12
--H 2 DIDS
+H 2 DIDS
HCO~
CV
HCO~
Cl-
1.47 2.02
1.24 1.51
1.50 2.11
.1.32 1.57
are t h o u g h t to represent separate and distinct enzymes [ 17]. When the ouabain concentration was raised from 10-4 to 10 -3 M in the assay media, no effect was seen on the anion-sensitive Mg2÷-ATPase activity. This result, in addition to the absence of K + in the assay media, excludes a relationship between the enzyme activity and (Na ÷ + K+)ATPase activity. Relation with (Ca 2+ + Mg2÷)ATPase The enzyme could, however, very well be related to the (Ca2+ + Mg2÷)ATPase activity of the erythrocyte membrane (Fig. 4). With increasing concentrations of CaC12 the enzyme activity increases in both the HCO3 medium and the C1- medium, whereas the ratio of these activities remains fairly constant. The lowered activity in the presence of EGTA and the small increase between 1 and 10 pM Ca 2÷ may indicate the presence of some Ca 2+ in assay medium or membrane preparation. The slight decrease in the ratio of the activities in the HCO~ and C1- media at higher Ca 2÷ concentrations may be due to precipitation or complexation of calcium in the HCO~ medium. There thus appears to be a strict coupling between the ATPase activities in both media at increasing calcium concentration. The activity is inhibited by ruthenium red and even stronger by chlorpromazine (Table IV), which are both t h o u g h t to be rather specific inhibitors of erythrocyte (Ca2+ + Mg2÷)ATPase activity [18]. Addition of 100/~M EGTA, a calcium-chelating agent, also lowers the activity. The HCO~-stimulated part of the ATPase activity is inhibited 38, 94 and 73% by ruthenium red, chlorpromazine and EGTA, respectively. When 100 pM CaC12 is added to the media, the enzyme activity increases in both media and is inhibited again by ruthenium red and chlorpromazine. Relation with carbonic anhydrase Since carbonic anhydrase was reported to stimulate the anion-sensitive Mg2÷ATPase activity of renal brush border membranes [19], we have tested its effects on the erythrocyte activity. The erythrocyte enzyme appears to be stimulated somewhat by the addition of carbonic anhydrase (Table V). However, the stimulation is not prevented by acetazolamide or dichlorphenamide, which are both strong inhibitors of carbonic anhydrase activity [20]. Furthermore, the effect is exerted also by denatured (boiled) carbonic anhydrase and it
90
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F i g . 4 . E f f e c t o f CaCI 2 o n t h e M g 2 + - A T P a s e a c t i v i t y i n H C O ~ m e d i u m ( o ) a n d in C I - m e d i u m ( e ) a n d o n t h e r a t i o o f A T P a s e a c t i v i t y in b o t h m e d i a ( n ) . E G T A is t e s t e d at a c o n c e n t r a t i o n of 1 0 0 #M. M e a n r a t i o s o v e r t h e a c t i v i t y i n t h e a b s e n c e o f a d d e d CaC12 ( ~ 1 0 0 % ) , w i t h s t a n d a r d e r r o r s , are s h o w n f o r five e x p e r i ments.
is abolished by addition of EGTA. Bovine serum albumin has no effect on the anion-sensitive Mg2+-ATPase activity (not shown), which suggests that the stimulation is not due to a general "protein" effect. Zinc, a cofactor of carbonic anhydrase, is ineffective and at higher concentrations even inhibitory. TABLE
IV
EFFECTS GHOSTS
OF VARIOUS
AGENTS
ON ANION-SENSITIVE
ATPase
ACTIVITIES
IN ERYTHROCYTE
C a C I 2 , r u t h e n i u m r e d , c h l o r p r o m a z i n e a n d E G T A are t e s t e d at 1 0 - 4 M. S p e c i f i c a c t i v i t y is e x p r e s s e d in p m o l A T P • h -1 • m g -1 p r o t e i n w i t h t h e s t a n d a r d e r r o r ; n is t h e n u m b e r o f e x p e r i m e n t s . CaC12 added
Agent
Specific activity HCO3 medium
Clmedium
Difference HCO 3 -- Cl-
Ratio HCO3/C1
-----
Control Ruthenium red Chlorpromazine EGTA
1.07 0.90 0.58 0.81
_+ 0 . 0 6 ± 0.06 + 0.02 ± 0.06
0 . 8 1 -+ 0 . 0 4 0 . 7 5 _+ 0 . 0 4 0.56 ± 0.02 0.75 t 0.06
0.26 ± 0.04 0.16 ± 0.03 0.02 ± 0.02 0 . 0 7 -+ 0 . 0 2
1.32 1.21 1.03 1.09
± ± ± ±
0.04 0.03 0.04 0.03
6 6 6 5
+ + +
Control Ruthenium red Chlorpromazine
1.73 ± 0.08 1.39 + 0.07 1.08 + 0.03
1.24 ± 0.06 1.23 ± 0.10 0 . 9 8 +_ 0 . 0 5
0.49 ± 0.03 0 . 1 6 *~ 0 . 0 4 0.10 ± 0.03
1.39 ± 0.02 1.14 ± 0.04 1 . 1 9 _+ 0 . 0 9
6 6 6
91 TABLE V EFFECTS OF A CARBONIC ANHYDRASE ACTIVITIES IN ERYTHROCYTE GHOSTS
PREPARATION
ON
ANION-SENSITIVE
Mg2+-ATPase
S p e c i f i c a c t i v i t i e s are e x p r e s s e d i n p m o l A T P • h - 1 . r a g - 1 p r o t e i n w i t h t h e s t a n d a r d e r r o r f o r f i v e e x p e r i m e n t s i n b o t h cases. A c e t a z o l a m i d e a n d E G T A w e r e t e s t e d a t a c o n c e n t r a t i o n o f 1 0 - 3 a n d 1 0 - 4 M, respectively. Agent
Specific activity
Difference
Ratio
HCO3 -- Cl-
HCO3/CI-
ncoi
el-
medium
medium
Control Carbonic anhydrase, 200 units/ml Carbonic anhydrase + acetazolamide * Acetazolamide
1.57 2.99 3.35 1.60
+ 0.03 _+ 0 . 2 4 ± 0.29 + 0.07
1.20 1.93 2.13 1.24
~ 0.03 _+ 0 . 0 6 ± 0.14 + 0.05
0.37 1.06 1.23 0.35
± ± ± ±
0.01 0.19 0.18 0.05
1.31 1.54 1.57 1.29
± ± ± ±
0.02 0.08 0.06 0.05
Control Carbonic anhydrase, 100 units/ml Carbonic anhydrase denaturated Carbonic anhydrase + EGTA
1.41 2.10 1.99 1.02
± 0.12 _+ 0 . 1 6 ~ 0.15 ± 0.06
1.08 1.41 1.34 0.91
_+ 0 . 0 9 ± 0.06 ± 0.09 ± 0.04
0.34 0.69 0.66 0.11
± 0.04 _+ 0 . 1 3 ± 0.08 ± 0.04
1.31 1.48 1.49 1.12
* ± ± ±
0.02 0.09 0.05 0.05
* D i c h l o r p h e n a m i d e , a n o t h e r i n h i b i t o r o f c a r b o n i c a n h y d r a s e (n = 2) o r h i g h e r c o n c e n t r a t i o n s a c e t a z o l a m i d e (5 m M ) do n o t a b o l i s h t h e c a r b o n i c a n h y d r a s e effect either.
of
Hence, the stimulatory effect might be due to contamination of the commercial carbonic anhydrase preparation with either calcium or an activator of erythrocyte (Ca2++ Mg2÷)ATPase [21,22]. The first possibility, a stimulatory effect of calcium, cannot fully explain the carbonic anhydrase effect, since the activity ratio also increases (Table V) which is in conflict with the negligible effect of CaC12 on this ratio (Fig. 4). The second possibility, stimulation by an activator in the carbonic anhydrase preparation, is not ruled out by the persistence of the effect upon boiling the preparation, since the activator protein seems to be relatively heat stable [21]. On the other hand, simultaneous addition of carbonic anhydrase and EGTA abolishes the stimulation by carbonic anhydrase (Table V). Thus both calcium and the activator protein could, either together or separately, be responsible for the observed effect.
Effect of ionophore The question arises, whether the anion-sensitivity of the (Ca2++ Mg2+)ATPase activity of the erythrocyte membrane is a direct effect of HCO~ on the enzyme, or whether HCO~ acts by increasing the Ca 2÷ permeability of membrane vesicles. Although vesicle formation is rather improbable in the lyophilized preparation, we have tested the effect of the calcium ionophore A-23187. At 2 • 10 -s M this substance is w i t h o u t effect on the enzyme activity in both the HCO5 medium and the C1- medium, with or w i t h o u t added 100 gM CaC12. This makes a direct effect of HCO~ on the (Ca 2÷ + Mg2÷)ATPase more likely. Discussion T h e p u r p o s e o f our s t u d y was to d e t e r m i n e w h e t h e r the e r y t h r o c y t e anionsensitive Mg2÷-ATPase activity can be a p l a s m a m e m b r a n e - l o c a t e d a n i o n trans-
92 port system. In our previous studies of various tissues we could only detect a mitochondrial localization of the enzyme in all tissues studied, viz. gastric mucosa [2], gill [2], kidney [3] and pancreas [4], thus making a role of the enzyme in anion transport across the plasma membrane in these tissues highly unlikely. However, since erythrocytes do not contain mitochondria, the reported HCO~-stimulated Mg2÷-ATPase activity in this cell [5] could still play a role in such a process. The absence of HCO~ stimulation in the presence of Tris buffer and the pH behaviour emphasize that this enzyme activity differs quite markedly from that in gastric mucosa [2] and kidney [19]. The erythrocyte activity does not only differ from the others by being relatively insensitive towards inhibition by SCN-, as previously reported by Duncan [5] and recently by Izutsu et al. [23], but also by the slight effects of sulfite and azide. Its substrate specificity resembles that of the gastric mucosal anion-sensitive Mg2÷-ATPase [2], except for the high AMPase activity in the C1- medium. It has a much lower sensitivity towards oligomycin compared to the enzyme activity from other tissues [2--4]. Triton X-100 strongly inhibits the erythrocyte activity at the concentration, which optimally solubilizes the gastric mucosal enzyme [8,14]. It is obvious that the erythrocyte enzyme differs in many ways from other anion-sensitive Mg2÷-ATPase activities. The ineffectiveness of H2DIDS appears to exclude a relationship between the anion-exchange protein in the erythrocyte membrane and the enzyme activity. Ouabain, a known inhibitor of (Na÷+ K~}ATPase activity, is also without effect, indicating that the activity does not represent an anion sensitivity of the (Na ÷ + K÷)ATPase system. The inhibition of the enzyme activity by chlorpromazine, ruthenium red and EGTA and its stimulation by Ca 2÷ strongly suggest that it is a part of the (Ca 2÷ + Mg2÷)ATPase system of the erythrocyte membrane. Increasing concentrations of CaC12 result in a parallel increase of the enzyme activity in both the HCO~ medium and the C1- medium, indicating a direct relationship between these two activities. Finally, the activity increase evoked by carbonic anhydrase, which was thought to form a part of the anion transport system, can be attributed to a contamination of the carbonic anhydrase preparation. Boiled carbonic anhydrase also stimulates the enzyme activity, whereas acetazolamide and dichlorphenamide do n o t abolish the carbonic anhydrase effect. EGTA also abolishes the effect, whereas Zn 2÷, a cofactor of carbonic anhydrase, is ineffective. The ineffectiveness of the Ca 2÷ ionophore A-23187 suggests that the HCO~ stimulation is n o t caused by an increase in calcium permeability of membrane vesicles, b u t is rather a direct effect of HCO~ on the (Ca 2÷ + Mg2÷)ATPase activity, such as reported by Ahlers and GSnther [24] for (Ca 2÷ + Mg2÷)ATPase from Escherichia coli in the presence of C1-. We must therefore conclude that the HCOS-stimulated Mg2÷-ATPase of the rabbit erythrocyte membrane does n o t only greatly differ in its properties from the anion-sensitive Mg2÷-ATPase found in other tissues, but that it actually forms part of the (Ca2÷ + Mg2÷)ATPase activity in the erythrocyte membrane and is n o t a separate enzyme. A role of this activity in anion transport across this membrane appears very improbable. In other tissues with anion-sensitive Mg2÷-ATPase activity, the enzyme does not seem to be located in the plasma
93
membrane, but rather in the mitochondria, which also precludes a role of the enzyme in anion transport. Acknowledgements
The skilful technical assistance of Mrs. Carolien van Dreumel-Hermens is gratefully acknowledged. We thank Professor Dr. H. Passow (Max-PlanckInstitut ffir Biophysik, Frankfurt a/M, G.F.R.) for the gift of H2DIDS, which was synthesized by Professor Dr. H. Fasold. This work was supported in part by the Netherlands Organisation for Basic Research (Z.W.O.) through the Netherlands Foundation for Biophysics. References 1 S o u m a r m o n , A., L e w i n , M., Cheret, A.M. a n d Bonffls, S. ( 1 9 7 4 ) Biochim. B i o p h y s . A c t a 3 3 9 , 4 0 3 - 414 2 V a n A m e l s v o o r t , J.M.M., de P o n t , J . J . H . H . M . a n d Bonting, S.L. ( 1 9 7 7 ) Biochim. Biophys. A c t a 4 6 6 , 283--301 3 Van A m e l s v o o r t , J.M.M., de P o n t , J . J . H . H . M . , Stols, A . L . H . a n d Bonting, S.L. ( 1 9 7 7 ) Biochim. Biophys. Acta 471, 78--91 4 V a n A m e l s v o o r t , J.M.M., De P o n t , J . J . H . H . M . a n d Bonting, S.L. ( 1 9 7 7 ) 1 1 t h FEBS Meet. C o p e n hagen, A b s t r . A - 4 - 1 5 - 6 0 3 - 3 5 D u n c a n , C.J. ( 1 9 7 5 ) Life Sei. 1 6 , 9 5 5 - - 9 6 6 6 S c h a r f f , O. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 4 3 , 2 0 6 - - 2 1 8 7 L o w r y , O.H., R o s e b r o u g h , N.J., F a r r , A.L. a n d R a n d a l l , R.J. ( 1 9 5 1 ) J. Biol. C h e m . 193, 2 6 5 - - 2 7 5 8 B l u m , A.L., S h a h , G., Pierre, T. St., H e l a n d e r , H . F . , Sung, C.P., Wiebelhaus, V.D. a n d Sachs, G. ( 1 9 7 1 ) Biochim. B i o p h y s . A c t a 2 4 9 , 1 0 1 - - 1 1 3 9 S i m o n , B. a n d T h o m a s , L. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 8 8 , 4 3 4 - - 4 4 2 10 S i m o n , B., K i n n e , R. a n d K n a u f , H. ( 1 9 7 2 ) Pfliigers A r c h . 3 3 7 , 1 7 7 - - 1 8 4 11 K a s b e k a r , D.K., D u r b i n , R.P. a n d L i n d l e y , D. ( 1 9 6 5 ) Biochim. Biophys. A c t a 1 0 5 , 4 7 2 - - 4 8 2 12 Sachs, G., Wiebelhaus, V.D., B l u m , A.L. a n d H i r s c h o w i t z , B.I. ( 1 9 7 2 ) in Gastric S e c r e t i o n (Sachs, G., Heinz, E. a n d Ullrich, K.J., eds.), pp. 3 2 1 - - 3 4 3 , A c a d e m i c Press, L o n d o n 13 D r u m m o n d , G.I. a n d Y a m a m o t o , M. ( 1 9 7 1 ) in The E n z y m e s ( B o y e r , P.D., ed.), Vol. IV, pp. 3 3 7 - 3 5 4 , A c a d e m i c press, L o n d o n 14 Wiebelhaus, V.D., S u n g , C.P., H e l a n d e r , H.F., S h a h , G., Blum, A.L. a n d Sachs, G. ( 1 9 7 1 ) Biochim. Biophys. Acta 241, 49--56 15 L e p k e , S., F a s o l d , H., Pring, M. a n d Passow, H. ( 1 9 7 6 ) J. M e m b r a n e Biol. 29, 1 4 7 - - 1 7 7 16 R o t h s t e i n , A., C a b a n t c h i k , Z.I. a n d K n a u f , P. ( 1 9 7 6 ) Fed. Proc. 35, 3 - - 1 0 17 S c h a t z m a n n , H.J. ( 1 9 7 5 ) in Calcium T r a n s p o r t in C o n t r a c t i o n a n d S e c r e t i o n (Carafoli, E., Clementi, F., D r a b i k o w s k i , W. a n d M a r g r e t h , A., eds.), p p . 4 5 - - 4 9 , N o r t h - H o l l . Publ. Co., A m s t e r d a m 18 B o n t i n g , S.L. a n d de P o n t , J . J . H . H . M . ( 1 9 7 7 ) in M a m m a l i a n Cell M e m b r a n e s ( J a m i e s o n , G.A. a n d R o b i n s o n , D.M., eds.), Vol. 4, pp. 1 4 5 - - 1 8 3 , B u t t e r w o r t h , L o n d o n 19 Liang, C.T. a n d S a c k t o r , B. ( 1 9 7 6 ) A r c h . B i o c h e m . B i o p h y s . 1 7 6 , 2 8 5 - - 2 9 7 20 Maren, T h . H . , ParceU, A.L. a n d Malik, M.N. ( 1 9 6 0 ) J. P h a r m a c o l . Exp. Ther. 1 3 0 , 3 8 9 - - 4 0 0 21 L u t h r a , M.G., H i l d e n b r a n d t , G.R. a n d H a n a h a n , D.J. ( 1 9 7 6 ) Biochim. B i o p h y s . A c t a 4 1 9 , 1 6 4 - - 1 7 9 22 B o n d , G.H. a n d C l o u g h , D.L. ( 1 9 7 3 ) Biochim. B i o p h y s . A c t a 3 2 3 , 5 9 2 - - 5 9 9 23 Izutsu, K.T., M a d d e n , P.R., Watson, E.L. a n d Siegel, I.A. ( 1 9 7 7 ) Pfltigers A r c h . 3 6 9 , 1 1 9 - - 1 2 4 24 Ahlers, J. a n d G i i n t h e r , Th. ( 1 9 7 5 ) A r c h . B i o c h e m . Biophys. 1 7 1 , 1 6 3 - - 1 6 9
