Biochemical Characterization of Plasma Membrane Vesicles of Cyanophora paradoxa *

Comp. Biochem. Physiol.Vol. 100B, No. 4, pp. 753-758, 1991 Printed in Great Britain

0305-0491/91 $3.00 + 0.00 © 1991 Pergamon Press pie

BIOCHEMICAL CHARACTERIZATION OF THE PLASMA M E M B R A N E C a 2+ P U M P I N G ATPase ACTIVITY PRESENT IN THE GILL CELLS OF M Y T I L U S G A L L O P R O V I N C I A L I S LAM A. VIARENGO,*tM. PERTICA,*G. MANCINELLI,*G. DAMONTE~and M. ORUNESU* *Institute of General Physiology and *Institute of Biochemistry, University of Genoa, 16132 Genoa, Italy (Tel: 010 3538241)

(Received 29 May 1991) Abstract--1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca2+-activated ATPase activity is present. The ATPase has high Ca 2+ affinity (K~ = 0.3 #M). 2. The optimum assay conditions to evaluate the enzymatic activity of the Ca2+-stimulated ATPase at 19°C are: 120-300 mM KCI ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca 2+ ATPase activity is stimulated by DTT (0.5-1 mM) and it is inhibited by low concentrations of vanadate (10-50#M) and -SH inhibitors such as PCMB and PCMBS (10gM); the enzyme appears to be calmodulin insensitive. 3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca 2+ at n-/zM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La3+; (c) the tool. wt of Ca 2+ ATPase is about 140kDa. 4. Low Ca 2+ concentrations (n-gM) are sufficient to stimulate the ATP-dependent Ca 2+ uptake by plasma membrane inside-out vesicles. 5. The results indicate that the Ca 2+ pump present in the gill plasma membranes could be responsible for Ca 2+ extrusion and therefore involved in maintaining the cytosolic Ca 2+ concentration within physiological levels.

INTRODUCTION In the plasma membrane of vertebrate cells a (Ca 2+, Mg 2+) ATPase is present whose activity is related to the transport of Ca 2+ from cytosol to the extracellular medium (Inesi, 1985; Carafoli, 1987, 1991). As is known, this enzyme is involved in restoring physiological cytosolic free calcium levels, although Ca 2+ can be also compartimentalized in endoplasmic reticulum vesicles and mitochondria (Carafoli, 1987). The physiological role of the Ca 2+ ATPase is of great interest, taking into account that free Ca 2+ is involved as a "second messenger" in coupling stimulus to response in a wide variety of cell types (Rasmussen and Barrett, 1984). The study of the biochemical mechanisms involved in Ca 2+ extrusion appears of particular importance for organisms, such as mussels, living in estuarine and coastal areas where the sea water salinity and the Ca 2+ content can widely fluctuate (Bayne, 1976). In fact, since Ca 2+ influx is an essentially passive process (Hille, 1984), a plasma membrane Ca 2+ ATPase activity may play a role in regulating the level of free cytosolic Ca 2+ in mussel cells, eventually preventing Ca 2+ accumulation in vesicles and mitochondria (Carafoli, 1987) as well as cytotoxicity due to alteration of Ca2+-activated metabolic pathways (Nicotera et al., 1988; Bellomo et al., 1989; Viarengo and Nicotera, 1990). tAuthor to whom correspondence should be addressed. 753

In this study we report data concerning the biochemical characterization of a Ca2+-activated ATPase activity located in the plasma membranes from the gills of mussels. MATERIALS AND M E T H O D S

Materials All reagents were of the highest purity available. ATP (disodium salt), ADP, AMP and ouabain were obtained from Boehringer Mannheim GmbH. Ethyleneglycol-bis (~-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), DL-dithiothreitol (DTT), 4-(2-hydroxyethyl)- l-piperazineethanesulfonic acid (Hepes), p-chloromercuribenzoate (PCMB), p-chloromercuribenzensulfonate (PCMBS) and trans-cicloexane-l,2-diamine-N,N,N',N'-tetraacetic acid (CDTA) were from Sigma. CaCI2.4H20 was from Merck. 45 CaCI2 00-40 mCi/mg) was obtained from Amersham International (Amersham, England) and (y-32 P) ATP (3000 Ci/ retool) from NEN Research Product; sodium orthovanadate was obtained from Pfaltz Bauer Inc. Plasma membrane preparation Plasma membranes were prepared from gills of mussels by separation on a sucrose density gradient essentially as described by Verma and Penniston (1981). The interphase between 41 and 55% sucrose was collected, suitably diluted and sedimented at 90,000 g for 60 rain. The final pellet represents an enriched plasma membrane preparation which was washed with 20 mM Hepes-2 mM EDTA pH 7.6, successively with 0.25 M sucrose-I mM EDTA-20 mM Hepes pH 7.6 and, finally, with 0.25 M sucrose-20 mM Hepes pH 7.6. After each washing the membranes were centrifuged at 30,000g for 20 min.

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trophorized on a discontinuous sodium dodecyl sulfate-polyacrylamide gel (5-10%) under the experimental conditions described by Sarkadi et al. (1986). Autoradiography of the stained and dried gels was performed with overnight exposure at -80°C using TRIMAX 3MX-D 18 x 24 cm.

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media containing 0.5 mM Tris-HCl, 50/aM dithiothreitol and 20/~M EDTA, pH 8.5. The ratio of sealed vesicles, determined by measuring latent and total acetylcholinesterase activity as described by Steck (1974), was about 70%.

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Fig. 1. Effect of temperature on the Ca 2÷ ATPase activity present in the plasma membrane of mussel gill cells. The experiments were carried out in the presence of 200#M Ca 2+ at 10, 19 and 30°C as described in Materials and Methods. The degree of purity of the plasma membrane preparation was biochemically tested. It was found to contain the highest Na+,K+-ATPase activity among the fractions obtained from the sucrose gradient (80%). Moreover, the evaluation of succinic dehydrogenase and acidic phosphatase activities as well as RNA and DNA content indicate only minimal contamination by mitochondria, lysosomes, RER and nuclei, respectively. Protein was measured by the method of Hartree (1972), with bovine serum albumin as a standard. Ca 2+ ATPase assay The Ca 2+ ATPase activity was measured by evaluation of inorganic phosphate release. The standard reaction mixture contained 30 mM Hepes pH 7, 240 mM KCI, 0.2 mM ouabain, 2 mM EGTA, 2 mM ATP and different CaCI2 and MgC12 concentrations, in a final volume of 1 ml. The free Ca 2+ concentration in the media was calculated using the computer program described by Fabiato and Fabiato (1979) which takes into account parameters such as ionic strength, concentration of Mg, EGTA, ATP and pH values. Incubations of about 70/zg of proteins were carried out at 19°C for 30 min and the released inorganic phosphate was determined by the extraction of the phosphomolybdate complex into the organic phase of benzen:isobutanol (1:1) as described by the method of Martin and Doty (1949). The calcium stimulated activity was evaluated by subtracting the amount of inorganic phosphate released in the absence of free calcium, i.e. in the presence of an excess of EGTA, from that obtained in the presence of added calcium. The specific activity of Ca 2+ ATPase was calculated as micromoles of Pi per milligram protein per minute.

Ca 2+ uptake by inside-out vesicles The Ca 2+ transport was measured by a standard filtration assay in 600/d of a medium containing 30 mM Hepes, 100mM KC1, 2mM MgCI2, 0.2raM ouabain, pH 6.8. In order to measure C a 2+ uptake, 50/~g of inside-out vesicles, 10/zM CaC12 and 2.85 #M 45 calcium chloride were added to the medium. The reaction was started by the addition of 4 mM Na-ATP. The samples were incubated at 19°C and the Ca 2+ uptake was carried out as described by Kessler et al. (1990). RESULTS Figure 1 shows the effect o f different temperatures on the kinetic of the ouabain insensitive Ca 2+ ATPase activity associated with the plasma membranes o f mussel gills in the presence of 200/~M free calcium. The enzyme activity shows a linear trend up to 60rain, at 10, 19 and 30°C, the enzymatic A T P hydrolysis increasing progressively up to 30°C. The effects o f ionic strength on the cationstimulated activity were measured in the presence of different amounts o f KCI (Fig. 2). The Ca 2+ ATPase activity shows optimal values in the 120--300 m M KCI range. In Fig. 3 the variations of the Ca 2+ ATPase activity as a function of p H are depicted. The enzyme activity is maximal at p H 7.0. The effect of different M g 2+ concentrations, in the absence of added calcium, on the ATPase activity of the gill cell plasma membranes is shown in Fig. 4. The enzyme activity was measured in an assay mixture containing different concentrations of M g 2+, from 0, obtained by adding 2 m M C D T A as Mg 2+ chelating agent, to 20 mM. As shown, the ATPase activity is clearly stimulated when the cation concentration is

Detection o f the phosphate intermediate (E ~ P)

The plasma membrane preparations (22#g of protein) were incubated at 4°C in the reaction mixtures (final volume of 100/zl) containing 30mM Hepes, pH7, 275nM KCI, 0.2mM ouabain and (~-32P) ATP (5/~M final concentration) and the E ~ P complex was stabilized by addition of La 3+ (37.5/z M). The reaction was stopped after 30 sec by the addition of ice-cold trichloroacetic acid (6%), containing 1 mM ATP and 10mM inorganic phosphate, and the samples were washed twice with the same trichloroacetic acid-ATP-Pi solution. The washed precipitates were dissolved in the electrophoresis sample buffer containing 0.25 M Tris-HC1, pH 6.8, 10 mM EDTA, 60% sucrose, 40 mM DTI', 2% SDS and Bromophenol Blue and aliquots were used either for 32 P determination in a Packard Tri-Carb liquid scintillation counter or for running an SDS-PAGE. The Ca 2+ ATPase--32 P complex was dec-

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raised up to a value of 2 mM, which represents the optimum concentration of Mg 2+ in the assay mixture. Figure 5 illustrates the Ca 2+ dependence of the plasma membrane ATPase activity in the presence and absence of added Mg 2+. When no Mg 2+ was added (Fig. 5a), two kinetic forms were found: one shows high Ca 2+ affinity (Kma for free Ca 2+ : 0.3 # M ) and low capacity of ATP hydrolysis (50nmol of P i m i n - ' m g p r o t e i n - ' ) ; the other has low Ca 2+ affinity ( K ~ for free Ca2+ : 100#M) and high capacity of ATP hydrolysis (450nmol of Pi min -~ mg protein-'). The effects of Ca 2+ on the ATPase activity in the presence of 0.2 mM MgCI2 (Fig. 5b) and 2 mM MgC12 (Fig. 5c) are also depicted. The results demonstrate that Ca2+-stimulated ATP hydrolysis also occurs in the presence of 0.2raM MgCI2 (Fig. 5b). When the Mg 2+ concentration is raised up to 2 mM (Fig. 5c) the enzyme activity is further stimulated by low (5-200~M)

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Fig. 5. Effect of increasing free Ca2+ concentrations on the ATPase activity. The experiment was carried out in the absence of Mg2+ (a) ('k), and in the presence of 0.2 mM Mg2+ (b) (A) or 2 mM Mg2+ (c) (B). The enzyme activity was evaluated as described in Materials and Methods. calcium concentrations; it is worth noting, however, that when Ca ions exceed the concentration of 200/~ M a slight inhibition of the enzyme activity can be detected. Figure 6 shows the effect of increasing ATP concentrations on the Ca2+-stimulated ATPase activity. The data indicate that the Kma for ATP is 0.025 mM. The phosphatase activity of the mussel gill plasma membrane preparation was tested in the presence of different substrates (ATP, ADP, AMP, GTP, G6P and//-glycerophosphate) (Table 1). ATP appears to be the preferential substrate but A D P and GTP were also hydrolyzed, although to a lesser extent (50 and 40%, respectively, of the maximum hydrolysis obtained when ATP was the substrate). On the contrary, G6P and//-glycerophosphate are not hydrolyzed by this plasma membrane enzymatic activity. Concerning the effects of compounds able to stimulate the Ca2+-dependent ATPase, it is important to point out that, in the presence of Ca 2+ (5-43 # M), no significant stimulation by calmodulin was observed. On the contrary, dithiothreitol (DTT 0.5-1 raM) causes a slight but significant increase (15%) of the Ca 2+ ATPase activity (data not shown). Data concerning the effects of three enzyme inhibitors such as vanadate (a well-known inhibitor of ATPase

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Fig. 6. Ca 2+ ATPase activity at various ATP concentrations in the presence of 200 #M Ca :+. The enzyme activity was evaluated as described in Materials and Methods.

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Table 1. Hydrolysis of different substrates by the plasma membrane preparations from mussel gills Ca2+ ATPase activity (%) Substrates Ca2+ 5/aM Ca2+ 200/aM ATP 100 100 ADP 50 48 GTP 32 34 AMP --G6P --fl-Glycerophosphate --Hydrolytic activity was evaluated at 5 or 200 #M free Ca2+ concentration. Maximal activity was obtained in the presence of ATP (100%). Ca2+ ATPase activities in the presence of 5 or 200/aM free Ca2÷ are 1 or 6/amol Pi mgprotein-I hr-I, respectively.

activities related to ion transport), p - c h l o r o m e r curibenzoate ( P C M B ) a n d p - c h l o r o m e r c u r i b e n z e n sulfonate (PCMBS), sulfhydryl reagents able to inhibit C a 2+ A T P a s e activities present in m a m m a l i a n cells are presented in Table 2. As shown, a 10/~M v a n a d a t e c o n c e n t r a t i o n is sufficient to significantly reduce the A T P a s e activity, a strong inhibition being achieved only when exogenous M g 2+ ( 2 m M ) is added to the reaction mixture ( B a r r a b i n et al., 1980). C o n c e r n i n g the effects of the sulfhydryl reagents, P C M B a n d P C M B S , it can be pointed o u t that, at very low c o n c e n t r a t i o n ( 1 0 # M ) , a n inhibition of a b o u t 5 0 - 7 0 % was observed, the total inhibition o f Ca 2+ A T P a s e activity being o b t a i n e d at 200 # M . Figure 7 shows the electrophoretic analysis o f the plasma m e m b r a n e proteins carried out to evaluate the possible E ,~ P f o r m a t i o n d u r i n g the CaE+-dependent A T P hydrolysis. The d a t a show t h a t A T P hydrolysis is also coupled to E ~ P complex f o r m a t i o n in the presence o f extremely low concentrations ( 1 0 0 n M ) o f Ca~+; o n the contrary, the e n z y m e - p h o s p h a t e complex is n o t formed when E G T A (which binds Ca ions) is present in the reaction mixture. It is i m p o r t a n t to point out, in addition, t h a t the E ~ P complex c a n n o t be detected in the absence o f La 3+, which inhibits the E ~ P hydrolysis. This fact d e m o n s t r a t e s that, as in the case of the t r a n s p o r t o f o t h e r ions due to o t h e r ATPases ( S c h u u r m a n s Stekhoven a n d Bonting, 1981), the p h o s p h o r y l a t i o n of the enzyme represents only a transient event p r o b a b l y related to the structural changes which occur d u r i n g the cation translocation. Moreover, the electrophoretic m i g r a t i o n o f the E ~ P

Table 2. Effects of vanadate, PCMB and PCMBS on Caz+ ATPase activity Ca2+ ATPase activity (%) Inhibitors Ca2+ 5#M Ca2+ 200#M Vanadate 0 100 100 Vanadate 10 #M -40 -45 Vanadate 100#M -90 -96 PCMB 0 100 100 PCMB 10/aM -50 -70 PCMB 200/aM - 100 -90 PCMBS 0 100 100 PCMBS 10/aM -65 -45 PCMBS 200gM -100 -94 The vanadatc inhibition was observed in the presence of 2 mM MgCI2. The enzyme activity was determined as described in Materials and Methods. Caz+ ATPase activities in the presence of 5 or 200/aM free Ca2+ are 1 or 6#molPlmg protein- i hr- i, respectively.

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Fig. 7. Autoradiography of the dried gel with the phosphorylated proteins. Phosphorylation with 0'- 32 P)ATP was carried out as described in Materials and Methods. Red cell membranes (22 #g) were phosphorylated in the presence of 1 0 0 # M C a 2+ plus 1 mM MgC12 (lanes I and 2); 22#g of proteins from mussel plasma membranes were phosphorylated in the presence of 120 nM Ca 2+ (lane 3), 43/~M Ca 2+ (lane 4), lmMMgC12 plus 2 m M E G T A (lane 5), 4 3 # M Ca 2+ without lanthanum (lane 6), 100#M Ca 2+ (lane 7), 4 3 # M C a 2+ plus 2mMMgC12 (lane 8), 100 # M Ca 2+ plus 2 mM MgC12 (lane 9), 200 # M Ca 2+ (lane 10). The calibration of the molecular masses was obtained from a simultaneously run gel lane containing the protein standards indicated.

complex on 5-10% polyacrylamide gradient gel in the presence of SDS shows that the mol. wt of the Ca z+ ATPase is about 140 kDa. In fact the C a 2 + - s t i m u lated ATPase of the mussel gill plasma membrane comigrates with the Ca 2+, M g2+ ATPase from human red cells which, as k n o w n , has a mol. wt o f 138 k D a (Sarkadi et al., 1986). Figure 8 d e m o n s t r a t e s t h a t in the presence o f free Ca 2+ ( 1 0 # M ) a n d A T P ( 4 m M ) the p l a s m a m e m b r a n e vesicles are able to accumulate the cation in their insides. C a 2+ u p t a k e is linear for a b o u t 10 m i n a n d it is a n A T P - d e p e n d e n t process. Moreover, as s h o w n in Fig. 9, Ca 2+, due to the high affinity o f the translocase for this cation, is accumulated in the gill p l a s m a m e m b r a n e vesicles also w h e n it is present in the reaction mixture in n - # M concentrations. DISCUSSION

The results demonstrate that an ouabain insensitive Ca 2+ stimulated ATPase activity, involved in the

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Fig. 8. ATP-dependent uptake of Ca z+ by inside-out vesicles from plasma membrane of mussel gill cells. The preparation of the inside-out vesicles and measurement of Ca :+ uptake in the presence ( 0 ) and absence ( I ) of ATP were carried out as described in Materials and Methods.

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Ca :+ transmembrane transport, is present in the plasma membrane of the mussel gill cells. The mol. wt of the enzyme is about 140 kDa, as judged by electrophoresis on polyacrylamide gel under SDS denaturating conditions. The Ca :+ ATPase shows high Ca :+ affinity (Km~ = 0.3/t M) and characteristics typical of a P-type ion pump, i.e. it forms a phosphoryl intermediate and it is inhibited by low concentrations of vanadate (Carafoli, 1987). In addition, inside-out vesicles from gill plasma membranes accumulate Ca :÷ in an ATP-dependent process, the uptake being stimulated by minimal concentrations of free Ca :÷. The results concerning the biochemical characteristics of the Ca2+-stimu lated ATPase show that the optimum activity is at pH 7.0, 120-300mM KCI and 2 mM ATP. The Kma of the enzyme for ATP was of 0.025 mM, similar to those of other Ca ~+, Mg 2+ ATPases present in the membranes of mammalian cells. The enzyme was activated by DTT (0.5-1.5 mM) and strongly inhibited by sulfhydrylic reagents such as PCMB and PCMBS, this demonstrating that the SH residues are important for the activity of this ATPase. Taken together, our data indicate that there are analogies between the biochemical behavior of the Ca :+ ATPase identified in the plasma membranes of mussel gill cells and that of the enzyme of mammalian red cells; it must be pointed out, however, that the enzyme of the mussel, similarly to the ATPase activity present in trout gill (Ma et al., 1974) and in rat hepatocyte (Carafoli, 1991) plasma membrane, does not seem to be activated by calmodulin; interestingly, a set of preliminary results seems to indicate that the amino acid sequence for calmodulin binding is present in the Ca :+ ATPase of mussel gill cells. In fact, the mussel gill Ca :+ ATPase can bind calmodulin, although only after SDS denaturation, as demonstrated by calmodulin overlay assay (Gazzotti and Viarengo, unpublished data). This latter finding indicates the need to take care in the interpretation of the results concerning the possible physiological regulation of this enzymatic activity. Concerning the specificity for the substrate, it has been demonstrated that the plasma membrane preparation from mussel gill is able to catalyze the hydrolysis not only of ATP but also of A D P and CBPB 100/4~H

757

GTP, although to a lesser extent (50 and 40%, respectively). It must be stressed, however, that when 0,-32 P) GTP is used as a substrate no phosphate intermediate is formed (data not presented). These data clearly suggest that the mussel gill plasma membrane preparation contains different Ca 2+stimulated phosphatase activities not all specifically involved in Ca 2÷ transport. In addition, it has been found that ATP hydrolysis by the mussel gill plasma membrane preparation can be enhanced by the addition of Mg 2+ to the reaction mixture. However, the Ca2+-stimulated ATPase of mussel gill plasma membrane, contrarily to the enzyme of human red cells but similarly to that present in trout gill, rat hepatocyte (Lin, 1985) and corpus luteum cell plasma membranes (Verma and Penniston, 1981), can also be activated by Ca ions in the absence of exogenous Mg :+. Moreover, the data reported in Fig. 5 demonstrate that, like the typical transmembrane Ca :+ transport ATPase (Ghijsen et al., 1980), the Ca :+ ATPase activity present in mussel gills is stimulated by free Ca:+; at n-/~ molar concentrations, also in the presence of an excess of Mg 2+. When only Ca 2+ is added to the reaction mixture, two kinetic forms were evidenced; one has high affinity for Ca :+ and low capacity of ATP hydrolysis, whereas the other shows low affinity for Ca :+ and high capacity of ATP hydrolysis. It is important to point out, however, that the results of the electrophoretic analysis show that only one Ca :+ translocase is present in the plasma membrane preparation, as evaluated by phosphoryl enzyme formation. In addition, the electrophoretic results demonstrate that, similarly to Ca :+ pumps of plasma membrane from different mammalian cells, in the presence of La 3+ the concentration of phosphoenzyme formed is greatly increased. In conclusion, the finding that the Ca 2+ ATPase present in the plasma membrane of mussel gill cells can be activated by minimal concentrations of free Ca :÷ (Kma0.3 #M), together with the demonstration that the E ~ P formation and Ca 2÷ uptake by plasma membrane vesicles occur in the presence of n-/z M free Ca ~+, strongly suggest that this enzyme may be involved in maintaining the physiological levels of free calcium in the cytosol of the gill cells. work was supported by a C.N.R. Grant (Target Project "Biotechnology and Bioinstrumentation"). Acknowledgement--This

REFERENCES

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