87

Btochzrmca et Btophystca Acta, 1026 (1990) 87-92 Elsewer

BBAMEM 74892

Similarities between the effects of dimethyl sulfoxide and calmodulin on the red blood cell Ca2+-ATPase Gustavo Benalm a and Leopoldo de Mels 2 I Centro de Btologta Celular, Faculdad de Cwnclas, UmversMad Central de Venezuela, Caracas (Venezuela) and e Insntuto de Cl~netas BtomJdmas, Departamento de Btoquimwa, Unwersldade Federal do Rto de Janetro, Rio de Janetro (Brasd)

(Received 5 December 1989)

Key words ATPase, Ca 2+-, Red blood cell, Calmoduhn, (Human)

The Ca2÷-ATPase of the erythrocyte plasma membrane can be activated by calmodulin, acidic phospholipids, limited proteolysis and self-association. Recently, it has been shown that different organic solvents increase both the //max and the Ca2+ affinity of the enzyme (Benalm, G. and De Meis, L. (1989) FEBS Lett. 244, 484-486). In this report the effects of ealmodulin and dimethyl sulfoxide ( 2 ~ , v / v ) on the Ca2+-ATPase are compared. Dimethyl sulfoxide also elicits the appearance of the low-affinity ATP binding site, which in this enzyme is strictly dependent on calmodulin. Dimethyl sulfoxide increases the Ca2+ affinity of the enzyme in a manner similar to that observed with the use of calmodulin and of acidic pbospholipids. This was tested using both native and partially trypsinized ATPase. When activated by calmodulin the enzyme is inhibited by compound 4 8 / 8 0 , trifluoperazine and calmidazolium. When activated by dimethyl sulfoxide the enzyme is still inhibited by calmidazolium but is no longer inhibited by either compound 4 8 / 8 0 or trifluoperazine. Activation of the ATPase promoted by either calmodulin or dimethyl sulfoxide is abolished when the Ca2+ concentration is raised from 10 pM to 2 mM. The effect of dimethyl sulfoxide is also abolished by 20 mM Pr In the presence of 1 to 10 mM Ca2+ the ATPase catalyzes an ATP ~ P, exchange. The rate of exchange increases several fold when dimethyl suifoxide is included in the assay medium.

Introduction The caloum-pumplng ATPase of the erythrocyte plasma membrane is responsible for calcium homeostasis in these cells [1] Tlus enzyme can be activated by the caloum calmoduhn complex [2,3], by acidic phosphohplds and long-chain, polyunsaturated fatty acids [4,5], by hmlted proteolysls [6-9], and by self-association [10,11] Data from different laboratones [12-15] indicate that the binding of calmodulm to the regulatory domain of the enzyme ~s mediated by hydrophobic interactions Modulation of the enzyme by acidic phosphohplds or by self-association also revolves hydrophoblc interactions [16] These flndmgs led us to Investigate the effect of organic solvents on the Ca 2+ATPase from red blood cells [17] In a previous work it

Abbrevaatlons Hepes, 4-(2-hydroxyethyl)-l-plperazaneethanesulfomc acid, EGTA, (ethylenebls-(oxyethylenemtnlo))tetraacetlc acid, Mops, 3-(N-morphohno)propanesulfomc acad Correspondence L de Mels, InsUtuto de C18ncaas Blom&hcas, Departamento de Bloqulrmca, Umversldade Federal do Rao de Janelro, Cldade Umvermhna, Rao de Janelro, RJ 21910, Brasd

was shown that different orgamc solvents increase both the Vmax and the Ca 2+ affinity of the enzyme to values that are smaflar to those attained with calmodulm [17] The effect of calmoduhn was best rmmtcked with the use of 20% (v/v) dlmethyl sulfoxlde In ttus report we compared the effects of dlmethyl sulfoxtde and calmoduhn on the apparent afflmty of the enzyme for ATP, on the trypslmzed ATPase, on the action of calmoduhn antagomsts and on the ATP ~ P, exchange reaction Methods Calmoduhn was isolated from bovine brain by phenyl-Sepharose chromatography [18,19] Human erythrocyte membranes deficient m calmodulm were prepared from recently outdated human blood [20] The erythrocyte Ca2+-ATPase was purified by affinity chromatography using a calmodulm affimty column [8] Routinely, 0 5-0 6 mg of ATPase was obtained from 500-600 mg of ghost protem. The purified ATPase was stored under N 2 at - 1 7 3 ° C at a concentration of 100-200 #g/ml, in a buffer containing 0 04% Triton X-100, 130 mM KC1, 20 mM Hepes-KOH (pH 7 4), 2

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--200 kDa

138 k D a -

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--66kDa

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for 30 nun Trypsin ( 5 0 / ~ g / m l ) was added to ahquots contatmng 100-200 /xg/ml of punfled enzyme suspended in the same buffer solution in which the enzyme was stored The digestion was arrested by addition of 10-fold excess soybean trypsin inhibitor After 10 man dlgestmn (Fig 1, lane 1) a small part of the ATPase was not digested (138 kDa) and a 90 k D a part was the dormnant fragment After 30 mln digestion (Fig 1 lane 2), the main polypeptldes of high molecular mass were those of 85 and 81 k D a which are not sttmulated by calmoduhn [8,26] The 90 k D a fragment whach still can be activated b y calmodulln and the 76 k D a peptlde were still wslble as faint bands Trypsin and soybean trypsin inhibitor were purchased from Sigma Chemical Co The protein concentration was determined by the method of Lowry et al [27,28], usmg bovine serum albumin as standard

Results

A TP dependence

1

2

3

Fig 1 Electrophoresls of the trypslmzed Ca z ÷-ATPase The purified ATPase (100-200 # g / m l ) was treated with trypsin (50 ~tg/ml) at 4 ° C The digestion was arrested by the addttlon of 10-fold excess soybean trypsin mlubltor after 10 mm (lane 1) and 30 nun (lane 2) Lane 3 are standards myosin (200 kDa), fl-galactosldase (116 2 kDa), phosphorylase b (92 5 kDa), bovine serum albuman (66 2 kDa) and ovalburmn (45 kDa)

m M EDTA, 2 m M MgCI2, 50 p M CaC12, 2 m M ditluothreitol, 5% glycerol ( v / v ) and 0 5 m g / m l phosphatidylcholme 32p~ was obtained from the Brazlhan Institute of Atormc Energy and purified as previously described [21] [-y-32p]ATP was prepared according to the method of Glynn and Chappell [22] ATPase activity was assayed by measunng the release of P, from [~/-32p]ATP at 3 5 ° C The reaction was quenched with two vol of a suspension of activated charcoal in 0 1 M HCl [23] After centnfugatlon, ahquots of the supernatant contaming [32p]p, were counted in a liquid sclntfllanon counter ATP ~ P, exchange was detemuned by measunng the incorporation o f [32P]Pt into A T P [21] Free calcmm concentrations were calculated as described by Fabiato and Fablato [24], taking Into account the concentration of A T P and MgCl 2 m the mecha and using the chssociation constant reported by Schwartzenbach et al [25] for the C a - E G T A complex Controlled trypsin proteolysls of the purified enzyme and SDS-electrophoresls were performed as prevmusly described [8,26] The chgestxons were carried out at 4 ° C

Previous reports have shown that calmoduhn mcreases the apparent affrmty of the enzyme for A T P and also ehclts the appearance of a second, low-affimty (regulatory) binding site for ATP [29,30] We now show that both of these effects can also be induced by dlmethyl sulforade (Fig 2) The concentration of orgamc solvent selected was that shown to maximally actwate the enzyme in a previous work [17] The effects of calmoduhn and dlmethyl sulfoxlde are not additive, since the same ATP dependence was observed m the presence of either calmoduhn, dlmethyl sulfoxide or calmoduhn plus dimethyl sulforade The degree of stimulation by calmoduhn and dlmethyl sulfoxlde vaned among the different enzyme

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Fig 2 Effect of dLmethyl sulfoxide on the enzyme affn'uty for ATP The assay mexhumcontmned 50 mM Mops-Tns buffer (pH 7 4), 100 mM KC1, 10 mM MgCl2 1 mM EGTA and 105 rnM CaC12 The calculated free C a 2+ concentraUon was 10 pM The reaction was started by the addmon of enzyme to a final concentration of 1-2 #g per ml and quenched after 30 nun at 35°C o, no addluons, e, 4 /~g/ml calmodulm, z~, 20% (v/v) dlmethyl sulfomde, a, 4 /Lg/ml calmodulm plus 20% (v/v) dLmethylsulfoxldc

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[COZ+] , FIm Fig 3 Effects of dlmethyl sulfoxtde and trypsin proteolysls on the enzyme affmtty for Ca 2+ Proteolysls was camed out for 30 mm as described m Methods and shown m Fig 1 lane 2 After proteolysls the enzyme was no longer activated by calmodulm (data not shown) Condmons and assay rnedm composition were as m Fig 2 except that the ATP concentration was 200 #M and chfferent CaC12 concentrations were added to obtain the free Ca 2+ concentrations shown m the figure o, untreated enzyme, @, trypstmzed enzyme, zx, untreated enzyme plus 20% (v/v) dlmethyl sulfoxtdes, A, trypsimzed enzyme plus 20% (v/v) chrnethyl sulfoxade

preparataons tested However, in each preparation the increment m Vmax ehcited by calmoduhn was the same as that ehcited by dlmethyl sulfoxtde Ca 2+ afftntty

Calmoduhn increases the enzyme afflmty for Ca 2÷ [29] An increase m afhmty is also observed after controlled proteolysls of the ATPase [31] In agreement with previous reports [8,31,32] it was found that after 30 mm proteolysls (Fag 1, lane 2) the enzyme is not further stimulated by calmodulm (data not shown) The effects of calmodulm and proteolysxs can be mumcked by addition of dimethyl sulfoxlde to the medium (Fig 3) In a previous report [17] it was shown that the effects of calmodulm and dunethyl sulfoxade on the calcium affinity of the enzyme are addmve We now show that an additive effect of the solvent is also observed with the trypsInlZed enzyme (Fig 3) dlmethyl sulfoxlde, but not calmoduhn [31,33], further increases the Ca/+ affinity of the trypsmlzed ATPase

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Fig 4 Intubmon by tnfluoperazme (TFP) The assay media composition and expertmental condmons were as m Fig 2 The ATP concentration was 200 /xM o, no adchtion, @, 4 # g / m l calrnodulm, ,% 20% (v/v) dlmethyl sulfoxade, A, 4/~g/ml calmodulm plus 20% (v/v) dtmethyl sulfoxade

dimethyl sulfoxade was included in the assay medium (Fig 4) Contrasting with these findings, the inhibitory actwlty of calnudazohum was not suppressed in the presence of &methyl sulfoxlde (Fig 6) The data of Figs 4 to 6 mdlcate that the drugs tested interact with different regions of the enzyme molecule Effects of 1", and high Ca 2 + concentratwn The actlvatton of the ATPase actlxaty promoted by dlmcthyl sulfoxade is antagonized by P, (Fig 7) and by high Ca 2+ concentrations (Fig 8) An inhibition of the ATPasc aclavlty is obscrved when the Ca 2+ concentration of the medium is raised to the nulhmolar range [29] In the presence of calmoduhn, the inhibition curve is shifted to lower Ca 2+ concentrations [29,39] A sunllar effect is observed when the enzyme is activated by

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Antt-calmoduhn drugs Tnfluoperazane and calrmdazohum inhibit the plasma membrane Ca2+-ATPase both m the absence (basal activity) and in the presence of calmoduhn. These drugs also mhihit the ATPase acuvated by e~ther proteolyls or acIchc phosphohplds [35,36] Compound 48/80 impairs only the activation promoted by calmodulm, It has no effect on the basal acUxaty [37,38] Here we show that when activated by dtmethyl sulfoxlde, the enzyme is no longer inhibited by either tnfluoper~me or by compound 48/80 (Figs 4 and 5) W~th the use of tnfluoperazme a small but slgmhcant activation, compared to the control containing &methyl sulfoxtde but not tnfluoperazme, was observed when

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Fig 5 Inlubmon by compound 48/80 The assay mecha composition and experimental conchUons were as m Fig 2 but with 200/~M ATP o, no adchaons, e, 4 lag/ml calmodulm, zx, 20% (v/v) dunethyl sulfoxade, A, 4/~g/ml calmodulm plus 20% (v/v) dlmethyl sulfoxade

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Fig 6 h~ub~Uon by caln~dazohum (CMZ) The assay media composition and experimental conditions were as m F~g 2 but w~th 200 #M ATP o, no add~tmns, o, 4 #g/ml calmodulm, rn, 20% (v/v) dlmethyl sulfox~de, I , 4 #g/ml calmoduhn plus 20% (v/v) d~methyl sulfox~de

d l m e t h y l sulfoxade U n d e r the c o n d i t i o n s of F i g 8, the c a l c i u m c o n c e n t r a t i o n n e e d e d for h a l f - m a x i m a l lnhlblu o n of the A T P a s e a c t l w t y in the a b s e n c e of c a l m o d u h n was 7 0 m M Tilts value decreased to 1 6 m M o n add m o n of c a l m o d u h n a n d to 1 0 m M w h e n 20% ( v / v ) d l m e t h y l sulfoxtde was present in the m e d m m

A TP ~ P, exchange In prexaous reports [10,40] it has b e e n s h o w n that the C a 2 + - A T P a s e catalyzes a r a p id A T P ~ P~ e x c h a n g e w h e n the C a 2+ c o n c e n t r a t i o n is r a s e d to the rmlhrnolar ra nge D u n n g thts exchange, the e n z y m e catalyzes sxmultaneously the hydrolysis of A T P a n d its synthesis f r o m A D P and P, W e n o w show that the rate of A T P synthesis increases w h e n d l m e t h y l sulfoxtde is i n c l u d e d in the assay m e d i u m (Figs 8B a n d 9B) T h e c o n c e n t r a tions of C a 2÷ and P, used were n o t sufficient to r each s a t u r a u o n H i g h e r c o n c e n t r a t i o n s of these ions co u l d not be tested due to the f o r m a t i o n of c a l c m m p h o s p h a t e precipitate Thus, f r o m the data of Figs 8 a n d 9 it is n o t possible to ascertain w h e t h e r d l m e t h y l sulfoxade in-



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[CaCl~,] ,mM Fig 8 Effect of tugh calcmm concentrations on the rates of ATP hydrolysis and of ATP ~ P, exchange The assay media contained 50 mM Mops-Tns buffer (pH 7 0), 100 mM KC1, 2 mM MgCI2, 50 #M ADP, 2 mM P,, 200 #M ATP, and different CaC12 concentrations The reaction was started by the addition of ATPase to a final concentraUon of 2-4 #g/ml and arrested after 10 nun incubation at 35 °C (A) ATPase actlwty [y-32p]ATP and non-radmactwe P, were used The reacuon was stopped by the addmon of tnchloroacetlc acid to a final concentraUon of 8% (w/v) (B) ATP ~ P, exchange [32p]p, and non-radioactive ATP were used and the reactson arrested vath a suspension of activated charcoal m 0 1 M HC1 o, no adchtlons, II, 4 #g/ml calmodulm, A, 20% (v/v) dlmethyl sulfoxade

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Fig 7 Effect of P, The assay medm composltaon and experimental condmons were as m Fig 2 but with 200 #M ATP zx, No addmons, A, 20% (v/v) dsmethyl sulfoxade

Fig 9 Effect of P~ on the rates of ATP hydrolysis and of ATP ~ P~ exchange The assay medm contained 50 mM Mops-Tns buffer (pH 7 0), 100 mM KC1, 2 mM MgC12, 50 #M ADP, 2 mM CaC12, 200 #M ATP and the PI concentrations shown m the figure Other conchtlons were as m Fig 8 (A) ATPase actlvtty, (B) ATP ~ P, exchange zx, no additions, A, 20% (v/v) dtmethyl sulfoxtde

91 creases the Vmax of exchange or decreases the concentrations of Ca 2+ and P~ needed for half-maxrmal exchange

Discussion Two different K m values for ATP are found in both the Ca 2÷-ATPase of sarcoplasnuc reUculum [41] and the ( N a ÷ + K ÷) ATPase of plasma membranes [42] The first K m refers to the binding of ATP to the catalytic site of these enzymes The second Km, detected at lugher ATP concentration (0 05 to 0 20 mM), promotes an increase m the Vmax and is thought to reflect the binding of ATP to a regulatory site of these enzymes For the erythrocyte calcium pump the second K m for ATP prevaously has only been detected in the presence of calmoduhn [29,30] In this work, using punfled enzyme, we show that addition of dimethyl sulfoxlde also promotes the appearance of the second K m for ATP This fm&ng supports the proposal that organic solvents can mimic the activation by calmoduhn of the plasma membrane Ca2 +-ATPase Pepudes with distinct afflmues for Ca 2÷ can be obtained depending on the conditions used for proteolysls of the ATPase [31] One of the peptides (81 kDa) has a higher affimty for Ca 2÷ than the native enzyme and is not activated by calmoduhn, but its afhmty for Ca 2÷ can be further increased by acidic phosphohplds The 76 kDa trypuc fragment has a higher Ca 2÷ affinity than either the native enzyme or the 81 kDa fragment, and is no longer activated by either calmoduhn or acidic phosphohplds The dominant pepUdes attained in the proteolysls conditions used were those with molecular weights of 81 and 85 kDa (Figs 1 and 3) Dlmethyl sulfoxlde can further increase the Ca 2÷ affimty of the trypslmzed ATPase to a level smular to that attained with acidic phosphohpids [31,33] Thus, it seems that organic solvent interacts with different domains of the protein and is able to simulate the effects of both calmoduhn and of acidic phosphohplds The mechamsm by which dimethyl sulfoxlde may abohsh the inlubltory effects of hydrophobic molecules such as trlfluoperazme and compound 4 8 / 8 0 has been discussed in detail m previous reports [43,44] Recently [32,45], it has been shown that phenothlazmes bind to the same 9 k D a peptide of the enzyme that interacts with calmoduhn [8,26,34] At present, we do not know why P~ (Fig 7) and high Ca 2÷ concentrations (Fig 8) impair the effect of dimethyl sulfoxade Perhaps tlus antagonism is related to the reversal of the catalytic cyle of the enzyme In fact, dimethyl sulfoxade was found to favor the synthesis of ATP observed during the ATP ~ P1 exchange reaction (Figs 8 and 9) Reversal of the catalytic cycle of the Ca2÷-ATPase from either erythrocytes [46] or sarcoplasrmc reUculum [44] is only observed after the enzyme has been phosphorylated by P1 and Ca 2÷ has bound to

a low-afftmty site on the enzyme (Kin, 1 - 2 mM) Phosphorylatlon of both Ca 2+ transport enzymes by P. is greatly faohtated by orgamc solvents such as dimethyl sulfoxide [44,46]

Acknowledgements This work was supported by the Fundaq~o Banco do Brasfl, F l n a n o a d o r a de Estudos e Projetos, Conselho N a o o n a l de Desenvolvamento Clentifico e Tecnologlco, and the Fundaq5o de Amparo a Pesqmsa do Rio de Janelro, Brazil, and Consejo de Desarrollo Clentifico y HumamsUco de la Umversidad Central de Venezuela, grant N o 03-10-2095-89

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92 27 Lowry, O H , Rosebrough, N J, Farr, A L and Randall, R J (1951) J Blol Chem 193, 265-275 28 Bensadoun, A and Wemstem, D (1976) Anal Blochem 70, 241-250 29 Schatzmann, H J (1982) m Membrane Transport of Calcmm (Carafoh, E, ed ), pp 41-108, Academac Press, New York 30 Muallem, S and Karhsh, S J D (1979) Nature (Lond) 277, 238240 31 Enyedy, A , Flura, D, Sarkadl, B, Gardos, G and Carafoh, E (1986) J Blol Chem 262, 6425-6430 32 Zunm, M, Krebs, J, Penmston, J T and Carafoh, E (1984) J Btol Chem 259, 618-627 33 Papp, B, Sarkadl, B, Enyedy, A, Cande, A J , Pennlston, J T and Gardos, G (1989)J Blol Chem 264, 4577-4582 34 Carafoh, E, Zunm, M and Benaam, G (1986) m Calcmm and the Cell, Clba Found Symp Vol 122, pp 58-72, Wiley & Sons, New York 35 Adunyah, E S, Ntggh, V and Carafoh, E (1982) FEBS Lett 143, 65-68 36 Vmcenza, F F, Adunyah, E A , Nlggh, V and Carafoh, E (1982) Cell Calcium 3, 545-559

37 Cnetzen, K , Adamczyk-Engelmann, P, Wuthnch, A , Kon~antmova, A and Bader, H (1983) BlochLrn Blophys Acta 736, 109-118 38 Ross1, J P F C, Rega, A F and Garrahan, P J (1985) Blochtm Blophys Acta 816, 379-386 39 Allen, B G , Katz, S and Roufogahs, B D (1987) Blochem J 244, 617-623 40 Mas-Ohva, J, De Mels, L and Inesl, G (1983) Blochermstry 22, 8522-5825 41 Inesl, G , Goodman, J J and Watanabe, S (1967) J Blol Chem 242, 4637-4643 42 Robmson, J D (1976) Bloctum Blophys Acta 429, 1006-1019 43 De Mels, L, Tuena de Gomez-Puyou, M and Gomez-Puyou, A (1988) Eur J Blochem 171,343-349 44 De Mels, L (1989) Blochlm Blophys Acta 973, 333-349 45 Brandt, P, Zurlm, M, Neve, R L, Rhoads, R E and Vanaman, T C (1988)Proc Natl Acad Scl U S A 85, 2914-2918 46 Chelsl, M, Zurlm, M and Carafoh, E (1984) Blochermstry 23, 2595-2600

Similarities between the effects of dimethyl sulfoxide and calmodulin on the red blood cell Ca2(+)-ATPase.

The Ca2(+)-ATPase of the erythrocyte plasma membrane can be activated by calmodulin, acidic phospholipids, limited proteolysis and self-association. R...
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