(Na', K') ATPase-

The UOEH Association of Health Sciences

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The UOEHAssociation ofHealth Sciences

J UOEH M(4): 253-260 (1992) 253

Reduction and Inactivation of the Sarcoplasmic

by 2-Mercaptoethanol-Contrast

to the (Na', K') ATPase-

[Original)

Ca2'-ATPase

Yoshinobu MUTOHi'2, Atsuko SUGINOt, Ken HIGASHI3, Masayuki TAKASUGI2,

Akio KUROIWA? and Masaru KAWAMURAiiDivision

ofBiotqgy,"Second

Dopartment of internal Medic'ine3Dapartment

ofBiochemistty, St,hoot of Medicine, Chtiversity of Occiipational and Envirenmental Hbatth,

.kipan. Ylahatanishi-ku, Kitalpashu 807, .J4pan

Abstrace :

Key words:

Tbe sarcoplusmic CaP''-ATPase was reduccd with 300 rnM 2-mercaptoethanol at elcvated

terriperatures (40 - 45℃ ) w{th a concornitant loss of iXTPase activity. Thc reductioii and

inaetivation of the Ca!+-ATPase proceeded rapidiy in the absence of Ca2'. '1'he

Ca'+-ATPase

was also inactivated with 2-mcrcaptocthanol in the presence of diluted SDS (O.4･ mg/ml) even at

20℃ . In contrast to thc (Na', KF') A'i'Pase, the inactivated CaUT-A'1'Pase in the prcsence of

diluted Sl)S was sedimented by the centrifugation at 100,OOO × g tbr 30 min.

Ca2F-ATPase, disutfide bond, 2-mercaptoethanol, (Nai, K') ATPase.

(Received 17 August 1992, accepted 1 october 1992)

Introduction

There is a close structural similarity between the Ca'"'-ATPase of sarcoplasmic

reticulum and the a-subunit of the (Nai, K") ATPase of plasma membrane, including

transmembrane topology as well as highly conserved domains fbr ATP binding and

phosphory]ation [1], On the other hand, while the CaL'-ATPase is composed solely of

110 kDa poiypeptide, the (Na', K') ATPasc requircs the glycosylated f?-subunit in addition

to thc cr-subunit. The B-subunit is indispensable in the biogenesis of thc (Na', Ki)

ATPase, but its role in the catalytic cycle is still unknown l2-51.

Kawamura and Nagano [6] and recently Kirlcy [7I have shown that the reduction ot' thc

disulfide bonds in the B-subunit resulted in the inactivation of the (Na', K') ATPasc.

The disulfide bonds in the a-subunit were lcft intact, It ufould be, interesting to discover

whether reducing agcnts, such as 2-mercaptoethanol, inactjvate Ca"-A'l'Pase without the B-subunit Here, we report that the Ca"-ATPase was reduced by 2-mercaptoethanol with a

concomitant loss of ATPasc activity.




･254 Y MuToH et at

Materials and Methods

Proparation ofsarcoplasmic reticulum

Sarcoplasmic'reticulum (SR) was preparcd from back muscles of New Zealand white

rabbits as dcscribed by Kanazawa et al [8], SR was further trcated with either

octacthylenedodecylethcr (C]!Es) or deoxycholate (DOC), Estimation of the disulfide

bond was carried out by using I)OC-solubilized enzyme that was composcd of a single band

of 1 10 kDa polypeptide on sodium dodecylsulfate polyacr>JIamide gcl.

7Yeatment u.:ith re(incing crgents at elevatecX temperatec'res

Ci2E"-solubilized Ca2'-ATPase (O.2-O.3 mg/ml) was incubated in 10 mM TES butrer

(pH 7.5) containing 100 mM KCI, 1 mM CaC17, and O.1 mg/ml Tween 80 in the presenceand absence of reducing agents (300 mNI 2-mercaptoethanol or 4 mM tributhylphosphine)

at elevated tempcratures, At the end of incubation, 100 gl aliquots of incubation mixturc

was withdrawn and added to 900 pt1 Ca?'-ATPase assay medium containing 50 mM

Tris/maleate (pH 7.0), 100 mM KCI, 5 mM MgCl?, lmM EGTA and O,47 mM CaC12.

ATPase activity was determined as the rate of rclcase of inorganic phosphate at 37℃ [81.For the controls, the rate of re]ease of inorganic phosphattr in thc absence of O.47 mM CaClp

was determined and subtracted tfom the above,

Labeling qf disuUide bonds

Labeling of disuliide bonds was carried out as described by Kirley [9]. DOC-

solubilized CaL'-ATI'ase (3 mg/ml) was alkylated with 4 mM lodoacetamide in e.5 ml of

200 mM TrisfHCI (pH 8.3), 5 mM EDTA, 5 M urea and 4.0% sodium dodecylsul{'ate

(SDS) in the dark at 37℃ I'or 1 h, 'I]he

alkylated Cti2'-ATPase was applied on a co}umn

of Bio-Gel P-6 DG and concentra{ed using (1]entricon 30 microconccntrators. 'Lihe

alk}ilation with iodoacetamide was repeated again. To 3.5 ml of Lhe alkylated protein, O.5

ml of a freshly mixed solution containing IM NaBOi (pH 8.0), 8 mlVI EDTA, 2 rnrvl tribu-

tylphosphine (TBP) and 2 mM 4-<aminosulfbnyl)-7-fluoro-2, 1, 3-benzoxadiazole (ABD-F)was added and heated at 60℃ for 1 h.

'FBP-reduced

and ABD-F-alkylated Ca'i-ATI'asc,

whose frec sulthydryl groups had been alkylated with iodeac;etamide, was then applied en a

column of TSK-GS"J-3000 ancl the peak fractions of (:a"'-ATPase wcrc pooled and assayed

fbr protein and ABD-F (absorbance at 385 nm). In another set of experiments, I)OC-

solubilized Ca"-ATPase (3 mg/ml) was incubated at 43℃ in 10 m)vr TrislHCI (pH 7.5),IOO mM KCI, 1 mM CaCIL, with or without 4 mM TBP in the presence of 2 mM ABD-

F. Aftcr 60 min, the paired samples were cooled to room tempcraturc and applied on a

column of Bio-Gel P-6 DG. Thc samp]es were solubilized with 4% SDS, separated by'IiSK-GSW-300e

chromatograph>, and assayed fbr protein and ABD-F as above,



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InacLivation of Ca2+-ATPase 2.55

Other methocts'

Protein was determined by the method of Lowry et al [10I with bovinc serum albumin

as a standard. SDS-polyacrylamidc ge] electrophoresis was carried out by the method of

Laemmli [l ll using a separating gcl of 1O%.

Results

inactivation ofthe Cke'i -A7T'ase lij, redatcing a.aents at etevated temperalures

The thermal inactivation of the Ca2'-ATPasc in the presence oJ' 300 mpt1 2-mercaptoe-

thanol was determined by measuring the activity at 37℃ after exposure to inactivating

temperatures. As it was rel)orted by Lepock et al I12] that Ca2i protects the Ca2'-ATPase

fi/om thermal inactivation, the sarnples were trcated with and without l mM Call'. Figure

1 illustrates the results obtaincd in the presencc of l mrv{ Ca2'. Figure l (A) shows the

time course of inactivation of the Ca"-ATPase activity at 43℃ . The inactivation by 2-

mercaptoethanol obeys pseudo first-order kinetics and was almost completed after 15 min

exposure to 43℃ . The other reducing agent, TBP (4 mM), induced similar inactivation

of thc Ca'F-AFIil]ase, though to a lesser extent. Without 2-mercaptoethanel, the rate of

inactivation at 43℃ was slow as shown in Fig, 1 (A). An adclition of ethanol (350 mM)

did not havc any signifieant efibet on thermal {nactivation. These results suggested Lhat

the inactivation due to the reducing agents resulted from the reduction of disulfide bond(s)

of the Ca?'-ATPase. Figure 1 (B & C) shows the efl'ec'ts oftemperature and concentration

of 2-mercaptoethanol, rcsl]cctively, on t,he itiactivation of ATPase activity,

" t' IE･ 6o 6o

"U

di

of' e

.

0 o 10 lto 30 35 40 4s O O.1 O.2 O.3

Time(min) TeinperatureC(;) 2-mercaptoethanol(Dvl)

Fig. 1. Inactivation of' Ca7+-ATPase with reducing agents at elevatcd temperaLures. CmEH-

solubilized enzymc (O.2-O,3 mg/ml) was incubated in the medium containing IU rntL'1 TES

huffer (pH 7.5}, 100 mM KCI, ] mM CIaCIL, and O,1 mg/m] Twcen 80 in thc presenctr and

absence of a reducing agent. For (iN), incuhation pcriods were varied with a uonstant

{toi]centrution of eiihcr 300 rnN{ 2-mercaptoethanot (O) er 4 mptt 'l'BP

(A) and without

reducing agents (()) at 43℃ . Fur {B), incubation temperaturcs wcre ehangeti in the

preseiice (O) and absence {C)) of 300 m)vl 2-mcrcaptoethanol. Incubatiun periods werc

15 min. For (C), 2-mercaptoethanol conccntrations "'ere var{ed and incubations Ns,erc

performed at 43 ℃ ibr 15 min,



The 　 UOEH 　 Assoolatlon 　 of 　 Health 　 Solenoes

YMUIOHetal256

　　　　In　the 　abscncc 　 of 　Cd2 ＋

（1n　the　prcsencc　of　l　 mM 　 EGTA ），　mactlvaUon 　of 　the　CaP＋ −

ATPase 　in　the　prescncc　ot 　300　mM 　2−mercaptoethanol 　was 　rapid 　and 　completed 　withm 　a　5

mln 　expo 〜ure 　to　43℃ （data　not 　shown ）

Labelzng　cf 　dtsulfide　bond・

　　　　In　order 　to　determlne　thc　number 　Qf　disulfide　bondg　of　a　molecule 　of　the　Ca2＋ATPase ，

DOC −solublllzed 　 cn7yme 　was 　labeled　wlth 　ABD −F　 ln 　thc　 absence 　and 　pregcncc　of 　a

纛巽

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H 麁dof 　2　mercaptoethanol 　ln　the　presence　 of 　dlluted　SDg 　　Mlcroromal　fractlon（［4− 2

mg ／m り p 正 epared 　from　plg　kldney ｛〕utcr 　mcdulla 〔upper ［）aIlels ）and 　rabbit 　back　musclcs

（lo“ er　panelr）“ cre 　mcub 飢 cd 　at　20℃ f（）r　 l　h　at　vancd （oncentrdttonb 　of 　2　mercaptoethanol

（0 − 06M ）m 且hq ）resenLe 　of　O　4　mg ／ml 　SD 〜 and 　Lentnfi ユged　at　100，000 × gfor　30　mm

Resulting　peHe〔s （lanes　1− t）and 　 supernatarlt （lanes　5

− 8）were 　absayed 　b》 po且v 肌 rylamide

gel　　el 〔 cLrophoIeslr 　　　Thc　　gtl〜　　w 唖 re　　btd ］ned 　　、〜 Lth 　　Coomass艮e　　bmlliant　　blue　　R250　　Arrowheddb　lndl 〔ate 　the α subumt 　ot 　 the （丶 a

＋ K ＋

）ATPare 　and 　the　Ca 片

へTPasem 山 euppe1 　 and 　lower　ptuiel，　iespecu 、 ely 　 2ME 　repr じ bents 　2　mertaptoethanol

N 工工一Eleotronlo 　Llbrary 　




Inactivation of Ca?'-ATPase 2:')7

reducing agent. Prior to labeling, the SH groups of the Ca2"-ATPase were alkylated with

iodoacetamide in the presence ofSDS, TBP was used as the reducing agent since it does

not react with ABD-F. Reduction with TBP and alkylation with .ABD-F should be

performed simultaneously so that reoxidation to disulfide during labeling can be avoided [9).The number of disulfide bonds, thus estimated, was 3,22 a molecule of the Ca?'-ATPase

(average of 3.13 ancl 3.31 in two separate determinations). X'Vhcn the native DOC-

solubilized enzyme was labeled with ABD-F in the presence ancl absencc of TBP at 48℃ for

60 min, the diflerencc in the number of ABD-F incorporated was O.97 (avcrage of O.94 and

1.0 in two separate determinations) between the two samples. (Since the inactivation by'IiBI'

was slow as shown in Fig. 1 (A), the enzyme was incubated for 60 min at 43℃ under

the condition where the inactivation was almost complete.) These results suggested that

at least one disulfide bond out of three in thc Ca?"-ATPase was clcaved upon treatment

with a reducing agent at elevated temperatures,

Effect of2-mercoptoethanol on the dn2'-ATPase in the presence of'diluted SDS

We have already observed that the (Na', K') ATPase lost its activity when treated

with 2-mercaptoethanol even at room temperature ifdiluted SDS was present [13, 14], and

that the inactivated enzyme was no longer sedimented by centrifugation at 100,OOO × g fbr

30 min [13], The C`solubilization"

of the (Na', K') AFIiPase was confirrned by using crude

microsomes from pig kidney outer meclulla (Fig. 2, upper panels). When microsomes

from rabbit skeletal muscles were treated with 2-mercaptoethanol in the presencc of O,4

mg/ml SDS at 20℃ for lh, the ATPase activity was nearly completely lost (data not

shown) and the `Csolubilization"

of the Ca?'-ATPase was not observed at all (Fig, 2, lower

panels).

Discussion

The SR Ca2+-ATPase is inactivated by reducing agents at elevated temperatures (Fig.1). The inactivation preceeds more rapidly in the absence of Ca'L than in its presence,

ln the ubsence of Ca'!' an irreversible confbrmatienal change is induced even at moderatc

temperatures in the region of the calcium binding sites ol' the Ca"-ATPase []5]. It is

interesting to assume that the contbrmational challge would expose disulfide bond(s) of the

CaU'-ATPase to 2-mercaptoethanol, which would result in the rapid reduction of disulfide

bond(s) leading to the rapid inactivation of the ATPase activity.

The Ca'LATPase is composed of a single polypeptide and lacks thc polypeptide

equivalent to the B-subunit of the (Na', K') ATPase. Under the conclition where the

Ca?'-AFI]Pase is inactivated with reducing agents, at least one disulfide bond of the Ca2'-

ATPase is reduced. It $cems likely that the reduction of the disulfide bond within a

molecule of the Ca"" -A[[iPase is rcspensible for the inactivation.

The (Na', K') ATPase whose a-subunit has a similar transmembrane topology to the




258 Y)v[uToH et al

Ca'"'-A[I]Pase, is also inactivated upon thermal treatment in the presence of reducing agents.

Contrast to the Ca2'-ATPase, no change in the disulfide bond ofthe cr-subunit ofthe (Na',Ki) ATPase occurs, but instead the B-subunit is reduced l6, 7]. That is, for the (NaT,Ki) ATPase, the reduction of the disulfide bonds of t,he B-subunit is responsible fbr the

thermal inactivation with rcduc;ing agents.

One possible exp]anation is that the two protcins arc difl'erent in structure with respect

to the locations of the disulfide bridge, even if both proteins contain the samc numbcr ol'

disulfide bonds, 'I'he

nurnbers of the disu}fidc bond of the a-subunit of thc (Na', K")

iNTPase so far reported arc at least one [16], two [17] and three l71. Thc disulfidc bonds of

thc cr-subunit of thc (Nai, K') ATPase may bc occludcd and not bc susceptiblc to 2-

rnercaptoethanol while, for the CaU'-ATI'ase, at least one of the disulfide bonds may be

cxposed and susceptiblc to the reducing agent, If this is the case, the CaU'-ATPase has

uniquc structural featurcs around thc disulflde bonds that are not shared by the (Na', K')

AFIiPase. Alternatcly, it may bc possib]c that the B-subunit pret,ects the a-subunit firom

reduction by forming the crP complex in the (Na', K") A'i'Pase, even if disulfidc bonds of

the a-subunit are present at identical locations te those in the Ca?'-ATPase. Thereforc,

the Ca2T' -ATPase without the protector might bc easily reduced, leading to inactivation. ･

Thc a-subunit of the (Na", K') ATPasc was "solubilized"

by diluted SDS in the

prescnce of 2-mercaptoethanol, whe,rcas the, Ca2"-ATPase rcmained in the membranc.

Thc mechanism by which thc reducin.cr. agents {'acilitate the `Csolubilization"

of the (Na",K") A'l'Pase with diluted SDS is interesting but still an open question. Howcvcr, these

rcsults may be taken as evidence, although circumstantial, that the Ca2'-ATI'ase and the

(Na', K') Arl'Pase a-subunit are somewhat diflerent in sttuctural fcatures. Recent

immunological and biochemical studies on the CaL''-ATPase draw similar conclusions [17,l8],

Acknowledgments

This work was supported by Grant-in-Aid lbr Scicntific Rcsearch to M. K. from the

Ministry of Eclucation, Science and Culture of Japan. 1,Vc thank Dr. S. Noguchi of

Kyushu Institute ofTechnology for his usefu1 discussions and suggestions.

References

1. Jergenscn P I. & Andcrseii j I' (l988): Structural basis for El-E2 conformational transttion in Na, K-

pump and Ca-pump preteins, J rvtembrane Biol 1011: 95-1202. Noguchi S, pt'Iishina rvt, Kawamura M et al C1987): Exprcssion of functional CNa', K')-ATPase fi'om

cloi'ied eDNAs. FEBS Lctt 225/ 27-32

3, lvlcl)c)nough A A, C;eering K & Farley R A (199U)/

'1'hc sodium pump lleeds iLs i3-suburiit. FASEBJ

4i 1598-1605



TheUOEHAssociation ofHealth Sciences

Tnactivation ofCR2'-ATPasc 259

4. Geering K (]990): Subunit assernbly and funcLional rrmturation of Na, K-ATPasc, J Membrar}e Biol

115: 109 -

E21 '

5. Kawamura M & Nog'uchi S (199I): Possible role of thc B-subunit in the expression of the sodium

purnp. In: The Sodium Purnp/ Stru{/turc, Mcchanism ancl Regulation (Kaplan J H, DeWecr P, ed).

The Rockcfcllcr University Press, New York pp 46-61

6. Kawamura pt'I & Nag'ano K (1984･): Evidence for essential disulfide bonds in the P-subunit of <Na', KT)-ATPase. Biochim Biophys Acta 774: 188-192

7. Kirley 'E' I. (1990): Inactivation of (Nai, K")-ATPase by "-mercaptocthanol. ,I Biol Chem

265i rl-227-,1･232

8. Kanazawa T, Ynmuda S, Yamamoto T et al (1971): Rcaction mechanism ot' the Ca? i-dependent

ATPasc

ofsarcoplasmic reticulurn frorTi skeletal musclc. j B{ochem 70: 95T12S

9. Kirley T L 0989): Determination of three disulfide bonds and one fi'ee sulfhydryL in thc P-subunit of'

(Na, K)-ATPasc. J Biol Chem 264･: 7185'7192

10. Lowry O H, Rosebrough .J J, Farr A L et at (1951): Protein measurement with the Folin phenol reagenL

J Biol Chem 193: 265-275

11. Laemirili U K (1970): Cleavage ofstructural protcins during the assembly of the hcad ofbacLeriophage

T4, Naturc (Lond) 227: 680r685

12, I,cpock J R, Rodahl A M, Zhang (: et al (I990): Thermal dcnaturation of the Ca!'i-fYl'1'ase of

sarcoplasinic Feticulurri reveals tN･vo thermoclynarriically irLdependent domains. Biochcn]istry

29: 68] -689

13. Kawamura }v(, Ohta 'I'

& paagano K (l980): Eil'ect of reclucing agcnts on the solubi]ization of rcnal

sodium and potassiurn ATPasc with cletcrgcnt. J Biecliem 87: 1327-1333

14･. Kawarnura pt{, Olimizo K, "torohashi ts4 et al (1985): Protcctive elll]ct of Na' and Ki against

illactivation of Na-, K+-ATPasc by high conccntrations oi' 2-mercaptoethanol at high ternpcratures.

Bioc,him Biophys Acta 821/ ll5-I20

15. (:heng K EI & Lepock J R (1992)/ Inactivation of calcium uptake b>, EGTA is due to an irreversibie

Lh(/,rmoLropic confbrmationa} chan.v.e in the calcium binding clomain of the Ca?'i-ATPasc.

Biochemistry 31:4074-4080

16. Gcvondyan N )v'I, Govonclyan V S, GavrilyeiJa E E et at (1989): Analysis of free sulfhydryl groups and

disulfide bonds in Nai, K'-ATI'ase. FEBS Lett 255: 265"'26817. Miller R P & J"arley R A (1990); B-subunit of CNa', KM)-ATPase contains thrcc disult"ide

bonds. Biochernistry 29: l52･1- -1532

18. rvtolnar E, Varga S & pt'tart,onosi A (1991): I)iflbrenccs in susceptibiliLy of various cation transport

A'I'1'ases te vanadntc-catalyzcd photoclcaN,agc, Biochim Biophys Acta 1068: 17-26I9. "･Iolnar E, Varg'a S,.)ona 1 et at (1992)/ lmmunologica] relatcdncss of thc sarcoplasmic ret{(/ulum Ca'i-

A'FPase and thc Na" , K

i -ATPasc. Biochim Biophys Acta 1 103: 2Hl-295



The 　 UOEH 　 Assooiation 　 of 　 Health 　 Soienoes

260 Y ．MUTOH 　et 　al

筋小胞体 Ca2＋ −ATP ア

ーゼの 2一メルカプトエタノールによる還元と失活

　（Na〒，　K

＋

）ATP アーゼとの対比

武藤　可信1・2

，杉野　厚子1

，東　　　監3

，高杉　昌幸z

，黒岩　昭夫2

川村　　越1

L産業医科大学生物学教室　

2産業医科大学第二内科学教室　

3産業医科大学生化学教室

要旨：　ウサギ骨格筋筋小胞体 Cai’＋．ATP ア

ーゼを 40 − 45℃ で 300 　mM 　2．メルカプトエタノール

　　　で還元すると，（Na＋，　K

＋

）ATP アーゼの場合と同様に，ATP アーゼ活性が消失した．　 Ca2＋ −

　　　 ATP アー

ゼの還元・失活は Ca2＋不在下で迅速に進行した．　 Ca2＋・ATP アーゼは 20℃ でも

　　　希薄な SDS （0，4　mg ／lnl ）が存在すると失活した．〔Na＋ K

−）ATP ア

ーゼも同条件下で失活す

　　　るが．失活した（Na＋

，　K＋

）ATP アー

ゼが 10000 ×g，30min の遠心で上清に回収されるの

　　　に対し，Ca2＋．ATP アーゼは沈澱に回収された、

　　　　　　　　　　　　　　　　　　　　JUOEH （産業医大誌），14〈4 ）： 253− 26D （1992＞

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(Na', K') ATPase-