Olyeepcn phaxpharylaxc b #I canccnfrrliuns ckxe lu fhaac lburirl in xkclcIttl mrilele infcrsclr wlfh surcaplusmic rclkulum mcmhraner, but nur

v&h iipnsomcr mtidc of lipids cxfraclcd from these. mcmhrrtntis, and ix inhibited upon bindinp to (ha mcmbrww. The infertwion af glyeagen phw phoryhtsc with the rurcuplutmic r@iieulum mcmkrw is modtrlrfcd by phsaphtrrybtion. rerr the 61km ar fhix cnqme shows w X0, BP Inbxae~len

rbaut IO-k&3 lower rhan the h Tan. Upon association IO fhc mcmbranc the lhfnrcseancc prcpcrlicr of fhc eacnxymc~Tglycagcn phospharylasc, pyridoxrl.~.phasFh#lc, arc a~roaply ultercd, ler fhe fIucrcsccne.cnf 535 nm is portiallr qucnchcd and rhe lIularcsccnecat 4~5==410 nm incrcascs. Using i’luorrsccin h&&cd s#rceaplasmicrcticnlum mcmbmnea WE hnvc I&.md fhrf fhc tfvcrn~r ciankxmatian carfhc CWJ* +M$*~ATPfuc is plae uhcrcd an binding or phaspharylrwc b. In conclusion, the rrsulta rcporfcd in this paper suggest fhaf glyeagen phespharylnac and Wf + MO’*ATPuse’dircc~ly interact under enperimsnral conditions rimllltr fu fhasc found in fhc sarcoplnsm, and fhrt fhis intcraetian is modulated by pherphcrylation of the phaspharylnsc. Glycagcn phnsphcrylasc; Sarceplasmic rsrienlunx Ca’+ +Mg’*-AYPasc;

1. INTRODUCTION In skeletal muscle glycogen is associat!d with’rcievant enzymes involved in its metabolism [l-3]. These glycogen particles also associate with C!ICsarcoplasrrric reticulum (SR)’ membranes [3,4]. The maj,or dcterminants of this association remain unclear, but there are functional gains. For example, stimulation of glycogen phosphorylase b kinase is faster when associated to the SR than in the soluble enzyme [4]. The integrity of glycogen plays an important role in maintaining an effective association of the glycogen particle with SR vesicular preparations [3]; but glycogen remain

phosphorylase tightly bound

and phosphorylase to preparations

b kinase

also

of the SR membrane, even when these are largely devoid of glycogen [3-z?].A large fraction (amounting to about 30-40%) of glycogen phosphorylase is likely to be tightly bound in vivo to the SR membrane and SR membrane preparations are ‘largely contaminated by giycogen phosphorylase [3,4]. Correspondence address: C, Gutierrez-Merino, Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, 06080 Badajoz, Spain

Abb,uvicttions: ATPase, 0” + Mg*’ -ATPasc; EGTA, ethyleneglycol-bis-(.&-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; FITC, fluorescein isothiocyanate; IU, pmols product per min per nig pmtein; PLP, pyridoxai-5 ‘-phosphate; SR, sarcoplasmic reticulum;.Tes,’ 2.[(2-hydroxy-1 ,I-bis-(hydroxymethyI) ethylamine] ethanosulfonic acid; Tris, tris(hydroxymethyl)-aminomethane

Published

by Elsevier

Science

Publishers

B. V.

Flnarcsccin; Rcyulrfion

In this communication we report experimental observations which show that glycogcn phosphorylase interacrs with the SN at ppf and ionic strength similar to those found in the sarcoplasm, and that this interaction is modulared by the phosphorylation state of phosphorylase and inhibits the activity of phosphorylase b. 2. MATERIALS AND METHODS SR vesicles and purified glycogen phosphorylase b hnve been prepared as described elsewhere [8,9]. Clycogen phosphorylase b was found to be more than 98% pure on the basis of SDS gel electrophoretic patterns, Because SR preparations usually contain a significant contanlination of glycogcn phospliorylase we have carefully tested that the preparations used in this study were devoidof fhe 97500Daband in SDS gel elcctrophoresis. The protein concentration was determined following the method of Lowry using bovine serum albumin as a standard [IO], and also spectraphotometrically using an extinction coefficient at 280 nm for glycogen phosphorylase of E’V4 = 13.2 at pH 6.9 [ll]. Lipids were extracted from SR memICI11 branes by repeated washing with HCCI~/HOCH~/HZO 2/ l/l v/v. The functional state of SR vesicles has been characterized as in [12]. Only preparations of SR showing, at least, 4-fold stimulation of the ATPase activity by calcimycin (1 Cl/ml) at W’C were used in this study, e.g. only well sealed SR vesicles. On average, the CaZC + Mg2+ATPase activity of leaky SR vesicles was 3-4 pmols ATP hydrolyzed per min per rng protein at 22°C. Phosphorylase u was prepared in a reaction mixture containing 8 mg*ml-’ phosphorylase 6, 70pg+ml-’ phosphorylasc kinase, 18 mM disodium glycerol-2-phosphate, 18 mM %mercaptoethanol, 1 mM ATP, 10 mM magnesium acetate (pH 6.8). The reaction was run until the phosphorylase activity ratio without AMP, Wf&iz plus AMP remained constant at about (~0.90). The reaction was stopped by an equal volume of 10 mM EDTA, 1 mM EGTA, 5 mM disodium glycerol-2.phosphate, 5 mM 2-mercaptoethanol (pH 6.8) 1131.

273

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ylyctx&gan, I). I Inki AMP. I m&CI l-xnk, 0. if bf WI, 16ilwf%S 7.41, t\n(t ~ho~~l~~ryl~~~ (0. I trt#- ml”‘!. kW#r: slrwirahW drlqpllt~n~d rc~ertliny 10 ~YIE l~~t~rt3ti.or Fbik~ t44ida~bhk~

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PlraapR~ryi&WtdVk~ h lb &WtkW~~Or #@WWI bO%IkdMI WH ~~~wrtltl II In [I $1, ttwinqthe ftWwl_n~ rtr~idn mlxrurc: IZ m&l

WtPr3&, 10 mM n~~ri&~rn wuw, fl.bf m&l YhDQ*, 50 m&t imI$iuolc, I m+MAMP, 0.41 a* I”’ gly$rdagan,I 11) ~h~~pb~~lu~~~t~tM~~. I IU prltleosaz6~6rRarph#~cdahydm~l,n#lr(gCi 6% Rwlo prtddcms ~1 rftptetPih&F* axwumprlon, in ~hcpwtcnco ef hi@ ~~ntrttt~t~~\~

aY ~lycagrn phasphorylnss wb! hare matrurtd ltx #ctlviry usinW the light %x~ltrtrln$ mclhod indiettted ht [vj, ltlc iighf wnttcrillg baif%i monlloretl by ttrrbidixy msawemcnls w~dar rhe c%prinwmul cc%b WIT, Intlicstodabavc The C~~~P~~CIIIA~npprerttr l’dlo~d was 4% urdincd In [VI, excapl thrr &~@xt ilsclr ww$ wxl ux primer and llle PCW/ORhnx been initinred by addiUon of AMP. i&crufe wc fclOnd char81trdditi0n of I nCMnMP pradwr 44rchivelr hl reueUon, l.c. the light aeatrcrlng chrnp stepped in about 3-4 min, WChvc: utod tl kwcr concenlfftIion 0r AMP In there WSJ~YI,in order 10 rliow Tar n much better ttccurrcy in our c~tinrtt~lanx olthc initlnl WC ofll#ht 8~~ teeing ehetrpc. The rear d the cwxnrraricmr td rrlcvimr kinetic xpecic~ in arxay medium wa8 idrntirrl Ia lhonc wxl in OutierrW Merino et nl 191. The lrbcling ef the SR vcxidrr with FITC nnd the d~lermisatian 0r the extent of labeling were rarricd out OS it~tlicrted elsewhrrc [8,i6), Pluaraccnre mensurcmenrs wcrc carPied out uJlng a JpeP. trofluerimeter HittP3+Pcrkin Elmer, model bfB-JO. cquippcd with A fhermoxtrred cell holder.

7.1I

Cltotllilwls

Bovine serum albumin, AMP, ATP, phosphucnoipyrtivrtc, EGTA, FLTC, phcnylmcthyl sulfonyi. flueridc. fi+ncrcaptoethanol, scphttdex G-50, aodium dodccyl sulfate, glycogcn, glucose l-phosphate, NADH, NADP”, Trix (TRIZMA bare), und TES were obt;lrtcd from Sigma, Calcimycin, pyruvatc kinare. lnctrtc dehydroglucosc.6.phosphntc phosphogluconlrlt~se and genasa, dehydrogenasc were purchased from Bochrinycr Mannhcim. All the other chcmicnls used in this study were: obtained from Merck.

3. RESULTS AND DiSCUS!%ON We have studied the effect of SR on the activity of glycogen phosphorylase. Relatively high concentrations of glycogen phosphorylase are required in order to form the complex with SR membranes and under these conditions the spectrophotometric assay of the activity of this enzyme becomes u.nsuitable, for NADP” ?s rapidly exhausted. Therefore, we have directly monitored the release of phosphate assaying the enzyme in the direction of plycogen s,ynthesis (see Section 2). As shown in Fig. 1, when phosphoryl,ase b is used the synthesis of glycogen is decreased by the presence of SR membranes in the reaction mixture. This regulatory effeet of SR membranes is mudh lower when measured with the a form. From these results we have estimated a value of. apparent Ko.5 of association between glycogen phosphorylase b and SR membranes of 0.8-1.0 mg/ml in these experimental conditions. The apparent &.5 of the D form is about IO-fold lower. Using uitracentrifugation (60 min at 35 000 x g) to separate membrane-bound from free enzyme we have obtained direct evidence that under these experimental conditions, at 1 mg phosphorylase 6*ml-’ and 1 mg mem-

274

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Fig< I. Depcndensc0r rhe 8twtlry al f&suirn phersglroryiare rrpafr the eon~:e~ruion of %I m@mbraner (arX,a)nnd uf Iipd~amcs of OH iiflidr (A) and al cgs lecithin (A), The stlnrcnfruflarr d SR nrcnb bfttncsitglvcnin fiq groleln,ml:“~anrlthrt orlipid~ln m~llpid~ml~‘~ an avctnpc the SH mcmbrrrnc e~rntalned0.6 nq lipid per m&tpretrir~ Etperlmentrl canditionr: IQ mM PES (p# 7.4jJ0.15 M tKl/lO nW ~l\~eoxc.i~pha~phrrc/ B.dS $.I” piyccl~wil I mM EBTA/ 0.1 mhl AMP, wnd 0.1 rnyl-ml”’ ar phuspharylitso # (a1 or b WI. Temperature lSpC.

brane protein- ml” only about 40% of rotat phospkorylase b is bound co the SR membrane, in good agrecmnr with the extent of inhibition produced under these experimental conditions. From the results preacntcd in Fig. 1, 8 maximum inhibition of ca, 80-90% of the glycogen phosphcrylase b activity is attained in the

pi%encc

of 4 mg SR,proteifl: ml”. This rcsu!t is in good agreement with earlier findings [4,6]. Fig. 1 also shows that neither liposomes made of lipids extracted from the SR membrane, nor liposomcs made of egg lecithin, significantly inhibit the phosphorylase in identical experimental conditions. Therefore, it follows from these results that the regulatory effect of the SR membrane is specific and not due to interaction of rhe phosphorylase b with the lipid bilayer. The results shown in Fig. 2 are consistent with the conclusions indicated above, atid in addition they show that the limiting size of glycogcn particles (monitored by the limiting scattering change) is decreased by the presence of SR membranes. The K0.s of this effect is only slightly dependent upon the temperature from 25 to 37OC. On the other hand, this parameter is not largely altered when measured with the a form in the presence of up to 1 mg SR protein. ml-‘, in good agreement with the results shown in Fig. 1. Furthermore, we have found no effect of erythrocyte membranes (up to 2 mg protein*ml-‘) on this parameter. On the basis of the structure of the SR membrane these results suggest a likely interaction between phosphorylase b and the Ca? I- Mg*+-ATPase, for this is the only protein of this membrane that is outwardly oriented [17]. TherefQre, the possibility that this interaction alters the conforma‘tion of both enzymatic systems has been explored. Upon mixing glycogen phosphorylase b with purified SR membranes the emission of fluorescence of PLP is,

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Fill, 2. Dependence upon tire ~:on¢ent~tlon of SR me~t~br~tne~or tire turbidity, ~han#e at 400 nm a~o¢iated to lllyeollen synthe~i~ catalyzed by phosphorylase b (~L,e} and ~s(+- 1. The mn,v;imt#mttJrbidlty ehanll!e (h.,~O h=t~been normalixed wlll~ respe¢l to the value (A~) me~sttretl in the absence of SR membrunes, The a~say rnedlunacontained: IO mM 'rEx tpH 9.4). 2..~ mM ~ly~ol~en(in 81ueo~etraits), t0 m M li!luco~eI.pho~phate, O.I mM AMP and phosphorylase ~5or ~ I m~l,ml~'t. Temperature 2:~ (O,&)anti 3'PC (@).

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readily evident from 1-2 rain after mixing. It can be observed that the fluorescence at 535 nm, a fluorescence characteristic of PLP bound to the active enzyme [181, is about 40% quenched (Panel B), and that a band centered at abottt 415-420 nm is largely increased (Panel A), This effect has also been observed in

the presence of allosteric ligands of glycogen phosphorylase b in the medium, such as AMP (50aM), Mg z* (10 raM), glucose (10 raM) or phosphate (10 raM) (results not shown). To check a putative effect o f glycogen phosphorylase on the c o n f o r m a t i o n of the SR Ca 2+ +MgZ+-ATPase we have labeled SR membranes with F I T C , a reagent that has been shown to specifically label the catalytic center o f this A T P a s e under certain experimental conditions [19,20], Covalently b o u n d fluorescein has been shown to monitor the E l / E 2 equilibrium distribution o f the ATPase [21]. In particular, the E2 c o n f o r m a t i o n shows an intensity o f the fluorescence o f fluorescein b o u n d to the catalytic center about 10-12070 higher than the E l c o n f o r m a t i o n [21]. The results o b t a i n e d i n the titration o f labeled SR membranes with glycogen p h o s p h o r y l a s e b are shown in Fig. 4. These results suggest that the c o n f o r m a t i o n o f the A T P a s e is altered upon binding o f glycogen phosphorylase to the SR membrane. The possibility that the fluorescence changes observed could be due to fluorescence energy transfer to P L P can be discarded, for P L P emits at longer wavelengths, and in this case fluorescein should be the d o n o r [22]. Therefore, it appears that glycogen phosphorylase b shifts the c o n f o r m a t i o n a l equilibrium o f t h e ATPase towards an E2-1ike conformational state, which is the c o n f o r m a t i o n showing the highest intensity of fluorescence o f fluorescein.

wavelenstlt, 335 am) or Illyeollen phosphorylase b in buffer (=1, and in the presenceorSR membranes (b); n,t=, stands for arbitrary units, Experimental conditions: 81y¢ollen pho~phorylase b ¢o,neentratlon (0. I ms/nail (a, bl~ and SR naennabranesconcentration (0, I roll pro. rein/m!) (b) in buffer: l0 mM Tris.~teetate (phi ?.0)/30 mM O" mereaptoethatlol."l~mperature: 2Y'C. Spectrum (b) was recorded a¢ approximately I0 rain after addition or SR membranes to Illy¢ogen phosphorylase b in buffer, Panel B: Emission spectra (excitation waveleng;h 425 nna)of glycogen phosphorylas¢ b In the absence(con. tinuous line) and in the presence of SR membranes (0,8 mg protein, ml =L)(dot ted line). Concentration or glycoiien phosphorylase b froln top to bottom: 2.5, 1,5 and 0,8 nag,ml" = Other experimental conditions: 10 mM TE$ (pH 7,4), 25"C. The scattering sii]nal from

SR membranes was loaded into ~ microcomputerand subtracted from the o r e rail fluorescencesignal obtained to yield the spectra showtl by dotted lines.

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Fig. 4, Dependence upon the concentration of glycogen phosphorylase b of the fluorescence of SR membranes labeled with FITC (emission wavelength: 518 am; excitation wavelength: 475 am). ExperimentaI conditions: SR (23 5:2 ~ug protein/ml) in buffer 50 mM TES (pH 7.45), 0.1 M KCl, 0.25 M sucrose and 2 mM 6'mercaptoethanol. Temperature: 20°C. The bars indicate the average fluctuations of the fluorescence change estimated from triplicate experimental series. 275

(unpubiithed rcrult$, and the Ca’* + ~g~~aATPas~ hsuabeen shown to be ~t~rn~lat~dto about the same level (iz. about N-4Usla)i by a variety of hydrophobic corn= pounds [X-27], Thcrefsrtc, it is likely thtrt glycogevr phosphurylas~ b i.~,effectively interacting. with this hydrophobic binding region of the ATPase at concenV trations close to these found in the sareoplasm. The negligible effect of 6~temgcraturo change from WC to 37’C q~~n the KQ,~ of interaction between phosphorylasc Ir and SR membranes clearly tuppertg this hypothesis, In canelusion, this report shows that 2 of the mast relevant protein components of the SR membrane glycogenolytic complex (glycogen phosptiorylasc and Ca” + Mg”-ATPase), interact under physicochemical conditions that do not largely deviate from physiological conditions, &I addition, this interaction appears to have clear functional relevance, because it induces an inactive form of the glycogcn phosphorylasc b, not strongly activatablc by AMP, and is controlled by phosphorylation of this enzyme. It is suggested chat this association can in vivo co-operate to potcntiate muscle relaxation. ncknowlergenlenrs:This no, PB87/1020

work has been supported by CICYT Grant

REFERENCES [l] Meyer, F., Heilmeyer Jr, L.M.G., Has&kc, R.H. and Fischer, E.H. (1970) J. Biol, Chem. 245, 6642-6648. (21 Heilmcyer Jr, L.M.G., Meyer, F., Haschke, R.H. and Fischer, G.H. (1970) 3, Biol, Chem, 245, 6649-6656.

276

112) ChW!rri!x~Mcrlne, C., Mellnr, A., Ps~trdercs,B,, Dtca, A. and taYnC&I&.. W89) Biochemiary 28,339~~3464. 1131‘krb, Q., %odor, A. und UsI, Ct. (1987) f3iachlm. Qiaphyr. Ac~n915, 19-27, [Id] L&irr4L.F. and Cardint, C.E. (19$7) Methods Enrymot. 3,

. (IS);#elm&h, E..andCart, CF. (1964) Proc. Narl. Acart, Sci, USA 51, I31-L38. [IQ WcnnO, F. onBGutierrexWxino, C, (1989) Biochinr, Diophys.

Acm 984, 135-142, [I71 Moller, J,V,, hnderwen, J.P, and Lc Maire, M. (1982) Mot, Cell. Biochrm. 42, 83-107. Ii81 Certija, M., Sfcinbcrg, I.% and Shnltiel, S. (1971) J. Biot, Chcm. 246, 933-938. [I91 Pick, U, and Kartish, S,J.D. (1980) Uiochim. Wiophys,AWI 626, 255-261a 1201Clorc, G.M., Groncnborn, A-M., Mirchinson, C. nntl Green, N.M. (1982) Eur. J. Biochcm. 128, 113-117, 1211 Pick, U. and Karlish, S.J,D. (1982) J. Biot. Chem. 257, 6120-6126. (221 Slrycr, I.. (1978) Annu. Rev. Biochcm, 47, 819-846. (231 Uhing, R,J., Janski, A.M. and Graves, D.J, (1979) J. Biol. Chem. 254, 3166-3169, 1241Sotiroudis, T-G., Cazianis, C.T., Oikonomakos, N.G, and Evangelopoulos, A.E. (1983) Eur. J. Biochem. 131, 625-631. [25] Jones, O.T, and Lee, A,G. (1985) Biochemistry 24, 2195-2202, [26] Jones, 0,T. and Lee, A-G, (1986) Pesticide Riochcm. and Physiol. 25, 420-430. [27] Fernander-Salguero, P., Hcnao, F., Layncz, J. and GutierrezMerino, C. (19900)Biochim. Riophys. Acta 1022, 33-40.