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Characterization of EGTA-washed synaptosomal melv/brane with emphasis on its calmodulin-binding proteins. Demonstration of possible reeonstitution with added ca|cium/ealmodufin N a o k i N a t s u k a r i i... T a d a y o s h i U e z a t o ~. H i d e h i k o O h t a -' a n d Michlya F u j i t a .~2 i National b:~tilute fiJr Ph).~iub~r~cal ~gcu:,~ces. Dir~e.t o] A cm e frang~rl. M)'r,daqt. Oka2~ki tJccpu~ and 2 Ddparlment r~f bl~t~:'hemi~tG, lhm~amut vr,~ Unit'cr~it.t Sc'he~e,/.f Medlt i,~e. H~raN~mal.~¢ f]apasD
(Recei~erJ 15 August It~qI}
Key words: Cahllt~dulin;Calmndulin bindingprotein; Adcnylatccyclas¢:/;-Adrcncrgicag~mis~;Synaplns~m~lmembrane: Ualmoclulin kii;ase II Endogenous calmodulin (CAM) in the EGTA-washed cerebral-cortical synaptosomal membrane ~SM) preparation was estimated below 3 / t g / m l protein by the semiquantitative ia~munob]at analysis (Natsukari, N., Ohta, 11. and F~ita, M, (1989) J. lmmunol, Metlmdg Ig.q. I59-166). Naembrane.bound CaM was insmunnelectran-micruscopieally demonstrated in EGTA-washed, non-treated (control), a~ad CaZ+-treated cerehral-cnrtical synaptosomal membranes (SM) as welt as for the SM enriched witk added CaM. The density of CaM increased in the above order. CaM.dependent adeaylate eyclase and CaM-del:~ndent protein ldnase I! (CaM-kinase il) activities were restored, whereas the phosphodiesterase (PILE) aetivib was no| affected by exogenous CaM over all the Ca 2+ concentrations tested. Adenylate ¢yelase at p C a @2 was synergistically activated either by GTP and CaM or by CaM and ~-adrenergle agonish ( +)-isopraterenoi, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein ki~ase A, CaM-kinase It, and their endogenous substrates in the SM. Based on SaP-antoradiography and tZsl-CaM overlay data eerta':,~ CaM-binding proteins .~eh as CaM-kinase !1 and synapsin I were identilied on SDS.Pi~GE. Caa+-de~ndent and -independent CaMBPs were distinguLshed by m I - C a M gel overlay with and without Ca ~+. The former had bigger molecular size ( >_49 kDa} than the [aSter ( < 34 kDa). Yield of CaZ+-depemlent CaMBPs was not affected by Ca a÷ cotlcentratiou during preparation of the SM while that of Ca 2÷-independent CaMBI's was reduced by exposure to 100/aM Ca z+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, miereviiioos membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings sL,,ggested that the EGTA-washed tM preFaration made a useful system for studying the role of CaM in the brain SM.
Introduction * Prescnl address: Department of Pharmaco~off/, Division of Neu-
~opha~macology, The Medical Co11¢g¢ of Pennsylvania/Eastern Pennsylvania Psychiatric Institute. Philadelphia. PA 19129. U.S.A. Abbreviations: BLM, basal-lateral membrane', CaM, calmodalia: CaMBP, CaM binding protein; CNBr, ¢yanol~¢~ b~omi:t¢; DTT, dithiolhreitoh MVM. mic~wZtuas mq:mbran¢; PI.;F.. phosphodiesterase; PMSF, phenylmeth~l:.ulphonylfhtoride;RBM, e,'-ythroc~;e membrane; SM(sLsynaptLr~ernatmembrane(s>. Correspondence: M. Fajila, Department of Bioehemig;r~ llamamalsu Oniv0rsity,Schoolof Medicii~.':., 3K~ |larlda-zh~;. Iiamumat:,u. 431-31. Japan.
Caimodulin (CAM) is a calcium-binding regulateD' protein enriched in neuronal tissues (see review, Reg. 1). As :,s welt known, it regulates the cyclic AMP (cAMP) metabolism linked to the calcium signaling (also, review Ref. 2). Malnoi~ et al. [3] previously demonstrate~i a synergistic effect of CaM and a /3-adrenergic agonistfs) on a.,-lenyiat¢ cyclasc activity in the EGTA-washed lysed synaptosomal me,.,,~brane (SM) obtained from eerebel-
194 lure. Gnegy eta]. also showed a synergistic activation of the cyclase by CaM and a dopaminergic agonist(s) in the EGTA-washcd particulate fraction from slriatum [41 and retina [5]. In agreement with these studies we observed a synergistic activation of adcny[ate cyelase hy GTP and CaM, by CaM and DI-specifie agonist, SKF 38393, and by CaM and (+)-isoprotcrenol in Ih¢ EGTA-washcd SMs from rat brat,+ cortex and striatum [61. Some investigators, however, found only addqive effects for these effectors using highty purified mem~ branes [7-Ill]. The discrepancy might be explained by differences o,f membrane preparations used. For instance, the integrity of interactions among components of signal transduction such as receptor, GTP-binding protein (G protein) and effector might be impaired by exhaustive purification of the membrane. The present and previous [6I data showrd that the EOTA-washcd membrane depleted of endogenous CaM restored the functions related with signal tram:duction with added Ca~'+/CzM Therefore, this preparation was assumed to be a useful model system to investigate the CaZ+/CaM-mediated cellular events. In the present work, we evaluated lhc prot~qn composition of ~i;e membrane preparafion with special atteiltion to their CaM-binding proteins (CaMI]Ps) as well as some enzymic activities. We have alsc~ demonstrated a functional reconstitution of CaM-dependent activities with added CaM A rc~atim-,.ship of types of CaMBPs distinguished by their Ca:*..;ependenee and +heh status fn the membrane has been noticed. The present findings seem to suggest the usefulness of the EGTA-washed lysed SM for elucidating the molecular mechnisms of signal transduction and its regulation by C a : + / C a M in neuronal cell activities. Materials and M e t h o d s
Materials I8-'~H]cAMP (20-311 C i / m m o l ) was purchased from the Radiochemical Centre, Amersham. [a-'~2P]ATP (80fl C i / m m o l ) and [?-32p]ATP (32 C i / m m o l ) were from New England Nuclear. The scintillation counting fluid, ACS I1, was a product ~f Amersham and Scintosol of Dojin Kagaku Co. (Tokyo). NaeATP, cAMP, Gql?+ lorskolin, D'IT, theophylline, creatine pb,osphate, r:GTA, (_+)-isoproterenol hydrochlorid¢, snake venom (Crotalus alrox) and ueutral alumina were obtained from Sigma. Creatine phosphokinase was from Boehringer-Mannhcim+ Nitrocellubse filters of pore size, 0.45 u.m were from Avantec Toyo Co. (Tokyo} and those of pore size 0.1 tzm from Schleicker & Schue!l (Da~el). Dowex resin ion-exchanger (AG 5 0 w - x g , 200-400 mesh) was fr.~ma the Bio-Rad. lmmunostaining kit (Immunostaining H R P kit IS-50B) was purchased from Konica Corp. (Tokyo). Solubiliza-
lion detergent kit (Dotite) which was composed of CHAPS, CHAPSO, MEGA-8, MEGA-9+ MEGA-10, tt-¢,etyl-fl-o-glucoside, tt-octyl-19ro-thio-glucoside and n-heptyl-/3-o-thioglucoside was from Wako Junyaku (Osaka). All other chemicals were of reagent grade. Autoradiography was performed on X-ray film (Fuji AIF, Fuji Color Co., Tokyo). Male Sprague-Dawley rat (125-150 g), C3FI mice (30-5t1 g, either sex) and male New Zealand White rabbit (2.0 kg) were obtained from Shizuoka Laboratory Animal Center (Hamamatsu). Porcine brain was from a local slaughterhouse.
Protein .~01¢1T.¢ CaM was purified from pot'cine hrain as previously described [2(I], 1251-CAM (92.5 I.*Ci/~g, bovine brain) was from New England Nuclear. Bovine brain CaM, bovine serum albumin (BSA), monoclonal antl-r-factor and anti-/3-tubulin mouse aseites fluid were purchased from Sigma, Anti-ff-ealspectin and anti-ealdesmonl50 monoclonal antibodies were generous gifts from Nippon Shin-yaku Co. (Kyoto). Anti-calcineurin antibody was raised in rabbit with bovine brain caleineurin (Sigma) as an antigen according to the immunization procedures described by Yakoyama et al. [14]. lmmunoglobulins fracfionated by 5(1% (NH4)2SO 4saturation [211] was used for Western blot analysis for ealeineuriu. Ho~eradish peroxidase-eonjugated antirabbit and anti-mouse IgGs were from Cappel laboratories. Protein kinase inhibitor, H-8, was obtained from Seikagaku Kogyo (Tokyo) and CaM antagonist, W-7, from MBL Co. (Nagoya).
Preparation of membrane and partial purification of membrane-bound CaMBPs Brain regions such as cerebral cortex, striata and cerebellum were dissected from rat or porcine brain. EGTA-washed lysed SM was prepared by a modified method of Maloo~ et at. [21] as described in detail previously [2{I]. Briefly, the isolated tissues were homogenized in medium A (2 mM EGTA, 50 /~g/'ml PMSF, 0+2 /~g/ml leupeptin, 0.5 mM D T f , 5 mM Tris-HCI, pH 7.5) containing 0.32 M sucrose (medium A') and centrifuged at 1000 x g for 20 rain, The supernatant was further centrifuged at 10000 × g for 15 rain. The supernatant was designated 'S z+ fraction. The pellet was suspended in medium ,V, diluted with medium B (medium A at pH 8.11, an1 centrifuged at 27 000 × g for 30 rain. The pellet was fesuspended and centrifuged at 27000×g for 30 rain. The final pelle~ ('P4') was suspended in a medium appropriate for mt individual experiment, usually, in medium C ' (60 mM Tris-HC~ (pH 7,5), 5.0 mM MgSO4, 0 5 aIM DTI'), and stored E.t - 80 ° C. SMs referred to as 'non-treated" and 'Ca-'+-lreated" SMs were prepared in the absence of E G T A and in the presence of 100 .ttM CaCI z, respectively, but otherwise
in the same manner as EGTA-washcd SM. For rcconstitution of EGTA-washed SM with Ca ~ ~/CaM. the membrane (I .5 m g / m l pr( g h~r 20 rain and the pellet suspended in the medium containing 100 ~ M CaCI,. "Partially purified CaMBPs' were first solnbilizcd by incubating $M at 4 ~ C for 2 h in medium C' containing 50 /zg/ml PMSF, 0.5% (v/v) Triton X-Ill0 and i).1% (w/v) detergent kit (Dottle). After cenlrifugation at 27{Jl10×g for 30 rain, the supernatant was dialysed overnight against a sufficient w~tume ef medium C ' containing 50 /~g/mi PMSF and 100 # M CaCI~ (medium D). The sample was recentrlfuged at 2701)0 × g for 30 rain and the supernatant was applied on a CNBr-activated Sepharose 4B-CaM-affinity column (1 X4.5 cm) equilibrated with medium D at 4°C. CaMBPs were elated from the column with medium D containing 1.0 mM EGTA. The MVM and BLM from mouse intestinal mucosa were prepared as described previously [22]. "['he final pellets were suspended in redistilled water and stored at - 8(I ° C until use. The rabbit RBM was prepared by the method described by Stock (23]. Briefly, 1 ml of packed e r ~ t h m ~ t e s was mixed with 40 ml of ice-cold 5 mM sodium phosphate buffer (pH 8.0) and centrifuged at 201)OOxg for 15 min. The loose sediment was collected, resuspended and recentrifuged three times. The final ghosts were suspended in 20 mM sodium phosphate buffer (pH 7.5) and :ilored at - S 0 ° C.
lmmunoeleciron-microscopy of ('aM The precise t~chniques wilt be described in detail elsewhere (H. Obta, unpublished data). In brief, small pieces (] × 5 mm) of membrane filter for blot analysis were immersed at 4 ° C for 2 h in the suspension of freshly prepared SM (1.3 mg/ml). After being incubated for I h in a blocking buffer (1% BSA in PBS), the fillers were incubated w~!h anti-CaM antibod.u (1 /~g/ml) at 4 ° C for 3 h. The filter paper was further treated with protein A-go!d prepared according to Frens [25] and Geoghegan and Ackermar~ [26], fixed in glutaraldebyde and osmium tr~troxid¢, embedded in LR white resin, and examined under glee!run m~crt~scope (JEOL, JEM-1OO C).
~'~Caauzoradiograp~y Ca2+-binding proteins including CaM in S 2 and P~ fractions from three differently prepared SMs were estimated by 4sea-binding on the nitrocellulose filtzrs [17]. After the samples and standard CaM were clectrophoresed in the Laemmli system (14% gel), they were eleetrobfotted to the nitrocellnlo~; membrane (pore size, 0.1 #m). it was immersed in a solution containing 61) mM KCI, 5 mM MgSO 4 and l0 ;aM
imidazolc-HCl (pH f>.~), incubated in the same medium contaioin~ ~ C a (4 ~ C i / m l ) for 5 rain and rinsed ~,ith 5(v/~ ethanol several times. After drying the autoradiogr,phy or, X-r~y film was performed for 24 h.
P:'I-CaM gel ot'erlay ':51-CAM gel uvcrtay was performed by a modified pr~ccOnrc of Hertzberg et el. [28]. The membrane preparations (2 3 mg protein/roll were mixed with I / 3 vo[. tff the buffer consisting of 125 mM Tris-HCI (pH 6.6), 3% (w/v) 5DS, 10% (w/v) glycerol and 5% (v/v} 2-mercapt~ethanol. 15 to 30 tzg pro',cin of each membrane was immediately subjecled to SD$-PAGE {5.5%. 8~,~, 11)~,~, t25t gels) in the Laemmli system I24J wihout prior heat denaturation and the electrophoresi~ performed at room temperature for 45 to 9.t) min. The gels were fixed with ll)O ml 25% (v/v) ist~propanol-lil% (v/v) acetic acid which w~.s exchanged three times for fresh one in the course of 3 b, then washed three times with l{~0 ml medium E (50 mM Tris*HCI tpH 7.5), 0.2 M NaCI) in the course o[ 3 h. "T.12e gels were incubated fur 2 h in 10{I ml medium E containing 1 m g / m l BSA to bk~ck non-snecific binding, then incubated overnight in 12 ml medium E containing 0.02% NAN,, 10 nM 1_~5l-CaM ( 1.8 - 10" c p m / m l ) and either IUO ,aM CaCI., or 1.1) mM EGTA. They were washed three times with 100 m! of the same medium without ~Z~l-CaM in the course of 3 h, then stained with 0.1% (w/v) Coomassie brilliant blue R-250 in 10% (v/v) acetic acid-50% (v/v) methanol for 15 min, and destained with 5% methanol9% acetic acid (or 1 h. They were dried and auloradiographed on X-ray films for 24 !o 811 h with enhancing screen (Dupont Cronex Lightning-Plus HK). The developed X-ray films were seamted densitometrically at 530 am.
Assay of (No ~,K ")-ATPase (Na÷,K~)-ATPase activity was measured as described previously [29]. The final assay mixture contained 60 mM imidazole-HCl (pH 7,5), 5,G mM MgSO4, 140 mM NaCI, t4 mM KC1, 0.5 mM EDTA and 1.0 mM ATP. The inorganic phosphate released was determined colorimetricalty.
Asxay of adenylate cyclase The adeny[ale cyelase was assayed by the method of Liana; and Sacktor [30] with some modifications [6]. A 50 ~1 reaction mixh~re contained, it, medium A, 40 p.M D'Iq', 1.0 m M [~zP]ATP (1 p.Ci per assay), 2.1) mM cAMP, 2.0 m M '~hcophylline, 20 mM ereatioe phosphate, 25 ~ g creztlne phosphokinase, brain corticai SM (25-30 ~ g protein per a~say), 1.0 m M EGTA, 0.924 mM CaCI 2 (pCa 6.2} and, when indicated, 10 IzMGTP, 10 p.M tbrs~:Jlin, 0.fi tzM CaM, Ill ~tM isoprolercnol. The mixtures were incubated at 3 0 ° C for 10 rain and the reaction stopped by adding 150 p.I of the solution
196 containing 5 mM Tr[s-HCI (pH 7.5), ,g) mM A'I'P. 2.1} ml~ [3H]cAMP (25 nCi/assay) and 2% (w/v) SDS, followed by heating at IOO °C fi)r 3 rain. After (E8 ml of 5 mM Tris-HCI (p[[ 7.5), was added to Lhc *r:i~.~,aae, cAMP formed was recovered by the double-column procedure or1 Dowex resin and neutral alumina. The radioactivity in Ihe'eolumn eluents (4.3 ml each) ia 1).1 M imidazole-HCI (pH 7.5), was measured in 12 ml of ScintisoI by liquid scintillation spectrometry. The recoveTy of cAMP was 75-85%. Deterr~ina|ions were performed in triplicate.
Assay of phosphodiesterase (PDE) The activity of cAMP-PDE in the cortical SM was measured by a modified procedure of Hidaka and Endo I31]. The final reaction mixture contained, medium A, t).5 mM Dq"T, 0.2 rag/rid BSA, 4(I tzM [~H]cAMP (0.5 ¢tCi/assay), 0,6/xM CaM, 5 5 / t g membrane protein, either 1.0 mM EGTA or 1.0 mM EGTA plus 0.911 mM CaCI~ (pCa 6.2), in a total volume of 500 ~zt. The reaction was started by adding [3H]cAMP to the mixture, which was incubated at 30QC for 12 mla afsd stopped by placing the tubes in a boiling w-'_,ter i~ath for 3 rain, followed by cooling on ice. After the addition of 50 ~1 of snake venom (1 mg/ml), the reaction mixture was further incubated at 30'~C for 10 rain, then applied to a Dt~wex resin column. Washing the column with 20 ml of distilled water, adenosine derived from 5'-AMP was eluted with 4.5 ml of 3 M NH4OH and collected in a scinti[latinn vial. The radioactivity in the column eluent was determined in 10 ml of ACS 11 by liquid scintiliation spectrometry. Values were based on triplicate determinations.
t'hospt~orylaHon The reaction mixhtre contained 60 mM Tr:,a-HC1 (p1| 7.5), 5.1) mM MgSO~, 0.5 mM DTT, 100 fzM [T-~2P]ATP (1.0 Ci/mmol), 110-125 p,g protein of .cnt'rical or eetebellar SMs, 0.053-1 ;tM CaM, 1.0 mM EGTA, varying concentrations of CaCI 2 and other reagents in a total volume of 100/xl, The reaction at 30 ° C was initiated by adding 30 htl of ice-cold SM. 10 t.tl of the reaetior, mixture was recovered al 0, 1, 3, 5, 1D, 30 and 611 rain and mixed with 25 p.l of the stoppi,, 3 solution containing 62.5 mM Tris-HCI (pH 6.6), 2.3% SDS, 5% 2-mercaptoethanol, 10% glycerol, followed by beating at !00~'C for 2 min. A 20 /zl aliquot of each sample was subjected to SDS-PAGE (10% gel) in the Lacmmli system. The get was stained, destained, dried and autoradiographed on X-ray film overnight. The film was scanned densitometfieally lot the pho-~phorylated bands. In some experiments, the d e c trophore~';ed bands were quantitated by Bid-image Ann-
Iyzer BAS 20l]0 (Fuji Film Co., Tokyo) before thc auto~adiography was performed.
bnmunoblot assay.~for CaMBPs and cy;o.s'keletat compolmmunoblot assays for CaMBPs and cytoskeletal .~.omponents were performed in the same manner as described prev!ously for CaM [2(I] except that SDSPAGE was carried out in 5.5% (w/v) gel for calspectin and caldesm,~nl.~l, in 10% (w/v) ge] for/3-tubulin and r-factor, and in 14% (w/v) gel for ealcineurin, in the Laemmli system [24]. Besides, a longer period of eleetrophoreite transfer, i,e., overnight, at 4 ° C , was employed without fixative. The SDS-PAGE for cateineurin was performed without heat-denaluration. The blotted bands were st~tined with immunostaining kit.
Miscellaneo'.e~" procedures The concentration of free Ca 2÷ in EGTA buffizring system was calculated on the computer program by the method of Fablato and Fablato [32] modified after Tsien and Rink [33] using the binding constants of Sehwarzenbach et al. [34]. Protein content was determined by tt, c methed of Lowry e~ al. [35] using BSA as standard. Results
Immunoeleclron-microscopy fo~ CaM in cortical SM preparation The repeated EGTA-wash deprived the membrane preparation of major portion of endogenous CaM [20]. To visualize this depletion and examine the possible effect of the presence of Ca 2+ ions during preparation, we prepared the control, i.e., non-treated, and the Ca2+-treated cerebral-cortical $Ms be~ides the E G T A washed SM as described in Materials and Methods and performed immunoelectron-mieroscopy on lhese membranes (Fig. I). A demonstration of gold particles aleng the membrane structure (Fig. IA, arrows) presumably revealed the pre.~enee of membrane-bound CaM molecules in the EGTA-washed SM. No such gold particles were detected with the preimmune-serum-treated cortical specimen (data not shown). The density of demonstrated gold particles increased as the membranes were prepared in the absence of E G T A (Fig IB) and in the presence of 160 taM CaCIz (Fig. 1C). The density increased in the above order. The greatest density of gold particles was t~bserved for lhe sample incubated in the presence of 4.5 g M added CaM and lll~ # M CaCI_, (Fig. ID). The preliminary ~H-CaM bindil~g experiment had shown that CaM-binding sites of the EGTA-washed SM was" saturated at .4.5 p.M of CaM (data not shown).
197
Ca ~ + bindi~o,, proleins in three dif.l~'reutl)' prepared SM~" In order to compare the Ca=M conk~nt of lhc tl]rce differently prepared SMs (see above):~Ca-autorudio~araph~' was performed for both the :hree mcn, branes and the corresponding eytosol fraeti,,,ns (Pig. 2). The S, supernatant (see Materi.".l~ and Meth~ds) w~s -'t~sume~J to he reoresentalive of cylosol fraction on ~hc ground that approx. 92% of total solub!c protein was rec(wered in thk fraction. The ratio of membrane-bound to soluble CaM wa~ much lower ir~ the EG l~A-treatcd SM (Fig. 2, lanes 2 / 5 ) than in the other tw~ SMs (lanes 3 / 6 and 4 / 7 k This finding confirmed the depiction of endogenous CaM and suggested its presumed translocation to the soluble fraction on EGTA Ireatmc,t. Other Ca-'+-binding proteins (28, 5¢1 and 85 kDa) were also detected in the soluble fraction. The 2,", and 5(I
''L.
"k
f o. 5pm
Dem~,~i~ati,,,, ,,]' ('a '~ ~-drpende~,t/ ~o;d -independent ('aMBI~" i~ three dijOn,really prepared SMs CaM binding protcin:, of the three SM preparation~ were demonstrated using "-~f-CaM gel overlay method tFig 3). The apparent molecular marx ~f detectable CaMBPs in brain SMs was 235, 't35/127, 58, 40, 34. 31.5. 24. 22.5 and that (or tht~se) lower than 20 kDa (see als~ Fig. 6)_ Of these, the smaller components ( < 3 4 kDa) showed a Ca-'+-independent or EGTAstimulated CaM binding (rig. 3, compare A and B). In ccntr~,l. CaM ~findlng by the bigger components (49 to 235 kD=l) ; v t l s entirely Ca-"-dependent (compare A
%
)
i)
L.
\
,x
r"
41'
C
kDa components were speculated to be calbindin,t) an6 ~:d)ulin. respectively [17] ialso see Fig. 8b).
D q'
lb ,J
I,."
4P .t ~,
/ 1
B .~P
11
t
Q
Fig. t. C~M molto,ales i n the E G T A - w a ~ h e d cortical S M (Lmmuaoele¢lftm-mictograph). O r i g i n a l magn;ticalion: ×3U(I(Rh E G T A - w a s h c d ¢or:.ical S M (A), non-treal~.d Sial ( B ) and 1110 taM Ca-'*-treattzd SNI (,g) were ~.':.¢d on fih~:r p a p e r mutrices and treated with ~mti-CaM antibody. E G T A - w a s h e d S M was in¢ubaled w i l h 4.5 taM C~M in Ihe ~,fes*gcc¢ of l ( ) n / a M C.:I-'" ;.ir 3 0 ~ C for 211 rain and Ireate,:t as abtwc (D]. Arrows illdi¢ine t h e imlrluitoi'e;tClWC go]d pi*l'iic]us | i2 nan in diamctc't'l. F~.)r d*:t~.til~.~i¢¢ t,~xi.
198
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•
1
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~
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2 3 4
567
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Fie. 2. Ca '~ (~(:a)-hinding proteins in variously prepared ¢orticaf SMs and noluhle S. [re,clions (pn?nradin~r:]ph)_ 4(] ~g protein [ff EGTA-wa.~hed cetrlneal SM (lane 2)~ non.treated SM (lane 3), Ca z% trc~;cd SM (lane 4) and the corre'~ponding {S~) fra=tlons (lanes 5, ¢, and 7, r~spccliv~ly} was et¢ctrophores~d as w~ll a~ 0.3 ~_$ of CaM standard (lane D. Ele¢lroblolling and ~Ca-autoradiography c,=rc, peefozmcd a~. dc.~cribud in Mulerial.~ cad Methods. The apparent molec.,hn" mass of major CaZ"-b[nding proteins were shown wllh reference to slai~dard CaM [ 17 kDa),
and B). specific The seemed
100
pea
The 24 and 22.5 k D a componentswere rathe~ t o the c e r e b e l l u m . absence of EGTA during preparation of SMs to c a u s e l i t t l e d i f f e r e n c e in C a M - b i n d i n g p a l -
terns (Fig. 3A and B, lanes ] and 2; lanes 4 and 5), whereas the presence of added Ca 2* ions resulted in a prefeeenttetial decrease in the relative araount of the smaller components ( ___34 kDa), especially, in the cerebellum-derived SM (Fi~. 3A, lane 6).
Effect.~ of added to Ca-' +/ CaM on some enzymic aclicities
A d e n y l a l c cyclase, Ca~+/CaM-dcpendcnt
phosphodiesterase (PDE) and p r o t e i n k i n a s e !1 ( C a M - k i n a ~ e
° ' ..,....
t
Fig. 4. Ca:'-concentratior, dependence of adenylate cyclase. Ca"~*/CaM-kinasc I1 and pho~phodieslera~e (PDE} in I:GTAwashed cortical SM. Th~ a¢lJvilles or adenylal~ cyclas~ (,a, ~,), Ca ~'/CaM-kina~ [] (a s~=b,~nlt)(Ill, D) and PDE (BI to. ~ l wcr~ e~limated in the ,ar;ous ¢or~c~r+~/ralJons of Ca z" with (a,. II. a} or w~thout ( A, ~a+o l [L6 #M CaM. Th= activity . f the first tw. unn~ymus is represented relalivc to thai al pCa 6.2 tadenylate ~.yclasc)and p(,~ 5.5 (CaM-kina.~¢ Ill, The dat;~ arc a representative of more than three independent expeiimcnts. PDE uctwity (B) is r~prescnted as mean _+S.D.
B
A ~
~
~ .
~
~
-'2#5~127
q m b / m ~.~.~ ~
1
2
3
4
qil$ m IlIB~
-24 -22.5
5
6
1
2
3
4
a, ma' .......
'.5
6
Fig. 3. CaMBPs in the three SM~ differently prepared from cerebral cortex and eerebc]luro (l~l-~utoradiogeaphJ. ~'le Ef;TA wlls.hed $Ms q]am:.~ I ~nd 4). n~o~-txe~ted SMs (lanes 2 and 5}. and Ca2%trealcd SMs (lanes 3 and 6) were prepared from cerebral eorte~: tl:me~. I-3) ann I-'~rebell,arn (lane~ 4-61 :=s described in Materials and Methods. It, ~g protein of each membrane was subjecled lo SDS-PAGE (129E) and n~"].CaM gel overlay. Tire lultcr was performed in the Orese.q¢c o[ either 100 ~M CaC]2 IA) or ) mM EG'I'A (B), followed by autoradk~graphy. The apparent m~Jl~ctdar raass t!f CaMISP~ ar~: indicated (also. Figs, 6 and 7), The dala are a representative of ,~ix u;q~rirnunts with lhre¢ dffferon! SM preparation;,,
19t~ It) w e r e assayed with the E G T A - w a s h e d mort,brahe p r e p a r e d f r o m c e r e b r a l cortex (Fig. 4). T h e a d c n y l a t c cyclase activity reecho-,1 m a x i m u m at p e a 6.2 -rid m a r k e d l y d e c r e a s e d at h i g h e r C a -~* c o n c c n t r a l i o n s ~Fig. 4A, triangles), while the a u t o p h o s o h o r y l a t i o n of C a M kinase i i w a s maximal ,~t pCa 5.5 (Fig. 4A, sqtlare'~). A l t h o u g h the P D E activity s h o w e d ~ slight p e a k b e t w e e n a c e 7 a n d 5 (Fig. 4B). Iberc was ntr significant d i f f e r e n c e in the activity in the p r c s c n c e or a b s e n c e of C a M over the C a ~'+ c o n c e n t r a t i o n s tested. It ~ a s conc l u d e d , t h e r e f o r e , t h a t E G T A - w a s h e d cortical SM contained, if any, very little C a M - d e p e n d e n t P D E isozyme ( f u r t h e r d a t a to s u p p o r t this c o n c l u s i o n are p r e s e n t e d below).
cAMP- and Ca 2 +/CaM-dependent phosphvryhtrion c A M P - d e p e n d e n t p h o s p h o r y l a t i o n of the possibie e n d o g e n o u s s u b s t r a t e s w a s tested with E G T A - w a s h e d cortical a n d c e r e b e l l a r SMs. T h e cortical SM w a s maximally p h o s p h o r y l a t e d 1 rain a f t e r initiation o f the reaction followed by r a p i d d e p h o s p h o r y l a t l o n (Table i). Fig. 5 (A, lane 2) shows a p r o t e i n kinase A-specific p h o s p h o r y l u t i o n o f the cortical SM. T h e 52 anti 73 k D a b a n d s a n d t h o s e of h i g h e r m o l e c u l a r magg w e r e a p p a r ently p h o s p h o r y l a t c d b y e n d o g e n o u s p r o t e i n kinase A.
T h e specificity of the r e a c t i o n was shown by its dec r e a s e d acti',ity e i t h e r in the a b s e n c e o f c A M P (lane 1 ) or in the p r e s e n c e of the specific inhibitor, H - g (lane 3). in the reaction mixture, R e p o r t e d l y , C a M - P I ) E is c o m p o s e d of two identical 60 k D a s u b a n i t s ~vbich a r e themselves C a M B P s , e a c h of which i n c o r p o r a t e s 2 reel of o h o s p h m e in c A M P - d e p e n d e n t m a n n e r [18]. T h e r e w a s no p r o m i n e n t c o m p o nent of the c o r r e s p o n d i n g size w h i c h was o h o s p h o r,/lated p r e f e r e n t i a l l y by p r o t e i n kinase A in the cortical SM (Fig. 5A. lane 2; B, lane I). A l t h o u g h the 52 k D a p r o t e i n b a n d (Fig. 5 A , lane 2; 13. lane 1, arrowb e a d ) s e e m e d to be an e n d o g e n o u s s u b s t r a t a of p r o t e i n kinasc A, it did not show C a M - b i n d i n g activity in the p r e s e n c e or a b s c u c e o f C a -'+ (Figs. 3 a n d 7). T h e c e r e b e l l a r SM s h o w e d a p h o s p h o r y l a t i o n p a t t e r n slightly d i f f e r e n t f r o m t h a t o f the cortical SM, especially, in the lower m o l e c u l a r mass r a n g e (Fig. 5B a n d C, c o m p a r e l a n e s I a n d 2). In the p r e s e n c e of C a - ' ~ / C a M t h e r e w a s a m a r k e d p h o s 0 h o r y l a t i o n of 49, 58 a n d 73 k D a b a n d s a n d s o m e h i g h e r m o l e c u l a r m a s s c o m p o n e n t s for b o t h the cortical a n d the c e r e b c l l a r S M s (Fig. 5C, l a n e s 1 a n d 2). T h e p h o s p h o r y l a t i o n w a s d e p r e s s e d in the p r e s e n c e of C a M a n t a g o n i s t , W-7 (Fig. 5C, lane 3), indicating the
T+~ 8LF_, I
,~ome t'ngvmiL"acticiriesof EGTA-wask.:d corticalSM Adetlylate ¢~,.iase, PDE and pmteln kina~ were assayed at pCa 6.2 (pH 7.51as described in Mate:ials and Method¢. Because the specific aclivily t]f adenylate cyclase was variable from preparation to preparation, values (means±S.D3 ~htmn were a rep:¢seatative of three separate preparatious, Figures in the parellthests are the numbers of separate: txptriment~. Statistical significance of cyclase activities ir~ Ihe:pre~enee and absence or CaM was estimated by paired t-test. The stimulated acl[vily is dtfr,ed ax the difference between the activity assayed [n If: presence of 10 ~M stimulant(s) IGTP, (_+)-isoproterenol and forskolin) in addilion to 0.6 AM CaM and that in the pro.seato of CaM a]orte, Phosphoqdation of the endogenous suhstrates (49/51 kDa and 51,;/~.1~'kDa) was c~timated ,~t t and 5 mix afler the ,.-'action war, ini,.iated. A prellralnary study had shown that maxinmm ph~phoolation was reached at I mix. Enzyme
Activity + CgM
- CaM 6,5+ ~6(0)
(Na +,K ~")-ATPase t
Adcnylase cy¢la~u ~
111,2_+ h_6(3}
basal * GTP-sl. * l~prolerenol-st. * Forskolin-st. Phosphodiesttrasu ~ Phosphowiatioo 4 (suhslrat,) 58 kDa 40 kDa
14b.3+ 40.2 2.~7.'-)+ "t3,2 1}5.6-1-13,[ 2186.6:1:61.3 10.9+ 0.3
98.2__. 17.4 31,0+ 1%9 q60.5: 62 9,9-- 0 3 (3)
- Ca"*
t miu
5 min
I mix
5 mi~
16.6 27.4
7.9 13,6
13.2 15.4
10.8 12.4
Units used: ~mal P i / h per mg protein. 2 pmQI cyclic A M P formcd/mln per rnE prote;n~ pmol cyclic A M P hydrolyzed/mi~ per m B pratt:in. 4 fmol PL incorporated/,u.g proleln.
* Values in the pl.csence and absence of C a M st.. stimulated.
w e r e statisticltliy
signiftcant bY p a r r e d
l-lc~| ( P < 0 J ) l : t l = 3).
20~J
cyelase either by CaM and GTP or by CaM and isoproterenoI in the presence of lO #M GTP which supported the activation resuhed in an aetivatiort more than additive. These findings are ~n agreement with those previously reported [6].
presence of CaM-dependent p r o l e i n kinasc i i in the brain SMs, The 49 kDa component ~a~ l~ho.,,ph~r,'la..'cd to a much lower level relat;ve to the 58 kDa component in the cerebellar than in the cortical SM (Fig. 5C, compare lanes 1 and 2), consistent with the lower level of tZ~l-CaM binding by the 49 kDa component relative to that of the 58 kDa component in the cerebellum-derived membrane (Fig. 3A, lanes 4 to 6).
Comparison of CaMBt~.~ o[ brah~ SM, and enterocyte and eryzhrocyte plasma membranes by means of t"'~l-CaM gel ocerlay
Synergistic actfi'ation of ade/zylate cyctase by isoproterenol and CaM, and s~me other enzymic properties of cortical SM
Fig. 6A(b) and B(bl shows the results ~f ~z~l-CaM gel overlay cxpcrhnc.nls with brain SMs and cnterocytc (MVM and BLM~ and erythrneyte (RBM) plasma membranes (see Fig. 7 for densitometric lracings). The rat brain membranes, cerebral-cortical, striatal and ccrebellar, seemed to share essentially the same CaMBPs. Only the 49 kDa ccmponent was much less conspicuous in the cerebellar preparation (Fig. 6B(b), lane "CER'; also, Fig. 3A, lanes 4 to O). Between rat and porcine cortical SMs slight differences were noticeable with regard to some CaM-bindlng compo-
Some enzymic aclivit[es of the cortical SM are summarized in Table I. The high (No *,K' )-ATPasc activity confirmed the plasma membrane na:ure of the present preparations. The activity of CaM-PDE was virtually absent. Several agents :,ucl= as CaM, GTP. forskolia and (+ ~it;~protcrcnol slimulated the adcnylate eydase at pea 6.2. Simultaneous stimulation of the adenylate
A
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Fig. ~.. cAMP- and C:~-~-/CaM-dependcn! pht)~ph~lation of EG"l'A-washed ~lrlicat and cercbeLlar SM~ (~-~P-autoradkP-raph). The ~.'AMF-dcpezndcnl (;~, awd B} anLLCa 2"/Cal'.¢-deDe~denl (C) pho~;phorylalionl reactions w~rv p~fform=d fur t rnia as de%'rihed in Materials and Me|hods
wilh mernha ,,..~ nrovcil~s as endogenous substralcri. CTX. EGTA-wa~hcd cortical; {'ER, ¢erebellar SM~. Experimental condilions arc bld~calcd in the pan,~L I ~ I IT,:;l~ cl]~ i'~clu.~ion of lhu ruagen'~ ill th~ r~aCliOn nli~ture. The ~pucificaity phosphor-clawed bands (armwhc:ads) and Celalb,'e rnolccuiar m~ls,~;arc indlc;dud.
2111 heats. The 711, 35 and 32 kDa comp,~nents, especially the last one. bound more CaM while the 24 kDa component bound much less CaM in the rat lhan in the porcine cerebral-cortical SM (Fig+ 6B(b). compare lanes "CTX' and "POR CTX'}. The 235+ 135 and 58 kDa c o m p o n e n t s whicl~ were clutable [tom a CaM-alfinity c o l u m n with I mM E G T A as described in Materials and Methods showed a Ca-~+-dcpcndent Cam-binding (Fig. 7(d): also, Fig. 3A and B). Interestingly, the 4 (k kDa c o m p o n e n t which was not recovered t'rerm the column with 1 mM E G T A (Fig. 7(d)) als¢) required Ca-'" ions for binding CaM i.FLg. 3A and B). Enterocytc p l a s m a membranes. BLM and MVM, had binding p a t t e r n s markedly different from those of brain SMs (Fig. 6A(b) and B(b), lanes "BLM" and ' M V M ' ; also, Fig. 7(e} and (f)). Two major CaMBPs, 5,R and 49 kDa components, of cerebral-cortical SM fotmd hardly any counterparts in the BLM and were entirely absent in the MVM, The 32 k D a component, one ¢}f the major C a M B P s in brain SMs, was not dclcc!cd in A
5.5++ {a)
gel
t h e i n t e s t i n a l m e m b r a n e s , I n c o n t r a s t , a 105 k D a c o m p o n e n t was a d o m i n a n t C a m B P in t h e c n t e r o c y t c m e m branch, e~pccially, in t h e M V M (Fig. 6 A ( b ) , laae
-MVM': B(b). lane,; "r4LM" and *MVM'). Rcwa~ka+~ly. there was no 11)5 k l ) a C a M B P in ~he brain SMs. A m o n g t~lher C a M B P s peculiar to the intest ne a 36 kDa cumpo;~cnt was prcsen~ in the R I M and 88 kDa componen! in the MVM. I t should be nt~tic.:d that the L35 kDa compuncnt was the most or the next most promin¢~l. C a M P , P throughout the membr~ nes tesled except lor the MVM (Fig. 6(b) and Fig. 7).
Western hh~l ana/y,~ls +Jr C\~MBt~ ~and cytosk"letat co#~zp¢:ttelffa it! t'r)r/ica./ SM To identify the ( ' a M B P s and cytosketetal ,,:omponents in E(}TA-wa~hed SMs we perti~fmed Western blot analysis t~.;r calcincurin, ealdesmon =~=~,r-factor and /5-tuhnlin. The membrane proteins weakly l"ut posilivLly hnmuno-rt.:tctcd with anti-calcineurm tFig. 8a. lanes I =rid 3), anti-calspcctin (data not sh,~wn) and B
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Fig. ft, Prolcin and =-'~I.CuM gel ~]verlay p; P,filcs t)f brain and enletocyte mcmhranc~. SDS-PA.EiE v,as performed in 5.5% I A I a~d II1":~- (B) g¢l.~ in the Laemmll system. After =-'sI-CaM gel Lwurlay (b) was pcr[~lrm~:,.I in the pt¢-.cn¢c nf I;;'1/ ~ M CaCla 1he /~els were stained Ior prol~in (a). Detail~ are d=scribed in Matcrial,~ and M¢thod~. A ~ ~te. ~rotein :tliq,.mt ~:~s applied {tw EGTA-washed SM I-mat ra" ccrebc!lum leER). striatura (ST). cerebral cortex [f~q'X), pristine cerebral corti:x (POR-C'I'X) and basal-I;Rc~al IBLM) and micr~'iltuus (MVM) n~cmbranes of mouse cntero~yt¢ h~[cl.hc[ will'= molecul~x~,nass standards (SIT~). Arabic nurm.-,rals repr~.':-t:nt the appueent molecular ma~';
Iq3
~i;, igV2ElsevierScience PublishersB.V. AlL righls reserved 01~7-48gt~/92/505 IHI
BBAMCR L3D%
Characterization of EGTA-washed synaptosomal melv/brane with emphasis on its calmodulin-binding proteins. Demonstration of possible reeonstitution with added ca|cium/ealmodufin N a o k i N a t s u k a r i i... T a d a y o s h i U e z a t o ~. H i d e h i k o O h t a -' a n d Michlya F u j i t a .~2 i National b:~tilute fiJr Ph).~iub~r~cal ~gcu:,~ces. Dir~e.t o] A cm e frang~rl. M)'r,daqt. Oka2~ki tJccpu~ and 2 Ddparlment r~f bl~t~:'hemi~tG, lhm~amut vr,~ Unit'cr~it.t Sc'he~e,/.f Medlt i,~e. H~raN~mal.~¢ f]apasD
(Recei~erJ 15 August It~qI}
Key words: Cahllt~dulin;Calmndulin bindingprotein; Adcnylatccyclas¢:/;-Adrcncrgicag~mis~;Synaplns~m~lmembrane: Ualmoclulin kii;ase II Endogenous calmodulin (CAM) in the EGTA-washed cerebral-cortical synaptosomal membrane ~SM) preparation was estimated below 3 / t g / m l protein by the semiquantitative ia~munob]at analysis (Natsukari, N., Ohta, 11. and F~ita, M, (1989) J. lmmunol, Metlmdg Ig.q. I59-166). Naembrane.bound CaM was insmunnelectran-micruscopieally demonstrated in EGTA-washed, non-treated (control), a~ad CaZ+-treated cerehral-cnrtical synaptosomal membranes (SM) as welt as for the SM enriched witk added CaM. The density of CaM increased in the above order. CaM.dependent adeaylate eyclase and CaM-del:~ndent protein ldnase I! (CaM-kinase il) activities were restored, whereas the phosphodiesterase (PILE) aetivib was no| affected by exogenous CaM over all the Ca 2+ concentrations tested. Adenylate ¢yelase at p C a @2 was synergistically activated either by GTP and CaM or by CaM and ~-adrenergle agonish ( +)-isopraterenoi, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein ki~ase A, CaM-kinase It, and their endogenous substrates in the SM. Based on SaP-antoradiography and tZsl-CaM overlay data eerta':,~ CaM-binding proteins .~eh as CaM-kinase !1 and synapsin I were identilied on SDS.Pi~GE. Caa+-de~ndent and -independent CaMBPs were distinguLshed by m I - C a M gel overlay with and without Ca ~+. The former had bigger molecular size ( >_49 kDa} than the [aSter ( < 34 kDa). Yield of CaZ+-depemlent CaMBPs was not affected by Ca a÷ cotlcentratiou during preparation of the SM while that of Ca 2÷-independent CaMBI's was reduced by exposure to 100/aM Ca z+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, miereviiioos membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings sL,,ggested that the EGTA-washed tM preFaration made a useful system for studying the role of CaM in the brain SM.
Introduction * Prescnl address: Department of Pharmaco~off/, Division of Neu-
~opha~macology, The Medical Co11¢g¢ of Pennsylvania/Eastern Pennsylvania Psychiatric Institute. Philadelphia. PA 19129. U.S.A. Abbreviations: BLM, basal-lateral membrane', CaM, calmodalia: CaMBP, CaM binding protein; CNBr, ¢yanol~¢~ b~omi:t¢; DTT, dithiolhreitoh MVM. mic~wZtuas mq:mbran¢; PI.;F.. phosphodiesterase; PMSF, phenylmeth~l:.ulphonylfhtoride;RBM, e,'-ythroc~;e membrane; SM(sLsynaptLr~ernatmembrane(s>. Correspondence: M. Fajila, Department of Bioehemig;r~ llamamalsu Oniv0rsity,Schoolof Medicii~.':., 3K~ |larlda-zh~;. Iiamumat:,u. 431-31. Japan.
Caimodulin (CAM) is a calcium-binding regulateD' protein enriched in neuronal tissues (see review, Reg. 1). As :,s welt known, it regulates the cyclic AMP (cAMP) metabolism linked to the calcium signaling (also, review Ref. 2). Malnoi~ et al. [3] previously demonstrate~i a synergistic effect of CaM and a /3-adrenergic agonistfs) on a.,-lenyiat¢ cyclasc activity in the EGTA-washed lysed synaptosomal me,.,,~brane (SM) obtained from eerebel-
194 lure. Gnegy eta]. also showed a synergistic activation of the cyclase by CaM and a dopaminergic agonist(s) in the EGTA-washcd particulate fraction from slriatum [41 and retina [5]. In agreement with these studies we observed a synergistic activation of adcny[ate cyelase hy GTP and CaM, by CaM and DI-specifie agonist, SKF 38393, and by CaM and (+)-isoprotcrenol in Ih¢ EGTA-washcd SMs from rat brat,+ cortex and striatum [61. Some investigators, however, found only addqive effects for these effectors using highty purified mem~ branes [7-Ill]. The discrepancy might be explained by differences o,f membrane preparations used. For instance, the integrity of interactions among components of signal transduction such as receptor, GTP-binding protein (G protein) and effector might be impaired by exhaustive purification of the membrane. The present and previous [6I data showrd that the EOTA-washcd membrane depleted of endogenous CaM restored the functions related with signal tram:duction with added Ca~'+/CzM Therefore, this preparation was assumed to be a useful model system to investigate the CaZ+/CaM-mediated cellular events. In the present work, we evaluated lhc prot~qn composition of ~i;e membrane preparafion with special atteiltion to their CaM-binding proteins (CaMI]Ps) as well as some enzymic activities. We have alsc~ demonstrated a functional reconstitution of CaM-dependent activities with added CaM A rc~atim-,.ship of types of CaMBPs distinguished by their Ca:*..;ependenee and +heh status fn the membrane has been noticed. The present findings seem to suggest the usefulness of the EGTA-washed lysed SM for elucidating the molecular mechnisms of signal transduction and its regulation by C a : + / C a M in neuronal cell activities. Materials and M e t h o d s
Materials I8-'~H]cAMP (20-311 C i / m m o l ) was purchased from the Radiochemical Centre, Amersham. [a-'~2P]ATP (80fl C i / m m o l ) and [?-32p]ATP (32 C i / m m o l ) were from New England Nuclear. The scintillation counting fluid, ACS I1, was a product ~f Amersham and Scintosol of Dojin Kagaku Co. (Tokyo). NaeATP, cAMP, Gql?+ lorskolin, D'IT, theophylline, creatine pb,osphate, r:GTA, (_+)-isoproterenol hydrochlorid¢, snake venom (Crotalus alrox) and ueutral alumina were obtained from Sigma. Creatine phosphokinase was from Boehringer-Mannhcim+ Nitrocellubse filters of pore size, 0.45 u.m were from Avantec Toyo Co. (Tokyo} and those of pore size 0.1 tzm from Schleicker & Schue!l (Da~el). Dowex resin ion-exchanger (AG 5 0 w - x g , 200-400 mesh) was fr.~ma the Bio-Rad. lmmunostaining kit (Immunostaining H R P kit IS-50B) was purchased from Konica Corp. (Tokyo). Solubiliza-
lion detergent kit (Dotite) which was composed of CHAPS, CHAPSO, MEGA-8, MEGA-9+ MEGA-10, tt-¢,etyl-fl-o-glucoside, tt-octyl-19ro-thio-glucoside and n-heptyl-/3-o-thioglucoside was from Wako Junyaku (Osaka). All other chemicals were of reagent grade. Autoradiography was performed on X-ray film (Fuji AIF, Fuji Color Co., Tokyo). Male Sprague-Dawley rat (125-150 g), C3FI mice (30-5t1 g, either sex) and male New Zealand White rabbit (2.0 kg) were obtained from Shizuoka Laboratory Animal Center (Hamamatsu). Porcine brain was from a local slaughterhouse.
Protein .~01¢1T.¢ CaM was purified from pot'cine hrain as previously described [2(I], 1251-CAM (92.5 I.*Ci/~g, bovine brain) was from New England Nuclear. Bovine brain CaM, bovine serum albumin (BSA), monoclonal antl-r-factor and anti-/3-tubulin mouse aseites fluid were purchased from Sigma, Anti-ff-ealspectin and anti-ealdesmonl50 monoclonal antibodies were generous gifts from Nippon Shin-yaku Co. (Kyoto). Anti-calcineurin antibody was raised in rabbit with bovine brain caleineurin (Sigma) as an antigen according to the immunization procedures described by Yakoyama et al. [14]. lmmunoglobulins fracfionated by 5(1% (NH4)2SO 4saturation [211] was used for Western blot analysis for ealeineuriu. Ho~eradish peroxidase-eonjugated antirabbit and anti-mouse IgGs were from Cappel laboratories. Protein kinase inhibitor, H-8, was obtained from Seikagaku Kogyo (Tokyo) and CaM antagonist, W-7, from MBL Co. (Nagoya).
Preparation of membrane and partial purification of membrane-bound CaMBPs Brain regions such as cerebral cortex, striata and cerebellum were dissected from rat or porcine brain. EGTA-washed lysed SM was prepared by a modified method of Maloo~ et at. [21] as described in detail previously [2{I]. Briefly, the isolated tissues were homogenized in medium A (2 mM EGTA, 50 /~g/'ml PMSF, 0+2 /~g/ml leupeptin, 0.5 mM D T f , 5 mM Tris-HCI, pH 7.5) containing 0.32 M sucrose (medium A') and centrifuged at 1000 x g for 20 rain, The supernatant was further centrifuged at 10000 × g for 15 rain. The supernatant was designated 'S z+ fraction. The pellet was suspended in medium ,V, diluted with medium B (medium A at pH 8.11, an1 centrifuged at 27 000 × g for 30 rain. The pellet was fesuspended and centrifuged at 27000×g for 30 rain. The final pelle~ ('P4') was suspended in a medium appropriate for mt individual experiment, usually, in medium C ' (60 mM Tris-HC~ (pH 7,5), 5.0 mM MgSO4, 0 5 aIM DTI'), and stored E.t - 80 ° C. SMs referred to as 'non-treated" and 'Ca-'+-lreated" SMs were prepared in the absence of E G T A and in the presence of 100 .ttM CaCI z, respectively, but otherwise
in the same manner as EGTA-washcd SM. For rcconstitution of EGTA-washed SM with Ca ~ ~/CaM. the membrane (I .5 m g / m l pr( g h~r 20 rain and the pellet suspended in the medium containing 100 ~ M CaCI,. "Partially purified CaMBPs' were first solnbilizcd by incubating $M at 4 ~ C for 2 h in medium C' containing 50 /zg/ml PMSF, 0.5% (v/v) Triton X-Ill0 and i).1% (w/v) detergent kit (Dottle). After cenlrifugation at 27{Jl10×g for 30 rain, the supernatant was dialysed overnight against a sufficient w~tume ef medium C ' containing 50 /~g/mi PMSF and 100 # M CaCI~ (medium D). The sample was recentrlfuged at 2701)0 × g for 30 rain and the supernatant was applied on a CNBr-activated Sepharose 4B-CaM-affinity column (1 X4.5 cm) equilibrated with medium D at 4°C. CaMBPs were elated from the column with medium D containing 1.0 mM EGTA. The MVM and BLM from mouse intestinal mucosa were prepared as described previously [22]. "['he final pellets were suspended in redistilled water and stored at - 8(I ° C until use. The rabbit RBM was prepared by the method described by Stock (23]. Briefly, 1 ml of packed e r ~ t h m ~ t e s was mixed with 40 ml of ice-cold 5 mM sodium phosphate buffer (pH 8.0) and centrifuged at 201)OOxg for 15 min. The loose sediment was collected, resuspended and recentrifuged three times. The final ghosts were suspended in 20 mM sodium phosphate buffer (pH 7.5) and :ilored at - S 0 ° C.
lmmunoeleciron-microscopy of ('aM The precise t~chniques wilt be described in detail elsewhere (H. Obta, unpublished data). In brief, small pieces (] × 5 mm) of membrane filter for blot analysis were immersed at 4 ° C for 2 h in the suspension of freshly prepared SM (1.3 mg/ml). After being incubated for I h in a blocking buffer (1% BSA in PBS), the fillers were incubated w~!h anti-CaM antibod.u (1 /~g/ml) at 4 ° C for 3 h. The filter paper was further treated with protein A-go!d prepared according to Frens [25] and Geoghegan and Ackermar~ [26], fixed in glutaraldebyde and osmium tr~troxid¢, embedded in LR white resin, and examined under glee!run m~crt~scope (JEOL, JEM-1OO C).
~'~Caauzoradiograp~y Ca2+-binding proteins including CaM in S 2 and P~ fractions from three differently prepared SMs were estimated by 4sea-binding on the nitrocellulose filtzrs [17]. After the samples and standard CaM were clectrophoresed in the Laemmli system (14% gel), they were eleetrobfotted to the nitrocellnlo~; membrane (pore size, 0.1 #m). it was immersed in a solution containing 61) mM KCI, 5 mM MgSO 4 and l0 ;aM
imidazolc-HCl (pH f>.~), incubated in the same medium contaioin~ ~ C a (4 ~ C i / m l ) for 5 rain and rinsed ~,ith 5(v/~ ethanol several times. After drying the autoradiogr,phy or, X-r~y film was performed for 24 h.
P:'I-CaM gel ot'erlay ':51-CAM gel uvcrtay was performed by a modified pr~ccOnrc of Hertzberg et el. [28]. The membrane preparations (2 3 mg protein/roll were mixed with I / 3 vo[. tff the buffer consisting of 125 mM Tris-HCI (pH 6.6), 3% (w/v) 5DS, 10% (w/v) glycerol and 5% (v/v} 2-mercapt~ethanol. 15 to 30 tzg pro',cin of each membrane was immediately subjecled to SD$-PAGE {5.5%. 8~,~, 11)~,~, t25t gels) in the Laemmli system I24J wihout prior heat denaturation and the electrophoresi~ performed at room temperature for 45 to 9.t) min. The gels were fixed with ll)O ml 25% (v/v) ist~propanol-lil% (v/v) acetic acid which w~.s exchanged three times for fresh one in the course of 3 b, then washed three times with l{~0 ml medium E (50 mM Tris*HCI tpH 7.5), 0.2 M NaCI) in the course o[ 3 h. "T.12e gels were incubated fur 2 h in 10{I ml medium E containing 1 m g / m l BSA to bk~ck non-snecific binding, then incubated overnight in 12 ml medium E containing 0.02% NAN,, 10 nM 1_~5l-CaM ( 1.8 - 10" c p m / m l ) and either IUO ,aM CaCI., or 1.1) mM EGTA. They were washed three times with 100 m! of the same medium without ~Z~l-CaM in the course of 3 h, then stained with 0.1% (w/v) Coomassie brilliant blue R-250 in 10% (v/v) acetic acid-50% (v/v) methanol for 15 min, and destained with 5% methanol9% acetic acid (or 1 h. They were dried and auloradiographed on X-ray films for 24 !o 811 h with enhancing screen (Dupont Cronex Lightning-Plus HK). The developed X-ray films were seamted densitometrically at 530 am.
Assay of (No ~,K ")-ATPase (Na÷,K~)-ATPase activity was measured as described previously [29]. The final assay mixture contained 60 mM imidazole-HCl (pH 7,5), 5,G mM MgSO4, 140 mM NaCI, t4 mM KC1, 0.5 mM EDTA and 1.0 mM ATP. The inorganic phosphate released was determined colorimetricalty.
Asxay of adenylate cyclase The adeny[ale cyelase was assayed by the method of Liana; and Sacktor [30] with some modifications [6]. A 50 ~1 reaction mixh~re contained, it, medium A, 40 p.M D'Iq', 1.0 m M [~zP]ATP (1 p.Ci per assay), 2.1) mM cAMP, 2.0 m M '~hcophylline, 20 mM ereatioe phosphate, 25 ~ g creztlne phosphokinase, brain corticai SM (25-30 ~ g protein per a~say), 1.0 m M EGTA, 0.924 mM CaCI 2 (pCa 6.2} and, when indicated, 10 IzMGTP, 10 p.M tbrs~:Jlin, 0.fi tzM CaM, Ill ~tM isoprolercnol. The mixtures were incubated at 3 0 ° C for 10 rain and the reaction stopped by adding 150 p.I of the solution
196 containing 5 mM Tr[s-HCI (pH 7.5), ,g) mM A'I'P. 2.1} ml~ [3H]cAMP (25 nCi/assay) and 2% (w/v) SDS, followed by heating at IOO °C fi)r 3 rain. After (E8 ml of 5 mM Tris-HCI (p[[ 7.5), was added to Lhc *r:i~.~,aae, cAMP formed was recovered by the double-column procedure or1 Dowex resin and neutral alumina. The radioactivity in Ihe'eolumn eluents (4.3 ml each) ia 1).1 M imidazole-HCI (pH 7.5), was measured in 12 ml of ScintisoI by liquid scintillation spectrometry. The recoveTy of cAMP was 75-85%. Deterr~ina|ions were performed in triplicate.
Assay of phosphodiesterase (PDE) The activity of cAMP-PDE in the cortical SM was measured by a modified procedure of Hidaka and Endo I31]. The final reaction mixture contained, medium A, t).5 mM Dq"T, 0.2 rag/rid BSA, 4(I tzM [~H]cAMP (0.5 ¢tCi/assay), 0,6/xM CaM, 5 5 / t g membrane protein, either 1.0 mM EGTA or 1.0 mM EGTA plus 0.911 mM CaCI~ (pCa 6.2), in a total volume of 500 ~zt. The reaction was started by adding [3H]cAMP to the mixture, which was incubated at 30QC for 12 mla afsd stopped by placing the tubes in a boiling w-'_,ter i~ath for 3 rain, followed by cooling on ice. After the addition of 50 ~1 of snake venom (1 mg/ml), the reaction mixture was further incubated at 30'~C for 10 rain, then applied to a Dt~wex resin column. Washing the column with 20 ml of distilled water, adenosine derived from 5'-AMP was eluted with 4.5 ml of 3 M NH4OH and collected in a scinti[latinn vial. The radioactivity in the column eluent was determined in 10 ml of ACS 11 by liquid scintiliation spectrometry. Values were based on triplicate determinations.
t'hospt~orylaHon The reaction mixhtre contained 60 mM Tr:,a-HC1 (p1| 7.5), 5.1) mM MgSO~, 0.5 mM DTT, 100 fzM [T-~2P]ATP (1.0 Ci/mmol), 110-125 p,g protein of .cnt'rical or eetebellar SMs, 0.053-1 ;tM CaM, 1.0 mM EGTA, varying concentrations of CaCI 2 and other reagents in a total volume of 100/xl, The reaction at 30 ° C was initiated by adding 30 htl of ice-cold SM. 10 t.tl of the reaetior, mixture was recovered al 0, 1, 3, 5, 1D, 30 and 611 rain and mixed with 25 p.l of the stoppi,, 3 solution containing 62.5 mM Tris-HCI (pH 6.6), 2.3% SDS, 5% 2-mercaptoethanol, 10% glycerol, followed by beating at !00~'C for 2 min. A 20 /zl aliquot of each sample was subjected to SDS-PAGE (10% gel) in the Lacmmli system. The get was stained, destained, dried and autoradiographed on X-ray film overnight. The film was scanned densitometfieally lot the pho-~phorylated bands. In some experiments, the d e c trophore~';ed bands were quantitated by Bid-image Ann-
Iyzer BAS 20l]0 (Fuji Film Co., Tokyo) before thc auto~adiography was performed.
bnmunoblot assay.~for CaMBPs and cy;o.s'keletat compolmmunoblot assays for CaMBPs and cytoskeletal .~.omponents were performed in the same manner as described prev!ously for CaM [2(I] except that SDSPAGE was carried out in 5.5% (w/v) gel for calspectin and caldesm,~nl.~l, in 10% (w/v) ge] for/3-tubulin and r-factor, and in 14% (w/v) gel for ealcineurin, in the Laemmli system [24]. Besides, a longer period of eleetrophoreite transfer, i,e., overnight, at 4 ° C , was employed without fixative. The SDS-PAGE for cateineurin was performed without heat-denaluration. The blotted bands were st~tined with immunostaining kit.
Miscellaneo'.e~" procedures The concentration of free Ca 2÷ in EGTA buffizring system was calculated on the computer program by the method of Fablato and Fablato [32] modified after Tsien and Rink [33] using the binding constants of Sehwarzenbach et al. [34]. Protein content was determined by tt, c methed of Lowry e~ al. [35] using BSA as standard. Results
Immunoeleclron-microscopy fo~ CaM in cortical SM preparation The repeated EGTA-wash deprived the membrane preparation of major portion of endogenous CaM [20]. To visualize this depletion and examine the possible effect of the presence of Ca 2+ ions during preparation, we prepared the control, i.e., non-treated, and the Ca2+-treated cerebral-cortical $Ms be~ides the E G T A washed SM as described in Materials and Methods and performed immunoelectron-mieroscopy on lhese membranes (Fig. I). A demonstration of gold particles aleng the membrane structure (Fig. IA, arrows) presumably revealed the pre.~enee of membrane-bound CaM molecules in the EGTA-washed SM. No such gold particles were detected with the preimmune-serum-treated cortical specimen (data not shown). The density of demonstrated gold particles increased as the membranes were prepared in the absence of E G T A (Fig IB) and in the presence of 160 taM CaCIz (Fig. 1C). The density increased in the above order. The greatest density of gold particles was t~bserved for lhe sample incubated in the presence of 4.5 g M added CaM and lll~ # M CaCI_, (Fig. ID). The preliminary ~H-CaM bindil~g experiment had shown that CaM-binding sites of the EGTA-washed SM was" saturated at .4.5 p.M of CaM (data not shown).
197
Ca ~ + bindi~o,, proleins in three dif.l~'reutl)' prepared SM~" In order to compare the Ca=M conk~nt of lhc tl]rce differently prepared SMs (see above):~Ca-autorudio~araph~' was performed for both the :hree mcn, branes and the corresponding eytosol fraeti,,,ns (Pig. 2). The S, supernatant (see Materi.".l~ and Meth~ds) w~s -'t~sume~J to he reoresentalive of cylosol fraction on ~hc ground that approx. 92% of total solub!c protein was rec(wered in thk fraction. The ratio of membrane-bound to soluble CaM wa~ much lower ir~ the EG l~A-treatcd SM (Fig. 2, lanes 2 / 5 ) than in the other tw~ SMs (lanes 3 / 6 and 4 / 7 k This finding confirmed the depiction of endogenous CaM and suggested its presumed translocation to the soluble fraction on EGTA Ireatmc,t. Other Ca-'+-binding proteins (28, 5¢1 and 85 kDa) were also detected in the soluble fraction. The 2,", and 5(I
''L.
"k
f o. 5pm
Dem~,~i~ati,,,, ,,]' ('a '~ ~-drpende~,t/ ~o;d -independent ('aMBI~" i~ three dijOn,really prepared SMs CaM binding protcin:, of the three SM preparation~ were demonstrated using "-~f-CaM gel overlay method tFig 3). The apparent molecular marx ~f detectable CaMBPs in brain SMs was 235, 't35/127, 58, 40, 34. 31.5. 24. 22.5 and that (or tht~se) lower than 20 kDa (see als~ Fig. 6)_ Of these, the smaller components ( < 3 4 kDa) showed a Ca-'+-independent or EGTAstimulated CaM binding (rig. 3, compare A and B). In ccntr~,l. CaM ~findlng by the bigger components (49 to 235 kD=l) ; v t l s entirely Ca-"-dependent (compare A
%
)
i)
L.
\
,x
r"
41'
C
kDa components were speculated to be calbindin,t) an6 ~:d)ulin. respectively [17] ialso see Fig. 8b).
D q'
lb ,J
I,."
4P .t ~,
/ 1
B .~P
11
t
Q
Fig. t. C~M molto,ales i n the E G T A - w a ~ h e d cortical S M (Lmmuaoele¢lftm-mictograph). O r i g i n a l magn;ticalion: ×3U(I(Rh E G T A - w a s h c d ¢or:.ical S M (A), non-treal~.d Sial ( B ) and 1110 taM Ca-'*-treattzd SNI (,g) were ~.':.¢d on fih~:r p a p e r mutrices and treated with ~mti-CaM antibody. E G T A - w a s h e d S M was in¢ubaled w i l h 4.5 taM C~M in Ihe ~,fes*gcc¢ of l ( ) n / a M C.:I-'" ;.ir 3 0 ~ C for 211 rain and Ireate,:t as abtwc (D]. Arrows illdi¢ine t h e imlrluitoi'e;tClWC go]d pi*l'iic]us | i2 nan in diamctc't'l. F~.)r d*:t~.til~.~i¢¢ t,~xi.
198
. . . .
•
1
-85 50
>
~
-28
2 3 4
567
/
re-
Fie. 2. Ca '~ (~(:a)-hinding proteins in variously prepared ¢orticaf SMs and noluhle S. [re,clions (pn?nradin~r:]ph)_ 4(] ~g protein [ff EGTA-wa.~hed cetrlneal SM (lane 2)~ non.treated SM (lane 3), Ca z% trc~;cd SM (lane 4) and the corre'~ponding {S~) fra=tlons (lanes 5, ¢, and 7, r~spccliv~ly} was et¢ctrophores~d as w~ll a~ 0.3 ~_$ of CaM standard (lane D. Ele¢lroblolling and ~Ca-autoradiography c,=rc, peefozmcd a~. dc.~cribud in Mulerial.~ cad Methods. The apparent molec.,hn" mass of major CaZ"-b[nding proteins were shown wllh reference to slai~dard CaM [ 17 kDa),
and B). specific The seemed
100
pea
The 24 and 22.5 k D a componentswere rathe~ t o the c e r e b e l l u m . absence of EGTA during preparation of SMs to c a u s e l i t t l e d i f f e r e n c e in C a M - b i n d i n g p a l -
terns (Fig. 3A and B, lanes ] and 2; lanes 4 and 5), whereas the presence of added Ca 2* ions resulted in a prefeeenttetial decrease in the relative araount of the smaller components ( ___34 kDa), especially, in the cerebellum-derived SM (Fi~. 3A, lane 6).
Effect.~ of added to Ca-' +/ CaM on some enzymic aclicities
A d e n y l a l c cyclase, Ca~+/CaM-dcpendcnt
phosphodiesterase (PDE) and p r o t e i n k i n a s e !1 ( C a M - k i n a ~ e
° ' ..,....
t
Fig. 4. Ca:'-concentratior, dependence of adenylate cyclase. Ca"~*/CaM-kinasc I1 and pho~phodieslera~e (PDE} in I:GTAwashed cortical SM. Th~ a¢lJvilles or adenylal~ cyclas~ (,a, ~,), Ca ~'/CaM-kina~ [] (a s~=b,~nlt)(Ill, D) and PDE (BI to. ~ l wcr~ e~limated in the ,ar;ous ¢or~c~r+~/ralJons of Ca z" with (a,. II. a} or w~thout ( A, ~a+o l [L6 #M CaM. Th= activity . f the first tw. unn~ymus is represented relalivc to thai al pCa 6.2 tadenylate ~.yclasc)and p(,~ 5.5 (CaM-kina.~¢ Ill, The dat;~ arc a representative of more than three independent expeiimcnts. PDE uctwity (B) is r~prescnted as mean _+S.D.
B
A ~
~
~ .
~
~
-'2#5~127
q m b / m ~.~.~ ~
1
2
3
4
qil$ m IlIB~
-24 -22.5
5
6
1
2
3
4
a, ma' .......
'.5
6
Fig. 3. CaMBPs in the three SM~ differently prepared from cerebral cortex and eerebc]luro (l~l-~utoradiogeaphJ. ~'le Ef;TA wlls.hed $Ms q]am:.~ I ~nd 4). n~o~-txe~ted SMs (lanes 2 and 5}. and Ca2%trealcd SMs (lanes 3 and 6) were prepared from cerebral eorte~: tl:me~. I-3) ann I-'~rebell,arn (lane~ 4-61 :=s described in Materials and Methods. It, ~g protein of each membrane was subjecled lo SDS-PAGE (129E) and n~"].CaM gel overlay. Tire lultcr was performed in the Orese.q¢c o[ either 100 ~M CaC]2 IA) or ) mM EG'I'A (B), followed by autoradk~graphy. The apparent m~Jl~ctdar raass t!f CaMISP~ ar~: indicated (also. Figs, 6 and 7), The dala are a representative of ,~ix u;q~rirnunts with lhre¢ dffferon! SM preparation;,,
19t~ It) w e r e assayed with the E G T A - w a s h e d mort,brahe p r e p a r e d f r o m c e r e b r a l cortex (Fig. 4). T h e a d c n y l a t c cyclase activity reecho-,1 m a x i m u m at p e a 6.2 -rid m a r k e d l y d e c r e a s e d at h i g h e r C a -~* c o n c c n t r a l i o n s ~Fig. 4A, triangles), while the a u t o p h o s o h o r y l a t i o n of C a M kinase i i w a s maximal ,~t pCa 5.5 (Fig. 4A, sqtlare'~). A l t h o u g h the P D E activity s h o w e d ~ slight p e a k b e t w e e n a c e 7 a n d 5 (Fig. 4B). Iberc was ntr significant d i f f e r e n c e in the activity in the p r c s c n c e or a b s e n c e of C a M over the C a ~'+ c o n c e n t r a t i o n s tested. It ~ a s conc l u d e d , t h e r e f o r e , t h a t E G T A - w a s h e d cortical SM contained, if any, very little C a M - d e p e n d e n t P D E isozyme ( f u r t h e r d a t a to s u p p o r t this c o n c l u s i o n are p r e s e n t e d below).
cAMP- and Ca 2 +/CaM-dependent phosphvryhtrion c A M P - d e p e n d e n t p h o s p h o r y l a t i o n of the possibie e n d o g e n o u s s u b s t r a t e s w a s tested with E G T A - w a s h e d cortical a n d c e r e b e l l a r SMs. T h e cortical SM w a s maximally p h o s p h o r y l a t e d 1 rain a f t e r initiation o f the reaction followed by r a p i d d e p h o s p h o r y l a t l o n (Table i). Fig. 5 (A, lane 2) shows a p r o t e i n kinase A-specific p h o s p h o r y l u t i o n o f the cortical SM. T h e 52 anti 73 k D a b a n d s a n d t h o s e of h i g h e r m o l e c u l a r magg w e r e a p p a r ently p h o s p h o r y l a t c d b y e n d o g e n o u s p r o t e i n kinase A.
T h e specificity of the r e a c t i o n was shown by its dec r e a s e d acti',ity e i t h e r in the a b s e n c e o f c A M P (lane 1 ) or in the p r e s e n c e of the specific inhibitor, H - g (lane 3). in the reaction mixture, R e p o r t e d l y , C a M - P I ) E is c o m p o s e d of two identical 60 k D a s u b a n i t s ~vbich a r e themselves C a M B P s , e a c h of which i n c o r p o r a t e s 2 reel of o h o s p h m e in c A M P - d e p e n d e n t m a n n e r [18]. T h e r e w a s no p r o m i n e n t c o m p o nent of the c o r r e s p o n d i n g size w h i c h was o h o s p h o r,/lated p r e f e r e n t i a l l y by p r o t e i n kinase A in the cortical SM (Fig. 5A. lane 2; B, lane I). A l t h o u g h the 52 k D a p r o t e i n b a n d (Fig. 5 A , lane 2; 13. lane 1, arrowb e a d ) s e e m e d to be an e n d o g e n o u s s u b s t r a t a of p r o t e i n kinasc A, it did not show C a M - b i n d i n g activity in the p r e s e n c e or a b s c u c e o f C a -'+ (Figs. 3 a n d 7). T h e c e r e b e l l a r SM s h o w e d a p h o s p h o r y l a t i o n p a t t e r n slightly d i f f e r e n t f r o m t h a t o f the cortical SM, especially, in the lower m o l e c u l a r mass r a n g e (Fig. 5B a n d C, c o m p a r e l a n e s I a n d 2). In the p r e s e n c e of C a - ' ~ / C a M t h e r e w a s a m a r k e d p h o s 0 h o r y l a t i o n of 49, 58 a n d 73 k D a b a n d s a n d s o m e h i g h e r m o l e c u l a r m a s s c o m p o n e n t s for b o t h the cortical a n d the c e r e b c l l a r S M s (Fig. 5C, l a n e s 1 a n d 2). T h e p h o s p h o r y l a t i o n w a s d e p r e s s e d in the p r e s e n c e of C a M a n t a g o n i s t , W-7 (Fig. 5C, lane 3), indicating the
T+~ 8LF_, I
,~ome t'ngvmiL"acticiriesof EGTA-wask.:d corticalSM Adetlylate ¢~,.iase, PDE and pmteln kina~ were assayed at pCa 6.2 (pH 7.51as described in Mate:ials and Method¢. Because the specific aclivily t]f adenylate cyclase was variable from preparation to preparation, values (means±S.D3 ~htmn were a rep:¢seatative of three separate preparatious, Figures in the parellthests are the numbers of separate: txptriment~. Statistical significance of cyclase activities ir~ Ihe:pre~enee and absence or CaM was estimated by paired t-test. The stimulated acl[vily is dtfr,ed ax the difference between the activity assayed [n If: presence of 10 ~M stimulant(s) IGTP, (_+)-isoproterenol and forskolin) in addilion to 0.6 AM CaM and that in the pro.seato of CaM a]orte, Phosphoqdation of the endogenous suhstrates (49/51 kDa and 51,;/~.1~'kDa) was c~timated ,~t t and 5 mix afler the ,.-'action war, ini,.iated. A prellralnary study had shown that maxinmm ph~phoolation was reached at I mix. Enzyme
Activity + CgM
- CaM 6,5+ ~6(0)
(Na +,K ~")-ATPase t
Adcnylase cy¢la~u ~
111,2_+ h_6(3}
basal * GTP-sl. * l~prolerenol-st. * Forskolin-st. Phosphodiesttrasu ~ Phosphowiatioo 4 (suhslrat,) 58 kDa 40 kDa
14b.3+ 40.2 2.~7.'-)+ "t3,2 1}5.6-1-13,[ 2186.6:1:61.3 10.9+ 0.3
98.2__. 17.4 31,0+ 1%9 q60.5: 62 9,9-- 0 3 (3)
- Ca"*
t miu
5 min
I mix
5 mi~
16.6 27.4
7.9 13,6
13.2 15.4
10.8 12.4
Units used: ~mal P i / h per mg protein. 2 pmQI cyclic A M P formcd/mln per rnE prote;n~ pmol cyclic A M P hydrolyzed/mi~ per m B pratt:in. 4 fmol PL incorporated/,u.g proleln.
* Values in the pl.csence and absence of C a M st.. stimulated.
w e r e statisticltliy
signiftcant bY p a r r e d
l-lc~| ( P < 0 J ) l : t l = 3).
20~J
cyelase either by CaM and GTP or by CaM and isoproterenoI in the presence of lO #M GTP which supported the activation resuhed in an aetivatiort more than additive. These findings are ~n agreement with those previously reported [6].
presence of CaM-dependent p r o l e i n kinasc i i in the brain SMs, The 49 kDa component ~a~ l~ho.,,ph~r,'la..'cd to a much lower level relat;ve to the 58 kDa component in the cerebellar than in the cortical SM (Fig. 5C, compare lanes 1 and 2), consistent with the lower level of tZ~l-CaM binding by the 49 kDa component relative to that of the 58 kDa component in the cerebellum-derived membrane (Fig. 3A, lanes 4 to 6).
Comparison of CaMBt~.~ o[ brah~ SM, and enterocyte and eryzhrocyte plasma membranes by means of t"'~l-CaM gel ocerlay
Synergistic actfi'ation of ade/zylate cyctase by isoproterenol and CaM, and s~me other enzymic properties of cortical SM
Fig. 6A(b) and B(bl shows the results ~f ~z~l-CaM gel overlay cxpcrhnc.nls with brain SMs and cnterocytc (MVM and BLM~ and erythrneyte (RBM) plasma membranes (see Fig. 7 for densitometric lracings). The rat brain membranes, cerebral-cortical, striatal and ccrebellar, seemed to share essentially the same CaMBPs. Only the 49 kDa ccmponent was much less conspicuous in the cerebellar preparation (Fig. 6B(b), lane "CER'; also, Fig. 3A, lanes 4 to O). Between rat and porcine cortical SMs slight differences were noticeable with regard to some CaM-bindlng compo-
Some enzymic aclivit[es of the cortical SM are summarized in Table I. The high (No *,K' )-ATPasc activity confirmed the plasma membrane na:ure of the present preparations. The activity of CaM-PDE was virtually absent. Several agents :,ucl= as CaM, GTP. forskolia and (+ ~it;~protcrcnol slimulated the adcnylate eydase at pea 6.2. Simultaneous stimulation of the adenylate
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Fig. ~.. cAMP- and C:~-~-/CaM-dependcn! pht)~ph~lation of EG"l'A-washed ~lrlicat and cercbeLlar SM~ (~-~P-autoradkP-raph). The ~.'AMF-dcpezndcnl (;~, awd B} anLLCa 2"/Cal'.¢-deDe~denl (C) pho~;phorylalionl reactions w~rv p~fform=d fur t rnia as de%'rihed in Materials and Me|hods
wilh mernha ,,..~ nrovcil~s as endogenous substralcri. CTX. EGTA-wa~hcd cortical; {'ER, ¢erebellar SM~. Experimental condilions arc bld~calcd in the pan,~L I ~ I IT,:;l~ cl]~ i'~clu.~ion of lhu ruagen'~ ill th~ r~aCliOn nli~ture. The ~pucificaity phosphor-clawed bands (armwhc:ads) and Celalb,'e rnolccuiar m~ls,~;arc indlc;dud.
2111 heats. The 711, 35 and 32 kDa comp,~nents, especially the last one. bound more CaM while the 24 kDa component bound much less CaM in the rat lhan in the porcine cerebral-cortical SM (Fig+ 6B(b). compare lanes "CTX' and "POR CTX'}. The 235+ 135 and 58 kDa c o m p o n e n t s whicl~ were clutable [tom a CaM-alfinity c o l u m n with I mM E G T A as described in Materials and Methods showed a Ca-~+-dcpcndent Cam-binding (Fig. 7(d): also, Fig. 3A and B). Interestingly, the 4 (k kDa c o m p o n e n t which was not recovered t'rerm the column with 1 mM E G T A (Fig. 7(d)) als¢) required Ca-'" ions for binding CaM i.FLg. 3A and B). Enterocytc p l a s m a membranes. BLM and MVM, had binding p a t t e r n s markedly different from those of brain SMs (Fig. 6A(b) and B(b), lanes "BLM" and ' M V M ' ; also, Fig. 7(e} and (f)). Two major CaMBPs, 5,R and 49 kDa components, of cerebral-cortical SM fotmd hardly any counterparts in the BLM and were entirely absent in the MVM, The 32 k D a component, one ¢}f the major C a M B P s in brain SMs, was not dclcc!cd in A
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t h e i n t e s t i n a l m e m b r a n e s , I n c o n t r a s t , a 105 k D a c o m p o n e n t was a d o m i n a n t C a m B P in t h e c n t e r o c y t c m e m branch, e~pccially, in t h e M V M (Fig. 6 A ( b ) , laae
-MVM': B(b). lane,; "r4LM" and *MVM'). Rcwa~ka+~ly. there was no 11)5 k l ) a C a M B P in ~he brain SMs. A m o n g t~lher C a M B P s peculiar to the intest ne a 36 kDa cumpo;~cnt was prcsen~ in the R I M and 88 kDa componen! in the MVM. I t should be nt~tic.:d that the L35 kDa compuncnt was the most or the next most promin¢~l. C a M P , P throughout the membr~ nes tesled except lor the MVM (Fig. 6(b) and Fig. 7).
Western hh~l ana/y,~ls +Jr C\~MBt~ ~and cytosk"letat co#~zp¢:ttelffa it! t'r)r/ica./ SM To identify the ( ' a M B P s and cytosketetal ,,:omponents in E(}TA-wa~hed SMs we perti~fmed Western blot analysis t~.;r calcincurin, ealdesmon =~=~,r-factor and /5-tuhnlin. The membrane proteins weakly l"ut posilivLly hnmuno-rt.:tctcd with anti-calcineurm tFig. 8a. lanes I =rid 3), anti-calspcctin (data not sh,~wn) and B
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Fig. ft, Prolcin and =-'~I.CuM gel ~]verlay p; P,filcs t)f brain and enletocyte mcmhranc~. SDS-PA.EiE v,as performed in 5.5% I A I a~d II1":~- (B) g¢l.~ in the Laemmll system. After =-'sI-CaM gel Lwurlay (b) was pcr[~lrm~:,.I in the pt¢-.cn¢c nf I;;'1/ ~ M CaCla 1he /~els were stained Ior prol~in (a). Detail~ are d=scribed in Matcrial,~ and M¢thod~. A ~ ~te. ~rotein :tliq,.mt ~:~s applied {tw EGTA-washed SM I-mat ra" ccrebc!lum leER). striatura (ST). cerebral cortex [f~q'X), pristine cerebral corti:x (POR-C'I'X) and basal-I;Rc~al IBLM) and micr~'iltuus (MVM) n~cmbranes of mouse cntero~yt¢ h~[cl.hc[ will'= molecul~x~,nass standards (SIT~). Arabic nurm.-,rals repr~.':-t:nt the appueent molecular ma~';
