isolation from rat diaphragm of a calcium-protein complex...

Isolation from Rat Diaphragmof a Calcium-Protein ComplexInvolved in Excitation-Contraction CouplingBy Stephen Hajdu, Christian J. Posner, and Edward J. Leonard

ABSTRACTRat diaphragms depleted of calcium involved in excitation-contraction (E-C

calcium) coupling were tagged with 4BCa. Seventy percent of the 45Ca wasextracted by a 25 HIM NaHCOs-50% glycerol solution. Treatment of themuscles with caffeine, shown previously to release E-C calcium, decreasedextractable 45Ca to about one-fourth of that of control muscles, indicating thata large fraction of the extracted 45Ca is involved in E-C coupling. On gelnitration, only lOSfc of the extracted 45Ca was bound to protein. This figureincreased to 30% when procaine (which stabilizes E-C calcium) was present inthe extraction solution; total extracted 4BCa remained unchanged. The resultssuggest that E-C calcium is extracted from the muscle as a calcium-proteincomplex and is readily dissociated from the protein unless prevented in part byprocaine. Cadmium-115, which is shown to replace 4BCa at the sites involved inE-C coupling, binds more strongly than 45Ca. When 11BCd-labeled muscleswere extracted with glycerol solution containing procaine, all the extracted11BCd was in protein-bound form. One of the extracted proteins formed aprecipitin line which was imrnunochemically identical with rat plasmacardioglobulin-C when tested in gel against an anu-cardioglobulin-C antiserum.This supports our hypothesis that cardioglobulin is the circulating form of a cellmembrane calcium transport system.

KEY WORDS calcium cadmiumcalcium-binding protein cardioglobulin

glycerol extraction procainecalcium transport system

• Cardioglobulin-C, the calcium-containingmember of the cardioglobulin system, hasbeen suggested as the source of calciuminvolved in the excitation-contraction (E-C)process in mammalian skeletal muscles and inthe potentiation of contraction in certainmammalian cardiac muscles (1).

In this paper, we report the extraction of acalcium complex from rat skeletal muscle,present evidence suggesting that the complextakes part in E-C coupling, and show that onecomponent of the extract is antigenicallyrelated to rat cardioglobulin-C.

From the Laboratory of Kidney and ElectrolyteMetabolism, National Heart and Lung Institute andthe Biology Branch, National Cancer Institute,Bethesda, Maryland 20014.

Received May 20, 1971. Accepted for publicationAugust 5, 1971.

MethodsPREPARATION OF MUSCLE FOR EXTRACTION

Although the cardioglobulin-C of differentspecies is functionally interchangeable, the chem-ical characteristics of each are different. Forexample, gel filtration on Sephadex G-200 showedthat human cardioglobulin-C is much smallerthan rat cardioglobulin-C (2). Since mostprevious work has been done on rat cardioglob-ulin-C (3), diaphragms excised from femaleSprague-Dawley rats (130-160 g) were used forthe extraction. The diaphragms were depleted ofE-C coupling calcium by 2—4 hours of constantstimulation in Ca-free Krebs solution. Suchmuscles became unexcitable to electrical orchemical stimulation. Addition of calcium to thebathing solution soon restored the muscle to fullactivity (1). A similar but faster depletion ofcalcium could be achieved if contracture wasinduced by the addition of caffeine (5 mg/ml ormore) in a Ca-free medium (4). After thespontaneous disappearance of contracture, whichoccurred after 20-60 minutes, the muscle wasunexcitable, but elimination of the caffeine and

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CALCIUM-PROTEIN COMPLEX OF E-C COUPLING 359

addition of calcium restored the contractility ofthe muscle. In the experiments described in thispaper, 45Ca (5-20 fic/ml in Ca-free Krebssolution) was added to the depleted muscle. Inthis way, high specific activity was achieved, butlarge quantities of radioactive material were nothandled. Because of the low total concentration ofcalcium in the solution, the calcium stores of themuscle were only partially replaced during aperiod of 20-25 minutes. This was reflected in thereturn of only 10-20$ of the original contractility.For complete restoration of contractility, wewould have had to handle 10—20 times moreisotope or accept a lower specific activity. Asimilar approach was used when the depletedmuscle was equilibrated with 11BCd. The taggedmuscles were washed for 50 minutes with Ca-freeKrebs solution before extraction was begun.

EXTRACTION

The presence of calcium or any of itssubstitutes is essential during extraction as amarker for the protein. In a previous study, theeffect of temperature, ions, and drugs on thestrength of the bond between calcium and thebinding protein was described (4). Low tempera-ture, low monovalent cation concentration, lowcalcium concentration, and pH greater than 8.0provided the strongest bond. To satisfy theseconditions, cold 25 mM NaHCOB was selected asthe basic extraction solution. Once a solution wasdesigned to stabilize the calcium-protein complex,it had to be modified to allow release of thiscomplex from the cell structure. For this purpose,a solution of 25 mM NaHCO3 in 50$ glycerol wasused, This allowed the solution to dissolve thephospholipid and cholesterol of the muscle mem-brane and to facilitate the release of the protein.The pH of the extraction solution was 8.7. Whenrequired, a more alkaline pH was achieved by re-placement of NaHCO3 with an equivalent amountof NaOH. Muscles tagged and ready for extrac-tion were cooled to 0°C with ice-cold Ca-freeKrebs solution for 5 minutes while still in the mus-cle bath (4), and then the bath contents werereplaced with ice-cold extraction solution foranother 20-30 minutes. Muscles were cut awayfrom their rib and tendon attachment, quickly putinto cold extraction solution, and taken into thecold room (4°C). They were blotted dry,weighed on a torsion balance, and homogenizedin a hand-operated glass tissue grinder (TenBroeck, 15 ml) for about 3 minutes with 1 ml ofextraction solution for every 20 mg of muscle.After removal of a 0.2-ml sample for counting, theremainder of the homogenate was centrifuged at48,000 g for 30 minutes. The liquid phasebetween the pellet on the bottom and a whitelipid layer floating on the top was separated with

Circulation Retard, Vol. XXIX, October 1971

a Pasteur pipette. The extract contained approxi-mately 1-3 X 106 count/ min ml-1. Extracts werestored in this form at —20°C without noticeablechemical change.

LARGE-SCALE EXTRACTION FOR IMMUNOCHEMICALSTUDIES

Ten grams of fresh ice-cold rat psoas and thighmuscle were ground in a Latapie tissue grinder(micro model, 15 ml). Eight grams of the groundmuscle were suspended in 160 ml of Ca-freeKrebs solution, stirred for 5 minutes, and thencentrifuged for 10 minutes at 48,000 g. Thesupernatant fluid was discarded and the musclepellet resuspended and treated as above. Theprocedure was repeated a third time, but thefinal centrifugation was for 30 minutes instead of10. The muscle pellet was suspended in 80 ml ofthe NaHCO8-glycerol solution and homogenizedin a glass tissue grinder in eight separate portions.The pooled homogenate was stirred for 30minutes and centrifuged for 50 minutes at 48,000g. The glycerol extract was separated from themuscle pellet and the lipid pellicle. Before gelfiltration, a 115Cd-tagged diaphragm extract,obtained as described above, was added as amarker, so that the whole extract containedapproximately 10* count/min imH. The proteinsof this crude glycerol extract were purified byfollowing the 11BCd marker, first on Sephadex G-200 columns. The 11BCd peak was cut andreconcentrated to the original volume by ultrafil-tration. This reconcentrated material was thenrechromatographed on Sepharose 2B columns,after which the 115Cd tag appeared in twodistinct peaks (Fig. 6). Although immunediffusion analysis detected a small amount ofcardioglobulin-C in the first peak (fraction 1),the titer was considerably higher in the secondpeak (fraction 2) and this fraction was thereforeused for the experiments.

CHROMATOGRAPHY

Sephadex G-20, G-50, and G-200 and Sepha-rose 2B were prepared according to the instruc-tions of the manufacturer (Pharmacia FineChemicals, Inc., Piscataway, N. J.). The finalequilibration of the gels was done in extractionsolution. Glass columns (2—4 cm i.d.) were used.The gel rested on Teflon felt (Armalon, Du Pont)and the same kind of felt was used to protect thetop of the column. The settled length of the gelvaried between 25 and 30 cm. The same solutionused for extraction was used for elution. BlueDextran 2000 (Pharmacia Fine Chemicals), 10mg/ml, was dissolved in 50S glycerol in a glasstissue grinder and centrifuged (48,000 g, 30minutes). The supernatant fluid was used as amarker during chromatography with Sephadexand Sepharose gels.



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360 HAJDU, POSNER, LEONARD

IMMUNOCHEMICAL METHODS

Antiserum to rat cardioglobulin-C was pro-duced by two subcutaneous injections into rabbitsof a rat cardioglobulin-C fraction in completeFreund's adjuvant as previously described (3).The gel used for precipitin analysis was 0.075$agarose (Seakem Company) in 0 .15M NaClbuffered at pH 7.3 with 0 .005M potassiumphosphate buffer. Frog hearts with boundcardioglobulin were used as a specific immunoad-sorbent for anti-rat cardioglobulin-C antibody aspreviously described (3). In brief, a 1/5 dilutionof rabbit anti-rat cardioglobulin antiserum wasadded in succession to a series of 6—9 frog heartscontaining bound cardioglobulin-C. The equili-bration time in each heart was 15 minutes.Antiserum was added to control hearts by thesame procedure. The sera were then ultrafilteredin cellophane membranes and the final volumeswere adjusted so that the optical density at 280nm for experimental and control sera was equal,at a value close to that of the original serum.

ISOTOPES AND DRUGS

The 4CCa1 had a specific activity of 5.5-6.7mc/mg. The n BCd2 had a half-life of 43 days andan activity of 0.25-0.38 mc/mg. The pH of thestock solution of isotope was adjusted toapproximately 6.0 with 1.0N NaOH. For experi-ments involving efflux measurements, diaphragmswere prepared and tagged as described aboveexcept that they were washed with Ca-free Krebssolution for only 20 minutes after tagging beforethe actual sampling began. Collecting for theefflux measurements took place in 16 separate 5-ml plastic baths exchanged every 10 minutes (1).Radioactivity of the individual bath contents wasmeasured in a liquid scintillation spectrometer(Packard Tricarb Model 574). One ml of samplewas added to 10 ml of Bray's solution (5). Theefflux rate constant was calculated by dividing thenumber of counts appearing in the bath over thecollection period by the number of counts in themuscle at the beginning of the same period. Sincethe measurements were made over 10-minuteperiods, this number was divided by 10 to expressthe rate constant in the units of min-1.

ResultsEXTRACTION OF THE «Co COMPLEX FROM MUSCLE

Rat diaphragms were depleted of E-Ccalcium by constant stimulation in Ca-freeKrebs solution, repleted with 4BCa, and thenextracted with NaHCGvglycerol. In 19 mus-cles, 65 ± 1% (mean ± sr>) of the muscle 46Ca

was extracted. An additional 5% was obtainedby two more extractions. Thus about 70% ofthe muscle 45Ca is extractable by 50% glycerol.

To determine whether the 4SCa was bound,extracts were chromatographed on SephadexG-20 and G-50. It was found that 11 ±3% (12columns, 9 extracts) of the 46Ca was excludedfrom the gel. This 45Ca, therefore, is probablybound to a molecule of large size. Since theremain ing 89%, entered the G-20 gel, it isprobably free 45Ca.

These results raised two questions: (1) Doeseither the bound or the free extracted calciumrepresent E-C calcium? (2) Did the free cal-cium exist as such in the muscle or was it re-leased from a complex during the extraction?The following experiments were designed toanswer these questions.

EXTRACTION OF THE "Co COMPLEXFROM CAFFEINE-TREATED MUSCLE

Since both caffeine (4, 6, 7) and ryanodine(lot no. 6259F)3 (1) release E-C calcium, theeffect of these drugs on glycerol-extractablecalcium was studied. Six pairs of 46Ca-labeledmuscles were used. The control member ofeach pair was equilibrated for 50 minutes inCa-free Krebs solution; the experimentalmuscle was exposed to an identical solutioncontaining either 5 mg/ml caffeine (threemuscles) or 5 /Ag/ml ryanodine (three mus-cles). The drug concentrations were selectedto achieve partial depletion of the E-Ccalcium, so that a diminished but measurableamount would remain. The glycerol extract ofthe caffeine-treated muscles contained 28% (26,26, 33%) of the 4BCa found in the pairedcontrol; the figure for the ryanodine muscleswas 44% (40, 44, 48%). The results of gelfiltration of the extracts on Sephadex G-50 areshown in Figure 1. Nine percent of the 45Ca inthe control muscle extract is excluded from thegel and appears as the first sharp peak. Theremainder is eluted in a second peak, whichrepresents the unbound calcium. The dottedcurve, obtained from the caffeine-treatedmuscles, shows that the fraction of bound andfree (first and second peaks, respectively)

1 Supplied by Union Carbide Corporation.2Supplied by New England Nuclear Corporation.

sSupplied by S. B. Penick and Company, NewYork.

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6

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e 4X

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-

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T30 60 90 120

ELUTION VOLUME m150 180

FIGURE 1

Elution patterns of *lCa obtained during gel filtrationon Sephadex G-SO of procaine-glycerol extracts (solidmuscles pretreated with caffeine (broken line) andcontrols without caffeine (solid line). Solution used forelution was identical with extraction medium. Tem-perature 4°C. Percents are the average of 6 extracts± SD.

for E-C coupling (9). Experiments were doneto see if this calcium-stabilizing action wasreflected in an altered ratio of bound to freecalcium when procaine4 was added to theglycerol extraction solution. Six 4BCa-taggeddiaphragm pairs were tested, the experimentalmember of each pair being extracted andchromatographed on Sephadex G-50 with aglycerol solution containing 20 HIM procaine.Figure 2 shows that the bound calcium in theprocaine extracts is about three times that ofcontrol. Thus the reported action of procainein stabilizing E-C calcium is reflected in ahigher fraction of the extracted calciumcomplex remaining in bound form.

REPLACEMENT OF E-C CALCIUM BY CADMIUM

Further evidence that the native form ofE-C calcium is bound was obtained by studieswith 115Cd. Three conclusions were drawn.(1) In a muscle depleted of E-C calcium,cadmium binds to the calcium binding sites.(2) Whereas ryanodine releases calcium fromE-C sites in the intact muscle, no release of

calcium is the same as for the controls. Twotentative conclusions were drawn from thisexperiment: (1) The glycerol was probablyextracting a complex involved in E-C cou-pling, since two different drugs which releasecalcium from the E-C system resulted in adecrease in the extracted calcium. (2) Sincethe ratio of first to second peak calcium in theextracts was the same for drug-treated andcontrol muscles, the only difference being thetotal amount of calcium, it appeared likelythat the calcium of both peaks was involved inE-C coupling. This finding also suggested thepossibility that one form of 45Ca is thenaturally occurring one and that the other is aconsequence of extraction or chromatography.If calcium could be stabilized in its originalform, one form might increase at the expenseof the other. Therefore the following experi-ments were done.

STABILIZATION OF THE CALCIUM COMPLEXBY PROCAINE

Local anesthetics antagonize the effects ofcaffeine (8) by stabilizing the calcium needed

4Supplied by Matheson, Coleman and Bell Co.

b 4

II 6*3-5%

-r—'30 60 90 120 150 180 210

ELUTION VOLUME ai

FIGURE 2

Elution diagrams of i}Ca obtained during gel filtrationon Sephadex G-50 of procaine-glycerol extracts (solidline) compared with NaHCOs + NaOH-glycerol(broken line); pH of both extracts was 9.7. Solutionsused for elution were identical with extraction media.Temperature 4°C. Percents are the average of sixprocaine and eight control extracts ± SD.

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0.8

0.6

0.4

0.2

i—r -| r

- a**PRETREATED

Ryonodine

30 50 70 90

MINUTES

FIGURE 3

HO 130 150

Effect of ryanodine (5 tig/ml) on ]>sCa efflux into Ca-free Krebs solution from rat diaphragmsexposed to 2.5 min cadmium before *5Ca tagging. Open circles, cadmium pretreated (5muscles); closed circles, controls (6 muscles). Temperature 23.5°C.

cadmium by ryanodine was observed.(3) Cadmium appears to bind more stronglythan calcium to E-C sites, and a high ratio ofbound to free cadmium in chromatographedglycerol extracts confirmed this observation.

Evidence that cadmium binds to the cal-cium E-C sites was obtained from fivediaphragms depleted of calcium and thenrepleted by bathing for 20 minutes in Krebssolution in which calcium was replaced byequimolar cadmium. After the muscles werewashed free of cadmium in Ca-free Krebssolution and labeled with 4SCa, efflux meas-urements were made. The efflux of controlmuscles, which were not exposed to cadmiumprior to 4BCa tagging, is shown in the topcurve of Figure 3. Addition of ryanodine at 60minutes causes a characteristic increasedrelease of calcium. This does not occur in thecadmium-treated muscles, suggesting thatcadmium blocked uptake of 4BCa by theryanodine-sensitive sites, presumably by bind-ing strongly to those sites. Furthermore, whenthe E-C calcium of four muscles was depletedand then repleted with 116Cd, efflux of 115Cdwas not increased by ryanodine (Fig. 4).

Additional evidence that cadmium wasbinding to E-C calcium sites was obtainedwith seven pairs of diaphragms. One member

of each pair was slightly depleted of calciumby a 50-minute period of stimulation onceevery 10 seconds in Ca-free medium, the othermember was profoundly depleted over thesame period of time by caffeine (5 mg/ml) inCa-free medium. The muscles were thenwashed for 10 minutes in Ca-free Krebssolution, exposed to llBCd, and extracted withglycerol. The seven control muscle extractshad a mean of 58,000 count/min mg-1. Thecaffeine-treated mean was 176,000 count/minmg-1 (t = 11.6, P<0.001), which shows thatmuscles in which a large part of the calciumwas released by caffeine took up three timesas much cadmium as the controls.

These experiments indicated that cadmiumcould occupy the E-C calcium sites. It wasthen shown that the glycerol-extractable cad-mium was quantitatively equal to the extract-able calcium. Seven diaphragm pairs weredepleted of calcium and then repleted inparallel with equal amounts of either 115Cd or4BCa (2.5 ^,c/ml 45Ca or 115Cd in Krebssolution containing 250 /XM calcium or cad-mium). Table 1 shows that amounts ofglycerol-extractable 115Cd and 4BCa are equal.(The whole muscle 115Cd counts are higherthan 45Ca counts, since 118Cd is apparently

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10 F ' i ' '

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Co++FREEKREBS SOL.

J L J L10 30 50 70 90

MINUTES

FIGURE 4

no 130 150

Effect of ryanodine (5 fig/ml) on lleCd efflux into Ca-free Krebs solution from rat diaphragms(aijerage of 4 muscles). No release of llsCd by the drug was observed, unlike results frommuscles tagged under similar conditions with isCa (see Fig. 3, control). Temperature 23.5°C.

bound by additional muscle sites not extract-able by glycerol.) Thus glycerol-extractablecadmium appears to be equivalent as a labelto glycerol-extractable calcium.

It was thus established that cadmium couldreplace glycerol-extractable calcium in a 1:1

I '

90 120 150

ELUTION VOLUME ml

FIGURE 5

Elution pattern of llsCd obtained during gel filtrationon Sephadex G-50 of NaHCOs-gUjcerol extracts ofmCd-tagged diaphragms (solid line). Broken lineindicates excluded volume as marked by Blue Dextran2000. Elution with NaHCOrglycerol. Temperature4°C.

CitcuUtion Research, Vol. XXIX, October 1971

ratio. The ryanodine experiments suggestedthat cadmium binds to the sites more stronglythan calcium. This was reflected in the resultsof chromatography of the cadmium glycerol-extracted cadmium. Figure 5 shows thatvirtually all the 11BCd is bound, being elutedfrom G-50 in the gel-exclusion peak. Figure 6shows results of chromatography on Sepharose2B, which allows larger molecules to enter thegel bed. About 70% of the total 115Cd forms awell-defined peak which entered the gel.About 15^ was excluded and another 15% wasrecovered as a very long tail. The major peakis probably the cadmium complex as it occursin the muscle, The first-peak material excludedby the gel is composed of either particles not

TABLE 1

Comparison of Uptake of aCa and lllCd by Calcium-Depleted Diaphragms and the ExlractabUity of TheseMarkers by Fifty Percent Glycerol

No. of musclesUptake by muscle

(count/min rag-1}Radioactivity in extract

(count/min mg-1)% extracted

7

3,875

2,37560.4

± 780

=*= 545± 4.5

7

8,863 ±

2,450 ±28.0 ±

1,130

2244.4

Values are means =*= SD.



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70 3 1 7 0 %

16 4+10 2%

60 90 210 240 270120 150 180

ELUTION VOLUME ml

FIGURE 6

Elution pattern of tzsCd obtained during gel filtration on Sepharose 2B of procaine-glycerolextracts of llsCd-tagged diaphragms. Elution with procaine-glycerol. Temperature 4°C.Percents are the average of 5 extracts ± SD.

brought down by centrifugation or of aggre-gates of second-peak material, the formationof which is common in other systems ofmacromolecules (10). The tail is probablycadrniurn that separated from the extractedcomplex (its amount was greater in extractsnot containing the stabilizing procaine) andcombined with a molecule which interactswith Sepharose, thus accounting for thedelayed elution. Inorganic 11BCd alone doesnot show this type of irregular behavior onSepharose. Thus the cadmium data supportthe notion stated earlier that glycerol extractsan unstable calcium macromolecule complexand that much of the calcium dissociates fromthe complex during extraction. The cadmiumcomplex is analogous, but much more stable.

ANTIGENIC RELATIONSHIP OF THE GLYCEROL-EXTRACTEDMATERIAL TO CARDIOGLOBULIN-C

We showed previously that there is acalcium transport protein system in rat plas-ma; one component of this system, calledcardioglobulin-C, is a calcium-protein com-plex. This plasma protein is bound to thelimiting membrane of certain cells, includingskeletal muscle (3). We therefore tried todetermine whether the glycerol-extracted cal-cium-protein complex of the muscle waschemically related to cardioglobulin-C.

Antiserum was raised in rabbits against ahigh molecular weight fraction of rat serum

proteins which included cardioglobulin-C (3).This will be called anti-C antiserum; itcontains an antibody against rat cardioglobu-lin-C as well as several other rat serumglobulins. The first step in the analysis isillustrated by the precipitin lines in Figure 7.

FIGURE 7

Photograph of immune precipitates from Ouchterlonydiffusion analysis demonstrating that line 3 (nearest toperipheral wells) identifies cardioglobulin-C. Centralwell contains whole rat serum. Peripheral well, dcontains antl-cardioglobvlin-C antiserum; the sameantiserum in well BC was absorbed by a series of froghearts which have rat cardioglobulin-C bound to themuscle membrane (note missing line 3); antiserum inwell C was absorbed similarly by frog hearts whichwere treated but did not retain cardioglobulin-C (fordetails see text).

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FIGURE 8

Immunodiffusion demonstrating the presence of cardio-globulin-C in the purified glycerol extract obtainedfrom rat skeletal muscle. Central well filled with anti-cardioglobulin-C antiserum. Peripheral wells contain:rat serum (1 o'clock), Sepharose 2B fraction 2 fromglycerol-extracted rat skeletal muscle (3 o'clock) andSephadex G-200 cardioglobulin-C fraction from ratserum (5 o'clock).

The central well contains whole rat serum,diluted 1:20. The peripheral well (d) containsanti-C antiserum. Three precipitin lines can beseen, which are numbered in order startingfrom the antigen well. The problem was toidentify the cardioglobulin-C precipitin line.This was accomplished by finding which linedisappeared when the antibody to cardioglob-ulin-C was specifically removed from theantiserum. The removal of this specific anti-body, which was described previously (3), isdone by equilibrating the antiserum with froghearts, which have rat cardioglobulin-C boundto the muscle membrane. If rat cardioglobulin-C is the only rat antigen bound to the frogheart, then antibody to this antigen alone willbe removed during equilibration with thehearts. When absorbed antiserum was placedin the peripheral well (BC in Fig. 7) line 3(the line closest to the antibody well in thecontrol, d) was missing from the resultantprecipitin pattern. From this evidence, cardio-globulin-C appears to be in precipitin line 3.Previous experiments (11) showed that thebinding of rat cardioglobulin-C to frog heart

CircttUlion Resutrcb, Vol. XXIX, October 1971

requires prior exposure to cardioglobulin-B,supplied in the form of diluted human plasma.When rat cardioglobulin-C is applied to froghearts without prior application of humanplasma and the hearts are then washed andused for absorption of antiserum, the ab-sorbed antiserum is still capable of developingthree precipitin lines. This is shown in Figure7; the absorbed antiserum was placed in thewell labeled C. Thus the frog heart is aneffective immunoabsorbent for the line 3antibody only under conditions in which ratcardioglobulin-C is present on the heartsurface.

Having established with reasonable certain-ty that cardioglobulin-C occurs in precipitinline 3, we proceeded with the next step inthe analysis, which is illustrated in Figure 8.The central well contains anti-C antiserum. Inthe peripheral wells at 1, 3 and 5 o'clock are a1:40 dilution of rat serum, a Sepharosefraction 2 from glycerol extract of rat skeletalmuscle, and a Sephadex G-200 cardioglobulin-C fraction from rat serum. Line 3 can beidentified in the rat serum precipitin reaction,and it is continuous with a corresponding linemade by the Sepharose muscle fraction andthe cardioglobulin-C antigens. There is nocrossing or spur formation where the linesmeet, so it can be concluded that the line 3proteins from the muscle calcium-proteincomplex and the cardioglobulin-C fractionsare immunochemically identical. The figureshows an additional muscle extract precipitinline which is also chemically related to a ratserum protein, since it is continuous with ratserum precipitin line 2. This is not acardioglobulin component, since the line stillforms with antiserum absorbed by cardioglob-ulin-B and cardioglobulin-C on frog hearts(Fig. 7).

Discussion

In the extraction of a protein-ion complexusing a radioactive ion to label the complex, itis always possible that the label will becomefree during homogenization and extraction.Although bound calcium is stable in restingmuscle, calcium release occurs during depolar-ization (1). The chemical configuration of the



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membrane characteristic of the polarized stateprobably cannot be sustained during homoge-nization. Therefore it was not surprising thatonly 1035 of the calcium was bound and 9095free after extraction with NaHCOa-glycerol,despite the fact that the composition of thissolution was based on a thorough physiologi-cal study that established conditions favoringstability of the bond between calcium and itsbinding constituent (4).

The first change in the ratio of bound tofree calcium in the extract was achieved bythe use of procaine, which is an antagonist ofcaffeine (8, 9). Thus procaine can be used forstabilization of the calcium bond. The secondimprovement was achieved by replacingcalcium with cadmium. The correctness of thissubstitution was evaluated in the exciseddiaphragm under physiological conditions. Inthese experiments, cadmium showed strongerbinding than calcium at the calcium sites.Thus the 116Cd peak on Sepharose 2B (Fig. 6)is due to a protein which very likely bindscalcium involved in E-C coupling.

Selection of 50% glycerol for the extractionsolution was fortunate, since despite the factthat it does not tend to denature proteins, itextracted 70% of the muscle 45Ca. Furthermoreglycerol extracted only a small amount of themuscle proteins, leaving behind most of thecontractile protein to which the good contrac-tile properties of glycerinated muscle attest.The crude glycerol extract obtained aftercentrifugation was a slightly pink, clearsolution and, except for a few lipid droplets,showed no microscopically recognizable sub-cellular structure at lOOOx magnification.

Our results indicate that the Sepharosefraction 2 skeletal muscle extract contains aprotein which is immunochemically identicalwith rat cardioglobulin-C, the calcium con-taining protein of a calcium transport systempreviously identified in rat plasma (11). Thisconclusion is based on the fact that one of theprecipitin lines formed by interaction ofSepharose fraction 2 with anti-cardioglobulin-C antiserum forms a line of identity with the

plasma cardioglobulin-C precipitin line. Thefindings suggest that the calcium-releasingprotein isolated from rat diaphragm is in factcardioglobulin-C. Additional weight is thusadded to the evidence that the rat diaphragmextract contains a calcium-releasing proteinsince the demonstration that cardioglobulin-Cis a calcium-releasing protein was madeindependently on a different model system(11). Of course until the diaphragm extractcontains only one protein, we cannot rule outthe possibility that there is another calcium-protein complex in the extract

References1. HAJDU, S.: Mechanism of the Woodworth

staircase phenomenon in heart and skeletalmuscle. Am J Physiol 216:206-214, 1969.

2. HAJDU, S., MAXTMIN, T.J., AND LEONARD, E.J.:Cardioglobulin. Separation, characterizationand assay of the individual components. CircRes 22:517-526, 1968.

3. LEONARD, E.J., MAXTMIN, T.J., AND HAJDU, S.:Cardioglobulin. Tissue localization and plasmaactivity with special reference to cardiovasculardisease and lupus erythematosus. Circ Res22:527-540, 1968.

4. HAJDU, S.: Effect of drugs, temperature and ionson Ca+ + of the coupling system of skeletalmuscle. Am J Physiol 218:968-972, 1970.

5. BBAY, G.A.: Simple efficient liquid scintillator forcounting aqueous solutions in a liquid scintilla-tion counter. Anal Biochem 1:279-285, 1960.

6. AXELSSON, J., AND THESLEFF, S.: Activation of thecontractile mechanism in striated muscle. ActaPhysiol Scand 44:55-66, 1968.

7. BIANCHI, C.P.: Effect of caffeine on radiocal-cium movement in frog sartorius. J Gen Physiol44:845-858,1961.

8. SCHOLLEH, J.: Uber den Antagonismus einigerLokalanasthetika gegeniiber dem Koffeineffektam Muskel. Arch Exp Path Pharmakol105:299-306, 1925.

9. FQNSTEIN, M.B.: Inhibition of caffeine rigor andradiocalcium movements by local anesthetics infrog sartorius muscle. J Gen Physiol 47:151-172, 1963.

10. PEDERSEN, K.O.: Exclusion chromatography.Arch Biochem Biophys, suppl. 1, pp 157—168,1962.

11. HAJDU, S., AND LEONAHD, E.: Binding ofcardioglobulin-C-Ca4B to cardiac muscle andrelease by cardioglobulin-A. Am J Physiol209:1-7, 1965.

Circulation Rtstsrcb, Vol. XXIX, Octobrr 1971



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Stephen Hajdu, Christian J. Posner and Edward J. LeonardExcitation-Contraction Coupling

Isolation from Rat Diaphragm of a Calcium-Protein Complex Involved in

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