was centrifuged in a Beckman JA-10 rotor for 10 min (frozen muscle) ...... the square seem to derive from a jack-like structure which is occasionally observed ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 01987 by The American Society of Biological Chemists, Inc.
Vol. 262, No. 4, Issue of February 5, pp. 1740-1747, 1987 Printed in U.S.A.
Purification of the Ryanodine Receptor and Identity with Feet Structures of Junctional Terminal Cisternae of Sarcoplasmic Reticulum fromFast Skeletal Muscle” (Received for publication, October 3, 1986)
Makoto InuiS, Akitsugu Saito, andSidney Fleischer From the Department of Molecular Bwbgy, Vanderbilt University, Nashville, Tennessee37235
The ryanodine receptor has been purified from junc- role in regulating the intrafiber free Ca2+concentration (1tional terminal cisternae of fast skeletal muscle sar- 4). Energized Ca2+ uptake in the SR is catalyzed by Ca2+ coplasmic reticulum (SR). The ryanodine receptor was pump protein which is the major component of the SR. The solubilized with 3-[(3-cholamidopropyl)dimethylam- structure and function of the Ca2+pump has been well charmoniol- 1-propanesulfonate (CHAPS) and stabilized by acterized (5-7). Release of Ca2+from the SR is triggered by addition of phospholipids. The solubilized receptor an action potential at the neuromuscular junction which is showed the same [3H]ryanodine binding propertiesas transferred to the SRby way of the transverse tubules, which the original SR vesicles in terms of affinity, Ca2+dependence, and salt dependence. Purification of the ry- invaginate from the sarcolemma. The transverse tubules form anodine receptor was performed by sequential column the “triad junction” with the terminal cisternae of SR via chromatography on heparin-agarose and hydroxylapa- “feet structures” (8-11). The signal is transferred across the tite in the presence of CHAPS. The purified receptor triad junction, which results in release of Ca2+from terminal bound 393 f 65 pmol of ryanodinelmg of protein (mean cisternae of SR. However, the molecular mechanism of signal f S.E., n = 5). The purified receptor showed three transmission and Ca2+release has not been resolved. Recent bands on sodium dodecyl sulfate-polyacrylamide gel studies suggest the presence of Caz+release channel in terelectrophoresis with M , of 360,000, 330,000, and minal cisternae of SR (11-18). The isolation of the channel is required for its molecular 175,000. Densitometry indicates that these are present in the ratio of 21111,suggesting a monomer M , of 1.225 characterization. Of many drugs which modulate the Ca2+ X 10‘ and supportedby gel exclusion chromatography release channel, ryanodine, a plant alkaloid, is a powerful in CHAPS. Electron microscopy of the purified prepa- biochemical tool to study the release of Ca2+ from the SR ration showed the squareshape of 210 A characteristic although the pharmacological effects of ryanodine on muscle of and comparable in size and shape to thefeet struc- are complex (19, 20). Two recent advances have charted the tures of junctional terminal cisternae of SR, indicating direction for isolation of the Ca2+release channels of SR. The that ryanodine binds directly to the feet structures. first is the isolation of a well-defined preparation of junctional From the ryanodine binding data, the stoichiometry terminal cisternae. Twenty percent of the membrane of this between ryanodine binding sitesto thenumber of feet structures is estimated tobe about 2. Since the ryano- fraction is junctional face membrane, containing well-defined junctional feet structures (11).In skeletal muscle fibers, in dine receptor is coupled to Ca2+ gating, the present finding suggests that the ryanodine receptor andCa2+ situ, the feet structures of this membrane are associated with release channel representa functional unit, the struc- the transverse tubules to form the triadjunction. It is via this tural unit being the foot structure which, in situ, is junction that the signal for Ca2+ release is expressed. The junctionally associated with the transversetubules. It second advance is the finding that ryanodine at low concenis across this triad junction that the signal for Ca2+ trations (apparent KI 50 nM) acts pharmacologically on release is expressed. Thus, the foot structure appears junctionalterminalcisternae, appearing to lock the Ca2+ to directly respond to the signal from transverse turelease channels in the “open state” (21, 22). Furthermore, bules, causing the release of Ca2+ from the junctional ryanodine binding was localized to the junctional terminal face membrane of the terminal cisternae of SR. cisternae in contradistinction with longitudinal cisternae of SR,andthe ryanodine binding affinity was in the same concentration range as its pharmacological action (21, 23). This study describes the purification of the ryanodine reThe sarcoplasmic reticulum (SR)’ in muscle serves a central ceptor from junctional terminal cisternae of SR. It provides * This work was supported in part by Grant AM14632 from the evidence that the purified receptor is identical with the feet National Institutes of Health and by a grant from the Muscular structures of junctional face membrane of terminal cisternae.
-
Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must EXPERIMENTALPROCEDURES therefore be hereby marked “advertisement” in accordance with 18 Materials-Ryanodine was obtained from Penick Corp. (Lindhurst, U.S.C. Section 1734 solelyto indicate this fact. $ Supported by an Investigatorship of the American Heart Asso- NJ) and [3H]ryanodine (70 Ci/mmol) was prepared and purified as described previously (21). CHAPS, sodium cholate, Triton X-100, ciation, Tennessee Affiliate. The abbreviations used are: SR, sarcoplasmic reticulum; CHAPS, Lubrol PX, and soybean lecithin (type IV-S) were obtained from 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate; Sigma. Zwittergent 3-12 and 3-14 and octyl P-D-glucopyranoside were CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-obtained from Behring Diagnostics, CHAPSO from Pierce Chemical propanesulfonate; HPLC, high performance liquid chromatography; Co., CI2E8from Nikko Chemicals (Tokyo, Japan), Tween 80 from SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electropho- Atlas Chemical (Wilmington, DE), and Lubrol WX fromI.C.L. Organics. TSK-G4000SW column (0.75 X 60 cm) was obtained from resis; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid.
1740
Purification of Ryanodim Receptor and Feet Structures LKB Instruments, Inc. (Gaithersburg, MD). Hydroxylapatite (BioGel HTP) was purchased from Bio-Rad, heparin-agarose was from Sigma, and Sephadex G-50 (fine) from Pharmacia P-L Biochemicals. Molecular weight standards were from Bio-Rad, Pharmacia, and Boehringer Mannheim (Mannheim, Federal Republic of Germany). Synthetic dioleoyl lecithin and dioleoyl phosphatidylserine were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Isolation of Junctional Terminal Cisternae Membranes-Membrane fractionsreferrable to junctional terminal cisternaeof SR were prepared from rabbit fast skeletalmuscle as described previously (11, 25) with some modifications. The white or pink appearing hind leg muscle or the frozen back muscle was ground in a meat grinder. The ground meat, in 50-g portions, was homogenized in 250 ml of 0.3 M sucrose, 5 mM imidazole HCl, pH 7.4 (homogenization medium), using a Waringblender at maximum speed for 60 s. The homogenate was centrifuged in a Beckman JA-10 rotorfor 10 min (frozen muscle) or 20 min (fresh muscle) at 8,000 rpm. The pellets were rehomogenized in 0.5 mM Mg-ATP containing homogenization medium for 90 s and recentrifuged as before for 20 min. The supernatantwas filtered through 3-4 layers of cheesecloth, and a microsomal pellet was obtained by centrifugation for 2 h at 14,000 rpm in a Beckman JA14 rotor or a Beckman 19 rotor. The microsomes were resuspended in 350 mlof homogenization medium and thenpoured into a Beckman Ti-15 rotor. The assembled rotor was then centrifuged at 2,000 rpm while the sucrose gradient was made by pumping 200 ml of 27, 32, 34,38, and45% sucrose, each buffered with 5 mM imidazole HCl, pH 7.4, and 150 ml of 60% sucrose. The rotor was centrifuged overnight a t 20,000 rpm.The junctional terminal cisternae sedimenting between 38.5 and 45% werecollected, diluted approximately 3-fold with 5 mM imidazole HCl, pH 7.4, and centrifuged in a Beckman 45Ti rotor for 2 h at 30,000 rpm. The pellets were then resuspended in homogenization medium and quick-frozen using liquid nitrogen and stored at -70 "C. A typical preparation uses 600 g of the ground meat and yields 250-300 mg of junctional terminal cisternae. [3H]Ryanodine Binding Assay-Solubilized samples (5-600 pg of protein, depending on the purity) were assayed at 24 "C in 0.2 ml of solution containing 20 mM Tris-HC1, pH 7.4, 0.3 M sucrose, 2 mM dithiothreitol, 25 p~ CaC12,1 M NaCl, 10-40 mg/ml CHAPS, and 520 mg/ml soybean lecithin with various concentrations of [3H]ryanodine (-50,000 cpm/pmol). In the routine assay, 300 nM [3H]ryanodine was included. After 40 min of incubation at 24 "C, bound and free [3H]ryanodinewas separated on a Sephadex G-50 mini-column (1.5 ml) pre-equilibrated with 10 mg/ml CHAPS, 5 mg/ml soybean lecithin, 0.3 M sucrose, 1 M NaCl, 2 mM dithiothreitol, 25 p~ CaC12,and 20 mM Tris/HCl, pH 7.4, which was centrifuged for 80 s a t 500 rpm in a Beckman TJ-6 centrifuge. A 100-pl aliquot of the eluate (about 0.6 ml) was mixed with 6 ml of ACS scintillation fluid (Amersham Corp.), and radioactivity was measured in a Searle Analytic 81 scintillation counter. The protein concentration of the eluate was estimated from a 10-100-pl aliquot using Amido Black 10B and 0.45-pm Millipore filters (type HA) by the method of Kaplan and Pedersen (26). In the standardassay condition, nonspecific binding, which was determined in the presence of an excess ryanodine (1.5mM), accounted for less than 5% of the counts (Table I). The junctional terminal cisternae membrane vesicles were also assayed using a Sephadex G-50 mini-column as described above. CHAPS and soybean lecithin were omitted in assay and column solutions. Forty to 60% of the applied protein was recovered in the eluate. Similar results were obtained when this method was compared with the Millipore filtration assay through 0.22-pm Millipore filters (type GSWP) described previously (21). Solubilization of the Ryanodine Receptor-Frozen SR membranes were thawed and suspended in 0.3 M sucrose, 1 M NaCl, 2 mM dithiothreitol, and 20 mM Tris/HCl, pH 7.4. Solubilization was initiated by adding the CHAPS-soybean lecithin mixture (100 mg/ml CHAPS and 50 mg/ml soybean lecithin) to make a CHAPS:protein weight ratio of 13.3. The final protein concentration was 1.5-3 mg/ ml. The sample was incubated a t 4 "C for 10 min and centrifuged at 46,000 rpm for 30 min in a Beckman 70.1Ti rotor or a t 95,000 rpm for 30 min in a Beckman TLlOO centrifuge using a TLA 100.2 rotor. The supernatantwas used for further study. Purification Procedure-The CHAPS extract (16.7 ml) from 50 mg of junctional terminal cisternae of SR was diluted with 150.3 ml of CHAPS-PL buffer (10 mg/ml CHAPS, 5 mg/ml soybean lecithin, 0.3 M sucrose, 2 mM dithiothreitol, 20 mM Tris/HCl, pH 7.4) and loaded onto aheparin-agarose column (1.5X 6 cm) pre-equilibrated with the above buffer containing 0.1 M NaCl. The column was washed with the same buffer until absorbance at 280 nm came to thebase line and
1741
eluted with a linear gradient between 0.1 and 0.8 M NaCl containing CHAPS-PL buffer (40 ml each). Aliquots (125pl) of the 4-ml fractions were assayed for ryanodine binding activity with 0.3 p~ [3H]ryanodine. In theassay, NaCl was supplemented to make the final concentration approximately 1 M. The peak fractions of the ryanodine binding activity were pooled(16 ml) and further purified on a hydroxylapatite column (1.3 X 3cm) pre-equilibrated with 10 mg/ml CHAPS, 0.6 M NaCl, 0.3 M sucrose, 2 mM dithiothreitol, and 5 mM sodium phosphate, pH 7.4. The receptor was eluted with 45 ml of linear sodium phosphate gradient (5-200 mM). Aliquots (150 pl) of the 2.3-ml fractions were assayed for ryanodine binding activity with 0.3 p~ [3H]ryanodineand 5 mg/ml soybean lecithin. The peak fractions of ryanodine binding activity were pooled, and 0.1 ml of 100 mg/ml soybean lecithin was added. The sample was dialyzed at 4 "C for 40 h against 4 liters of 0.6 M NaCl, 2 mM dithiothreitol, and 20 mM Tris/HCl, pH 7.4. The dialyzed sample was centrifuged in a Beckman 70.1Ti rotor for 1 h at 50,000 rpm. The pellets were resuspended in a small volume of 0.6 M NaC1, 0.3 M sucrose, 2 mM dithiothreitol, and 20 mM Tris/HCl, pH 7.4, in the presence or absence of 10 mg/ml CHAPS and 5 mg/ml soybean lecithin. Gel Filtration HPLC-The purified sample solubilized with 10 mg/ ml CHAPS was applied to a TSK-G4000SW column (0.75 X 60 cm) pre-equilibrated and eluted in a solution of 10 mg/ml CHAPS, 0.6 M NaCl, 2 mM dithiothreitol, and 20 mM imidazole HCl, pH 6.8. The column was eluted a t a flow rate of 0.5 ml/min. Fractions were assayed for ryanodine binding activity, supplemented with soybean lecithin and NaCl. Protein patterns of each fraction were checked by SDS-PAGE. Molecular weight standards were: blue dextran, M, 2,000,000; thyroglobulin, M, 669,000 ferritin, M, 440,000; catalase, M, 232,000; aldolase, M, 158,000. SDS-Polyacrylamide Gel Electrophoresis-Gel electrophoresis was performed in 5-15% polyacrylamide gradient gels in the buffer system of Laemmli (27), employing a mini-slab gel electrophoresis apparatus (gel size, 75-mm length, 0.75-mm thickness). Samples were denatured at 24 "C for 30 min in 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 50 mM Tris/HCl, pH 6.8, prior to electrophoresis. Gelswere stained with Coomassie Blue or silver, the latter according to Merril et al. (28), or both of them. In the double staining, gels were first stained with Coomassie Blue followed by the silver staining. Molecular weight standards were: a2-macroglobulin, M, 340,000 (nonreduced) and 170,000 (reduced); myosin, M, 200,000; @-galactosidase, M, 116,250; phosphorylase b, M, 92,500; bovine serum albumin, M, 66,200; ovalbumin, M,45,000; carbonic anhydrase, M, 31,000; soybean trypsin inhibitor, M, 21,500; lysozyme, M, 14,400. The densitometric scan of the stained gel wascarried out by an LKB Ultroscan XL Laser densitometer using LKB gelscan software. Protein Assay-Protein determination of SR vesicles was madeby the method of Lowry et al. (29). For solubilized samples, the method of Kaplan and Pedersen (26) was used with Amido Black 10B and 0.45-pm Millipore filters(type HA). In both cases, bovine serum albumin served as standard. Electron Microscopy-The samples were examined using a JEOL JEM 100s electron microscope. Fixation of junctional terminal cisternae vesicles in 2% glutaraldehyde, 1%tannic acid for thin section electron microscopy was performed as described previously (30, 31). The purified ryanodine receptor, devoid of phospholipids, was examined by electron microscopy using negative staining. Phospholipids were removed by gel filtration HPLC. Two microliters of the sample in 10 mg/ml CHAPS, 0.6 M NaC1, 2 mM dithiothreitol, and 20 mM imidazole HCl, pH 6.8, were applied to a supporting membrane of a very thin carbon-coated film over 400-mesh grid. The support grid was covered with the sample, and excess sample was removed by touching the grid with filter paper. One drop of 1%uranyl acetate (pH 4.2) was applied to the grid. After 1 min of incubation, the grid was washed with 5 drops of 1%uranyl acetate. Excess solution was removed by touching with filter paper, and the grid was dried under vacuum in the electron microscope. RESULTS
Ryanodine Binding by the Solubilized Receptor-In
order to purify the ryanodine receptor from junctional terminal cisternae of SR, we optimized the conditions for solubilization of the receptor to retain [3H]ryanodinebinding by the solubilized sample. For the ryanodine binding assay, rapid gel filtration on a Sephadex G-50 mini-column was used to separate protein-bound [3H]ryanodinefrom free [3H]ryanodine.The effect
Purification of Ryanodine Receptor and Feet Structures
1742
of the type of detergent on the ryanodine binding assay was first examined. Among a variety of detergents, Zwittergent 312 and 3-14, Lubrol WX and PX, CI2Es,Triton X-100, and Tween 80 had a relatively high nonspecific interaction with [3H]ryanodine, thereby interfering with the binding assay. With these detergents, when the mixture of 2% detergents and 0.3 PM [3H]ryanodine was applied to rapidfiltration minicolumns, a significant amount of the applied radioactivity eluted in the void volume apparently as a ryanodine-micelle complex (Table I). CHAPS, CHAPSO, and sodium cholate resulted in less than 0.02% of the applied radioactivity in the void volume (Table I). Among the latter detergents, the highest [3H]ryanodine binding in the detergent-treated SR was obtained with CHAPS (data notshown). The stability of the ryanodine receptor in high detergent concentrations was examined next since the receptor protein would be exposed to excess detergentduring the purification procedure. In the following studies, we used a high concentration of CHAPS (detergent to protein weight ratio of 13.3) to optimize the TABLEI
PHIRyanodine bindingassay in the presence of detergents Values are the mean t S.D. from five determinations. _
Conditions _
_
~
[3H]Ryanodine in eluate ~
pmol
A. Detergent controls" CHAPS CHAPSO Sodium cholic acid CHAPS + soybean lecithin Octyl glucoside Tween 80 Triton X-100 C&B Lubrol WX Lubrol PX Zwittergent 3-12 Zwittergent 3-14 B. SR CHAPS (-) Soybean lecithin (-)* C. CHAPS + SR Soybean lecithin (+)' Standard condition NaCl ( - I d 5 mM EGTA' 1.5 mM ryanodind
-
conditions for [3H]ryanodinebinding of solubilized sample. [3H]Ryan~dine binding and the stability of its association to thesolubilized SR asa function of time is shown in Fig. 1. Without added soybean lecithin, and in the presence of Ca2+, [3H]ryanodine binding increases at 10 min and thendecreases with the level of maximal binding being 7 pmol/mg protein or less. When the mixture was supplemented with soybean lecithin, the level of [3H]ryan~dine binding plateaued at about 20 pmol/mg protein after 20 min at 24 "C, which was about the same level as that in the original SR vesicles (Table I). Essentially similar results were obtained using synthetic phospholipids (dioleoyllecithin and dioleoylphosphatidylserine) (data notshown). With thesupplementation of phospholipids, about 90% of the maximal binding activity was Ca2+dependent (Fig. 1 and Table I). Thus, phospholipids are essential to maintain the function of the ryanodine receptor in the solubilized state. The saltconcentration dependence of [3H]ryanodine binding in solubilized SR with CHAPS and soybean lecithin is shown in Fig. 2. Maximal binding was observed at NaCl concentrations above 0.8 M . The same result was obtained with KC1 instead of NaCl (data not shown). The original SR vesicles also had the same salt dependence for [3H]ryan~dine binding (data not shown). Based on the above observations, the solubilization and ther3H1rvanodine bindine assav media included soybean lecithin io.; weight ratioto CHAPS) and 1 M NaC1. The solubility of the ryanodine receptor was examined with different concentrations of CHAPS in 1 M NaCl containing solution. The weight ratio of soybean lecithin to CHAPSwas maintained at 0.5. At CHAPS concentrations, weight ratio to protein above 10, about 80% of the ryanodine binding activity in the SR vesicles was solubilized (Fig. 3). The solubilized receptor at the CHAPSconcentration of 13.3 in weight ratio as thatin the to protein had essentially the same K d and Bmax original SR vesicles (Fig. 4). Thus, the solubilized receptor appears to retain the inherent binding propertiesof the receptor in the original SR vesicles. Purification of the Ryanodine Receptor-In the first step of purification, junctional terminal cisternae of SR (3 mgof I
pmollw
0.009 t 0.007 0.009 f 0.006 0.010 f 0.008 0.090 f 0.030 0.165 f 0.039 0.608 f 0.148 0.666 f 0.146 1.23 f 0.22 2.51 f 0.62 4.60 f 2.07 8.27 ? 1.07 12.67 f 0.50 2.40 f 0.20
19.46 t 1.70
2.80 k 0.22 0.36 t 0.05 0.27 f 0.03 0.08 t 0.01
22.85 f 1.76 2.98 f 0.43 2.24 f 0.27 0.60 f 0.09
~~~
~~~
Y
v
Detergents at a concentrationof 2% in water were incubated with 300 nM [3H]ryanodineat 24 "C for 40 min and thensubjected to a gel filtration mini-column as described under "Experimental Procedures." Radioactivity in the excluded volume was measured. Junctional terminal cisternae (1.5 mg of protein) was incubated at 24 "C for 40 min with 300 nM [3H]ryanodine in 0.3 M sucrose, 1 M NaCI, 25 p~ CaC12,and 20 mM Tris/HCl, pH 7.4, and subjected to a gel filtration mini-column. Protein and radioactivity in the excluded volume were determined. The mixture was devoidof (-) CHAPS and soybean lecithin. e The solubilized membranes (1.5 mg/ml protein) supplemented with (+) 20 mg/ml CHAPS and 10 mg/ml soybean lecithin were assayed for [3H]ryanodinebinding as described under "Experimental Procedures." In the standard condition, the incubation medium contained 300 nM [3H]ryanodine, 0.3 M sucrose, 1M NaCl, 25 pM CaCl,, 2 mM dithiothreitol, 20 mg/ml CHAPS, 10 mg/ml soybean lecithin, and 20 mM Tris/HCl, pH 7.4. The sample was subjected to a gel filtration mini-column. Protein and radioactivity in the excluded volume were measured. NaCl was omitted from the standard condition. e 5 mM EGTA was included in the standard incubation medium replacing 25 p M CaC12. f 1.5 m M unlabeled ryanodine was added to the standard incubation medium.
-
n
an0
0"-
-"""
" .
10 20 30 40 50 6 07 0
80 90
Time (mid
FIG. 1. Effect of soybean lecithin on ryanodine binding of solubilized junctional terminal cisternae of SR.Junctional terminal cisternae (1.5 mg/ml protein) were solubilized with 20 mg/ml CHAPS (m, 0 )or 20 mg/ml CHAPS plus 10 mg/ml soybean lecithin (0,0 ) in 0.3 M sucrose, 1 M NaCl, 2 mM dithiothreitol, and 20 mM Tris/HCl, pH 7.4. After 10 min of incubation at 4 "C, [3H]ryanodine and either CaC12 (0,m) or EGTA (0,0 ) were added to a final concentration of 300 nM and 25 p~ or 5 mM, respectively. The sample was incubated at 24 "C for 5-80 min. The [3H]ryanodinebinding was determined on separate aliquots with a gel filtration mini-column as described under "Experimental Procedures." Results shown are averages (+%E.) of three experiments performed in triplicate.
Purification of Ryanodine Receptor
andStructures Feet
- 0.3 I
-c
~..y
,
~~~\.
I ~~
m
g-0
0.2 -
Membrane bound ~ m a x -20.1 pmollmg Kd = 63 nM
-E v
W
?
~
-.r~
~%
Solubilized
E o.l
$
I
/
l o t
1743
~ m s= x 19.0 pmollmg
Kde.52 nM
3
m
0
U
0
5
0
-p-Q
Q-9-n
15
10
20
Bound (pmollmg)
FIG.4. Scatchard analysis of ryanodine bindingto original (0)and solubilized (0)junctional terminal cisternae of SR. NaCl (MI Solubilization of junctional terminal cisternae (1.5 mg/ml protein) FIG.2. Effect of salt concentrationon ryanodine binding of was performed with 20 mg/ml CHAPS and 10 mg/ml soybean lecithin solubilized junctional terminal cisternaeof SR. Junctional ter- in 0.3 M sucrose, 1 M NaCI, 2 mM dithiothreitol, and 20mM Tris/ minal cisternae (1.5 mg/ml protein) were solubilized with 20 mg/ml HCI, pH 7.4. [3H]Ryanodinebinding was measured using a gel filtraCHAPS and 10 mg/ml soybean lecithin in various concentrations tion mini-column as described under“Experimental Procedures.” (0.1-1 M) of NaCl, 0.3 M sucrose, 2 mM dithiothreitol, and 20mM Ryanodine binding to theoriginal vesicles was also determined using Tris/HCl, pH 7.4. After incubation for 10 min a t 4 “C, [3H]ryanodine a gel filtration mini-column without CHAPS and soybean lecithin. (300 nM) and either CaC1, (0)or EGTA (0) were added to a final The correlation coefficients of original and solubilized samples were concentration of25 p M or 5 mM, respectively. The sample was -0.975 and -0.978, respectively. maintained a t 24 “C for 40 min. Ryanodine binding was determined as described under “Experimental Procedures.” 0
0.2
0.4
0.6
1.0
0.0
100,
ri-
,100
HMW=I-; 80
S
Mr =-34Q
-200
1 r
L
“170 - 1 16 -92.5
v
60
g v
-66.2
.-cW 40
$
-45
n
-3 1
20 01
0.5
-2 1.5 - 14.4
‘0
a
1
2
5
(xlO3)
15 10
CHAPSlProtein ( w l w )
FIG.3. Solubilization of ryanodine binding activity from junctional terminal cisternae of SR. Junctional terminal cisternae (3 mg/ml protein) were solubilized with various concentrations of CHAPS in 0.3 M sucrose, 1 M NaCl, 0.5 weight ratio of soybean lecithin to CHAPS, 2 mM dithiothreitol, 20 mM Tris/HCl, pH 7.4. The weight ratio of CHAPS to protein was varied from 0.5 to 13.3. After incubation a t 4 “C for 10 min, the samples were centrifuged in Beckman TLA 100.2 rotor a t 95,000 rpm for 30 min. Protein concenand [3H]ryanodine binding (0)in the supernatant were tration (0) measured as described under “ExperimentalProcedures.” Results are expressed as percent of total protein and total binding, which were determined in the total mixture without centrifugation treated with 13.3 weight ratio of CHAPS to protein. The total protein and ryanodine binding were 3 mg/ml and 19.6 pmol/mg, respectively. Values are averages (+S.E.) of four different experiments performed in triplicate.
1
2
3
4
5
FIG.5. Purification of the ryanodine receptor complex from junctional terminal cisternae. Electrophoresis was carried out according to the method of Laemmli (27), buton a 5-15% polyacrylamide gradient gel. The gel was double-stained with Coomassie Blue of SR, lune 2, and silver. Lane 1, junctionalterminalcisternae solubilized junctional terminal cisternae with CHAPS and soybean lecithin; lune 3, pooled ryanodine binding fractions from a heparinagarose column; lune 4 , pooled fractions from a hydroxylapatite column; lune 5, purified ryanodine receptor. HMW, high molecular weight (330,000-360,000) doublet; CPP, Ca2+pump protein; CBP, Ca2+binding protein (calsequestrin).
protein and the Caz+bindingprotein(calsequestrin) were effectively removed (Fig. 5 , lane 3 and Fig. 6, inset). Further purification was performed on a hydroxylapatite column. In this chromatography (Fig. 7), soybean lecithin was omitted since in thepresence of the soybean lecithin fraction, aproper protein/ml) (Fig. 5, lane 1 ) were solubilized with CHAPS (40 base line would not be achieved. The ryanodine binding mg/ml or 13.3 mg/mg protein) and soybean lecithin (20 mg/ activity was eluted at the second peak after the start of a ml) in 1 M NaCI, 0.3 M sucrose, 2 mM dithiothreitol, and 20 linear gradient elution of phosphate (Fig. 7). The peak fracmM Tris/HCl, pH7.4. Under these conditions,75-90% of the tions of ryanodine binding activity from a hydroxylapatite 11). The column revealed the protein bands of M , 360,000 and 330,000 ryanodinebindingactivity is extracted(Table CHAPSextracthadthe sameprotein pattern as that of doublets, 200,000 and 175,000 on SDS-PAGE (Fig. 5, lune 4 original terminal cisternae (Fig. 5 , lunes 1 and 2). After and Fig.7, inset). Of those peptides, the 200,000 protein, dilution to reduce NaCl concentration to 0.1 M, the sample probably myosin, was removed by incorporating the receptor was applied to aheparin-agarose column. The ryanodine into phospholipid vesicles. With thesemanipulations, the binding activity absorbed to heparin-agarose and eluted as a ryanodine receptor was further purified (Fig. 5, lune 5 ) and single peak by increasing the NaCl concentration (Fig. 6). In concentrated into a small volume. Table I1 summarizes the purification of ryanodine receptor this step, the major SR components such as the Ca2+ pump
1744
and Feet Structures
Purification of Ryanodine Receptor TABLEI1
8 ,
Purification of ryanodine receptor The purification procedure is described under “Experimental Procedures.” The values indicated here are from a typical experiment. Ryanodine binding Protein
Yield
Fraction” Suecific activitv Total activitv Pmllmg
mg
1. Junctional terminal
50.0
19.3
x
pmol
%
965
100
cisternae 18.0 840 87.0 2. Solubilized junctional 46.7 terminal cisternae 13032.3 312 3. Heparin-agarose chro- 2.40 matography 25.8 chro- 249 286 4. Hydroxylapatite 0.87 matography 378b 163 16.9 5. Incorporation into 0.43 DhosDholiDid’ a The protein pattern after each step is shown in the SDS-PAGE gel in Fig. 5. The sDecific activitv in five different DreDarations averaged 393 f 65 pmol/mg (mean & S.E.). Methodology for incorporation into phospholipid is given under “Experimental Procedures.” ”
Y
5
E, v
0
4
._ c .n 0
3
0
.c
2
2
1
0 0 C
n
- .- - - - - - - - - - - - - - -. 20
0
40
LO
80
60
Time ( m i d
FIG.7. Hydroxylapatite column chromatography of ryanodine receptor. Approximately 16 ml of the pooled peak from a heparin-agarose column was applied to a column of hydroxylapatite pre-equilibrated with 10 mg/ml CHAPS, 0.6 M NaCl, 0.3 M sucrose, 2 mM dithiothreitol, and 5 mM sodium phosphate, pH 7.4. The receptor was eluted a t a flow rate of 1.5 ml/min with a 45-ml linear sodium phosphate gradient (5 to200 mM Na phosphate, indicated by was monitored. Aliquots dashed lines). Absorbance at 280 nm (-1 (150 pl) of the 2.3-ml fractions were assayed for ryanodine binding activity (0).Inset shows SDS-PAGE pattern of the pooled peak from a heparin-agarose column (0)and the fractions indicated by arrows (1-4). H M W , high molecular weight (330,000-360,000) doublet.
-
5
r
‘FE“
1
0
3
E,
v
Y
m
0
20
40
60
80
100 140 120
Time ( m i d
FIG.6. Enrichment of the ryanodine receptor by heparinagarose chromatography. The diluted CHAPS extract (167 ml) was applied to a column (1.5 X 6 cm) of heparin-agarose pre-equilibrated with 10 mg/ml CHAPS, 5 mg/ml soybean lecithin, 0.3 M sucrose, 0.1 M NaC1, 2 mM dithiothreitol, and 20 mM Tris/HCl, pH 7.4. The receptor was eluted at a flow rate of 2 ml/min with a linear NaCl gradient (0.1-0.8 M NaCl indicated by dashed lines) made up was in the pre-equilibrated buffer. Absorbance at 280 nm (-) monitored. Aliquots (125 pl) of the 4-ml fractions were assayed for ryanodine binding activity (0).Inset shows SDS-PAGE pattern of the startingCHAPS extract (0)and thefractions indicated by arrows (1-5). H M W , high molecular weight (330,000-360,000) doublet; CPP, Ca2+ pump protein;CBP, Ca2+ binding protein (calsequestrin).
protein. Approximately 0.4 mg of the receptor protein can be obtained from 50 mg of junctional terminal cisternae of SR. Overall recovery of ryanodine binding activitywas 17% of the initial activity. Characterization of the Purified Receptor-The ryanodine binding of purified receptor was 393 k 65 pmol/mg protein (mean 2 S.E.) in five different preparations (Table 11). This represents a 20-fold purification from junctional terminal cisternae. In Scatchard analysis of ryanodine binding to the purified receptor (Fig. 8),the Kd was 78 nM, similar to thatof original (63 nM) and solubilized (52 nM) junctional terminal cisternae of SR (Fig. 4),indicating that theryanodine receptor was purified unchanged with regard to ryanodine binding. The purified ryanodine receptorcontains threepolypeptide bands, a doublet at M , 360,000 and 330,000 and a 175,000
E
a
2
a 0
m
1
0
0
100
200
300
400
Bound (pmollmg)
FIG.8. Scatchard analysis of ryanodine binding to the purified receptor. The purified sample (25 pgof protein/ml) was incubated at 24 “C for 40 min with various concentrations (15-240 nM) of [3H]ryanodine in10 mg/ml CHAPS, 5 mg/ml soybean lecithin, 1 M NaCl, 0.3 M sucrose, 25 p M CaC12,and 20 mM Tris/HCl, pH 7.4. [3H]Ryanodine binding was measured using a gel filtration minicolumn as described under “Experimental Procedures.” The correlation coefficient was -0.919. band on SDS-PAGE (Fig. 9). The relative absorption ratio among three bands was 1006026 (360,000:330,000:175,000) when determined by densitometric gel scan of five different purified preparations. In gel filtration HPLC, the purified receptor eluted in the second peak which corresponds to M , of about one million (Fig. 10). A more accurate M , is limited by the lack of availability of appropriate high molecular weight standards. The first peak is in the void volume, and the third peak contains no protein, probably consisting of phospholipids in the preparation. An SDS-PAGE profile of the ryanodine binding fractions from the HPLC column revealed the presence of three polypeptide bands of M , 360,000, 330,000, and 175,000 (Fig. 10, inset). The elution of this
Purification of Ryanodine Receptor and Feet Structures
1745
complex of three peptides coincided with the ryanodine binding activity (Fig. 10). The ryanodine binding to the receptor requires phospholipid. The receptor purified by hydroxylapatite inthe presence of 10 mg/ml CHAPS, 0.6 M NaCl, 0.3 M sucrose, 2 mM dithiothreitol, and 175 mM sodium phosphate, pH 7.4, can be stored overnight a t 4 "C. Normal ryanodine binding is observed when phospholipid is added to the assay (5 mg/ml soybean lecithin and 10 mg/ml CHAPS in the presence of 1 M NaCl). However, there is little or no ryanodine binding (less than 10%) in the absence of phospholipid. Electron Microscopy of thePurified Receptor Complex-The morphology of the purified receptor was examined by negative staining electron microscopy (Fig. 11A). The purified receptor was observed as squares of 214 A sides 23 A (mean S.D., n = 208). Such squares are the unique characteristic of the feet structures of the junctional face membrane of terminal cisternae (11) (Fig. 11, B and C). Some squares form strings, being connected to one another at thecorners of the squares (Fig. 11A). This typeof connection gives rise to thecheckerboard-like lattice of the feet structures observed in the junctional face membrane (11)(Fig. 11C). Our results show that the reconstituted ryanodine receptor and the feet structures are morphologically identical.
*
C
0.2
I
I
I
0.4
0.6
0.8
1.0
Relative Mobility
FIG.9. Polypeptide composition of the purified ryanodine receptor. The purified sample (5 pgof protein) was run on SDSPAGE using a 5-15% polyacrylamide gradient gel. The gel stained with silver is shown in C. The tracing in B represents the densitometric scan of the stained gel. The graph in A shows a plot of M , of standards cY2-macroglobulin (340,000 (nonreduced) and 170,000 (reduced)), myosin (200,000), p-galactosidase (116,2501, phosphorylase b (92,500), and bovine serum albumin (66,200) as used to determine the M , of three bands in the purified sample: M , 360,000, 330,000, and 175,000. The top of the gel and the dye front are at 0 and 1.0 relative mobility, respectively. The dye front of the gel was approximately a t 6 cm.
*
DISCUSSION
The ryanodine receptor has been purified from junctional terminal cisternae SR of fast skeletal muscle. The purification procedure includes four steps: 1) solubilization with CHAPS in the presence of phospholipids: 2) heparin-agarose column chromatography; 3) hydroxylapatite column chromatography; and 4) incorporation into liposomes. The enrichment of the ryanodine receptor complex from junctional terminal cisternae is about 20-fold. The ryanodine ligand binding affinity is essentially the samefor the purified receptor as for the junctionalterminal cisternae vesicles. The purified ryanodine vo a b c d receptor has identical morphology to the feet structures of I I I I I 0.02 I 2 3 4 junctional face membrane of the terminal cisternae of SR. Y Y T T Thus, we conclude that the feet structures are functionally 1 2 3 4 and structurallyequivalent to theryanodine bindingcomplex. 3601 330K Further, the feet structures, which in situ are junctionally 175X associated with the transverse tubules, are now more directly implicated in the Ca2+release mechanism in excitation-con.!, traction coupling. 0.01 g In order to purify the receptor complex, we first examined N a the conditionsfor solubilization of the receptor to retain Q ryanodine binding by the solubilized sample. Among the detergents tested, CHAPS was found to be the best detergent for solubilization of the receptor and for the ryanodine binding assay (Table I and Fig. 3). Supplementation with phospholipids is essential to maintain theryanodine binding activity 0 of the solubilized receptor in thepresence of high concentra0 20 40 60 tion of detergent (13.3 mg of CHAPS/mg of protein) (Fig. 1). Time ( m i d The need for phospholipid appears to reflect the slow displaceFIG.10. Estimate of molecular size of the solubilized purified ryanodine receptor by gel filtration HPLC. The purified ment of residual phospholipid by detergent from the receptor sample (50 pg of protein) was applied to an HPLCcolumn of TSK- protein. Recently, Pessah et al. (24) reported the [3H]ryanoG4000SW (0.75 X 60 cm) pre-equilibrated and eluted with a solution dine bindingin solubilized heavy SR without supplementation of 10 mg/ml CHAPS, 0.6 M NaCl, 2 mM dithiothreitol, and 20 mM with phospholipid. However, they used a lower detergent imidazole HCI, pH 6.8. The column was eluted a t a flow rate of 0.5 was monitored, and fractions concentration (1-2 mg of CHAPS/mg of protein) compared ml/min. Absorbance a t 280 nm (-) were assayed for ryanodine binding activity (0)with supplement of with ours (13.3 mg of CHAPS/mg of protein), which may not soybean lecithin. Elution positions of M , standards areshown on the be enough to solubilize the receptor completely. They also top. V,, void volume as determined by blue dextran; a, thyroglobulin reported that ryanodine binding activity of solubilized SR was (669,000); 6, ferritin (440,000); c, catalase (232,000); d, aldolase markedly decreased after Sepharose 6B column chromatog(158,000). Inset, SDS-PAGE pattern of the fractions indicated arrowheads (1-4). Other fractions contained no protein. 360K, 330K, and raphy. This could be explained by the removal of phospho175K are the peptides with M , of 360,000, 330,000, and 175,000, lipids from the receptor during chromatography. The purified ryanodine receptor reveals three peptide bands respectively.
-
T
Purification of Ryanodine Receptor and Feet Structures
1746
.,..
,
...
FIG. 11. Comparison of the morphology of the purified ryanodine receptor (A) with the feet structures of junctional terminal cisternae vesicles ( B and C).A, negative*staining electron microscopy of the purified receptor. The purified receptor shows the square shapeof about 210-A sides (arrowheads). The corners of the square seem to derive from a jack-likestructure which is occasionally observed (see arrowheads). Some squares are connected to one another at the corners (arrows). B and C, thin sections of junctional terminal cisternae vesicles. The feet structures are obsqrved in thin section in cross-section ( B ) and in tangential section (C). The foot structures protrude about 120 A from the outer surface of the junctional face membrane (11).The square shape (about 200 A square) of the feet is observed (arrowheads) in the tangential sections. The magnification for A, B, and C is the same (X140,OOO).
of M , 360,000, 330,000, and 175,000 on SDS-PAGE (Fig. 9). In the report by Pessah et al. (24), the [3H]ryanodine-labeled fraction of solubilized SR from a Sepharose 6B column had polypeptides of M , > 200,000, Ca2+pump protein, and Ca2+ binding protein (calsequestrin). Our study clearly shows that Ca" pump protein and Ca2+ binding proteinare not components of the ryanodine receptor. The latter two proteins are removed from the ryanodinebindingactivityon heparinagarose column chromatography (Fig. 6). These threepeptides (360,000,330,000, and 175,000), corresponding to that of ryanodine binding activity,appear toexist in acomplex since they copurify together inthree differentchromatographic systems (Figs. 6,7, and 10). The relative intensity of the three bands, determined by densitometry, was 100:60:26 (360,000:330,000:175,000) (Fig. 9).The molar ratio of the three polypeptides is 1.85:1.22:1, taking into account their molecular size. On the assumption that the ratio is 2:l:l (360,000:330,000:175,000),the ryanodine receptor monomeric unit consists-of a combined molecular weight of 1,225,000. This is in the range of the M , obtained by gel filtration chromatography (Fig. 10). High molecular weight proteins with M , 360,000 and 330,000 have previously been suggested to be components of the feet structures of the junctionalface membrane of terminal cisternae (11, 25, 32). We find that the purified receptor complex forms the same square shape and size as the feet
structures of junctional face membrane of terminal cisternae. Furthermore,these observationsindicate that ryanodine binds directly to thefeet structures. The molecular weight ( M ) of the foot structure can be estimated from the dimensions of the foot structure using the following equation, M = N x p x V
where N = Avogadro's number; p = density of protein (g/ cm3),taken as1.37; V = the volume of the foot structyre. The 5ize of th? isolated foot structure is found to be 210 A X 210 A X 120 A (this paper and see also Ref. 11).We assume that the foot structure is anchoredwithin the membrane, fob example, by four {ransmembrane cybndrical pillars of e0 A diamete; and 70 Aelong. V is (210 A x 210 A x 120 A) 4[1r(25 A)* X 70 A)] = 5.84 X 10"' cm3. Therefore, the molecular size ( M )of the foot structure is 4.82 x lo6. The stoichiometry or molar ratio ( R )of ryanodine binding in pmol/mg protein to feet structures per mg sites (BmaX) protein can be calculated usingthe following equation.
+
R =~ 3 X ~ ,10-1*/(1 ~ x 10-3/~) R = B,,
X
M
X
10-9
From the ryanodine binding activity of the purified preparation (393 pmol/mg) (Table 11), R is. calculated to be 1.89. The predicted enrichment of ryanodine binding from junc-
Purification of Ryanodine Receptor
andStructures Feet
1747
Reticulum (Fleischer, S., and Tonomura, Y., eds) pp. 119-145, tional terminal cisternae (JTC) can be calculated from (the Academic Press, Orlando, FL percentage of junctional face membrane (JFM) in JTC) x 8. Franzini-Armstrong, C. (1980) Fed. Proc. 3 9 , 2403-2409 (the percentage of feet proteins in the JFM). The enrichment 9. Franzini-Armstrong, C., and Nunzi, C. (1983) J. Muscle Res. Cell is (100/21.3) X (100/25) = 4.69 X 4 = 18.8 (see Ref. 25). The Motil. 4 , 233-252 20 pmol/mg 10. Somlyo, A.V. (1979) J. Cell Bid. 80, 743-750 junctionalterminal cisternae have a B,,, protein. Therefore, a purified preparation of feet structures 11. Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J . Cell Biol. 99,875-885 wouldbe predicted to bind 376 pmol of ryanodine/mg of 12. Morii, H., and Tonomura, Y. (1983) J. Biochem. (Tokyo) 9 3 , protein if no inactivation occurs. Indeed, it would appear that 1271-1285 essentially all of the ryanodine binding activity has been 13. Kirino, Y.,Osakabe, M., and Shimizu, H. (1983) J. Biochem. recovered. (Tokyo) 94, 1111-1118 The size of the foot structure has been estimated to be 4.8 14. Nagasaki, K., and Kasai, M. (1983) J. Biochem. (Tokyo) 94, 1101-1109 x lo6 daltons which is about 3.9-fold higher than thevalue of 15. Fabiato, A. (1985) J. Gen. Physiol. 85, 291-320 the purified monomeric unit of the receptor (1,225,000 dal- 16. Ikemoto, N., Antoniu, B., and Mbszaros, L. G . (1985) J. Biol. tons, the sum of (360)2 330 175 kilodaltons). Taking into Chem. 2 6 0 , 14096-14100 account the 4-fold symmetry of the foot structure inprojection 17. Smith, J. S., Coronado, R., and Meissner, G . (1985) Nature 3 1 6 , 446-449 (Fig. l l ) , these results suggest that thefoot structure consists of an oligomer of four monomeric units of 1.2 x lo6 each. 18. Meissner, G., Darling, E., and Eveleth, J. (1986) Biochemistry 25,236-244 This calculation would also suggest that one binding site is 19. Jenden, D. J., and Fairhurst, A. S. (1969) Phurmacol. Reu. 2 1 , -1-25 composed of a dimer of 2.4 x lo6 daltons. Our present study shows that the foot structure is equiva- 20. Sutko, J. L., Ito, K., and Kenyon, J. L. (1985) Fed. Proc. 4 4 , lent to the ryanodine receptor. In turn, ryanodine in junc2984-2988 tional terminal cisternae was found to modulate ca2+release 21. Fleischer, S., Ogunbunmi, E. M., Dixon, M. C., and Fleer, E. A. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 7256-7259 suggesting that this structure in the junctional face membrane is directly involved in signal transduction and Ca2+release in 22. Meissner, G . (1986) J . Bid. Chem. 26196300-6306 23. Pessah, I.N., Waterhouse, A.L., and Casida, J. E. (1985) excitation-contraction coupling. Biochem. Bwphys. Res. Commun. 128,449-456
-
+
+
~~~
~~~
”
24. Pessah, I. N., Francini, A. O., Scales, D. J., Waterhouse, A. L., Acknowledgments-We thank William Wilson, Drs. Shan-Ping Shi and Casida, J. E. (1986) J. Biol. Chem. 2 6 1 , 8643-8648 and Sherry Wang for their help in preparing SR membrane fractions, 25. Costello, B., Chadwick, C., Saito, A., Chu, A., Maurer, A., and and Mark Dixon for his help inpreparing [3H]ryanodine. We also Fleischer, S. (1986) J. CellBiol. 103, 741-754 thank Dr. Christopher Chadwick for helpful discussion. 26. Kaplan, R. S., and Pedersen, P. L. (1985) Anal. Biochem. 1 5 0 , 97-104 27. Laemmli, U. K. (1970) Nature 227,680-685 REFERENCES 28. Merril, C. R., Goldmann, D., and Van Keuren, M. L. (1983) 1. Tada, M., Yamamoto, T., and Tonomura, Y. (1978) Physiol. Reu. Methods Enzymol. 96,230-239 58,l-79 29. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 2. Ikemoto, N. (1982) Annu. Reu. Physwl. 44, 297-317 (1951) J. Biol. Chem. 193, 265-275 3. Endo, M. (1977) Physiol. Reu. 5 7 , 71-108 30. Saito, A., Seiler, S., and Fleischer, S. (1984) J. Ultrastruct. Res. 4. Martonosi, A. (1984) Physwl. Reu. 6 4 , 1240-1320 86,277-293 5. de Meis, L., and Vianna, A. L. (1979) Annu. Reu. Biochem. 48, 31. Saito, A., Wang, C.-T., and Fleischer, S. (1978) J. Cell Biol. 7 9 , 275-292 601-616 6. Tanford, C. (1983) Annu. Reu. Biochem. 52,379-409 32. Caswell, A. H., and Brunschwig, J.-P. (1984) J. CellBiol. 99, 929-939 7. Fleischer, S. (1985) in Structure and Function of Sarcoplasmic
