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Interaction of ruthenium red with Ca2+-binding proteins
ANALYTICALBIOCHEMISTRY
188,123-131
Interaction Jeffrey
(1990)
of Ruthenium
H. M. Charuk,
Charles
Red with Ca*+-Binding
A. Pirraglia,
and Reinhart
MRC Group in Membrane Biology, Departments of Medicine University-of Toronto, Toronto, Ontario, Canada M5S IA8
Received
December
Academic
Press,
0003-2697/90 Copyright All rights
Clinical
Science Division,
4,1989
Inc.
The inorganic dye ruthenium red, [(NH&Ru-ORu(NH~)~-O-RU(NH~)~]C&. 4H20, was originally used as a histochemical stain for acidic glycosaminoglycans (1). The dye is relatively impermeable to intact cells but binds selectively to T-tubules and sarcoplasmic reticulum of skeletal and cardiac muscle (2-4). These membrane systems contain the dihydropyridineand ryanodine-sensitive calcium channels, respectively (5-9). Ruthenium red blocks Ca2+-induced Ca2+ release from both skinned slow or fast-twitch skeletal and cardiac muscle fibres (10-12) as well as sarcoplasmic reticulum vesicles isolated from either skeletal or cardiac muscle 1 To whom
A. F. Reithmeier’
and Biochemistry,
The interaction of ruthenium red, [(NH&Ru-ORu(NH~)~-O-RU(NH&,]C~~. 4Ha0, with various Ca’+binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (I& = 0.7 PM; B,, = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfatepolyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2+-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stainsall reaction. Ruthenium red bound to the same proteins detected by the 4sCa2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2+-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2+-binding proteins following electrophoresis. 0 1990
Proteins
correspondence
should
be addressed.
$3.00
0 1990 by Academic Press, of reproduction in any form
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tissue (13,14). Furthermore, both the ruthenium red inhibition and ryanodine sensitivity (15-20) of Ca2+-induced Ca2+ release are found associated with the heavy vesicle fraction derived from the terminal cisternae of sarcoplasmic reticulum (21). The ryanodine receptor of sarcoplasmic reticulum has now been purified (22) and its identity with the Ca2+release channel and feet structures of the junctional terminal cisternae confirmed (23,24). In addition to its interaction with the Ca2+-channel protein of sarcoplasmic reticulum, ruthenium red was reported to block energy-dependent Ca2+ uptake into isolated mitochondria (2526) and sarcoplasmic reticulum (27). Inhibition of ATP hydrolysis by ruthenium red has also been observed for the Ca2+ + Mg’+-dependent ATPases of muscle sarcoplasmic reticulum (27,28) and sarcolemma (29). The effect of ruthenium red is specific for Ca2+-ATPases since neither Na+/K+-ATPase nor Mg2+-ATPase activities in red blood cell membranes were affected (30). The reduced rates of ATP hydrolysis and Ca2+uptake by sarcoplasmic reticulum treated with ruthenium red was associated with altered phosphorylation kinetics of the Ca2+-ATPase enzyme (31). In addition to its effects on Ca2+ channel and Ca2’ATPase functions, ruthenium red reduced the Ca2+ binding to lipid-free protein extracts of sarcoplasmic reticulum (28). Calsequestrin is the major Ca2+-binding protein in sarcoplasmic reticulum (32). It is localized within the heavy vesicle fractions (33) derived from terminal cisternae where the highest density of ruthenium red staining was previously observed (2-4). We were therefore prompted to investigate the possible interaction of ruthenium red with calsequestrin and its effect on Ca2+binding. We report that ruthenium red inhibits Ca2+binding to calsequestrin and specifically stains this and other proteins known to bind Ca2+. METHODS
Muterids. Rabbit skeletal muscle calsequestrin was prepared by ammonium sulfate fractionation (34). Rab123
124
CHARUK,
PIRRAGLIA,
bit skeletal muscle sarcoplasmic reticulum was isolated by the method of Campbell and MacLennan (35). Bovine brain S-100 protein was a gift from Dr. Alexander Marks (Department of Medical Research Banting and Best Institute, University of Toronto). Cyanogen bromide-activated Sepharose 4B was obtained from Pharmacia Chemicals. Ruthenium red, Ponceau S, 3’,3’-diaminobenzidine, protein molecular weight standards, bovine brain calmodulin, and porcine skeletal muscle troponin were purchased from Sigma Chemical Co. Prestained protein molecular weight standards were obtained from Bio-Rad. Stains-all dye was an Eastman Kodak product. Endoglycosidase F (catalogue No. 878740) was purchased from Boehringer-Mannheim. Nitrocellulose was from Schleicher and Schuell. Immobilon was purchased from Millipore. Vectastain ABC and biotinylated antibody to rabbit IgG were Vector Laboratory products purchased from Dimension Labs. Calcium-45 was obtained from New England Nuclear. Purification of erythrocyte Band 3 and antibody production. Human red blood cell Band 3 protein was purified from osmotically lysed and stripped ghost membranes by amino ethyl and parachloromercurobenzoic acid-Sepharose chromatography (36). Band 3 protein (l-2 mg/ml) was deglycosylated by incubation at 22°C for 24 h with endoglycosidase F (0.5 unit/mg protein) in 0.1% 2-mercaptoethanol, 0.1% C&Es, 5 mM sodium phosphate (pH 7.5). A polyclonal rabbit antibody to purified human Band 3 protein was prepared as previously described (37). Immobilization of calsequestrin on Sepharose 4B. A 25-mg sample of calsequestrin was dissolved in 15 ml of coupling buffer (0.1 M NaHC03, 0.5 M NaCl (pH 8.3)), added to 6 ml (bed volume) of swollen, washed, cyanogen bromide-activated Sepharose 4B, and then allowed to react for 21 h at 4°C with gentle shaking. The resin was washed on a sintered-glass filter funnel three times with 10 ml of coupling buffer, once with 10 ml of 0.5 M NaCl, 0.1 M sodium acetate (pH 4.0), three times with 10 ml of 0.5 M NaCl, 0.1 M Tris-HCl (pH 8.0), and once with 10 ml of 10 mM Tris-HCl (pH 7.5). Calsequestrin-sepharose was stored as a 1:l suspension in 10 mM Tris-HCl (pH 7.5) at 4°C for no longer than 1 week prior to use. The quantity of protein attached to the resin was determined by hydrolysis in 6 N HCl at 110°C for 24 h followed by amino acid analysis. With this method, 4 mg of calsequestrin were routinely coupled to 1 ml of Sepharose 4B. Ca’+-binding assays. Aliquots (200 ~1) of calsequestrin-Sepharose (1:l suspension) were incubated in the presence or absence of various concentrations of MgClz, LaCL, or ruthenium red. A stock solution of 100 mM 45CaC12 (lo6 cpm/nmol) in 10 mM Tris-HCl (pH 7.5) was added to tubes along with appropriate amounts of 10 mM Tris-HCl (pH 7.5) to give various or fixed concentra-
AND
REITHMEIER
tions of 45Ca2+ in a total volume of 0.5 ml. The tubes were shaken briefly by hand, and after 10 min at 22°C three 50-~1 aliquots of each suspension were withdrawn and the samples including pipet tips were counted for radioactivity using a Beckman LS 7800 counter. The samples were then centrifuged briefly in a microfuge and three 50-~1 aliquots of each supernatant were withdrawn and counted for radioactivity. The amounts of Ca2+ bound to calsequestrin-Sepharose were determined from the differences in total and free radioactivities of 45Ca2f in the samples. Ruthenium red binding assays. Optical densities at 533 nm (38) of l-ml samples of 10 mM Tris-HCl (pH 7.5) containing various or fixed concentrations of ruthenium red in the presence or absence of various concentrations of CaCl,, MgC12, or LaCl, were measured using a Gilford spectrophotometer. Aliquots (25 ~1) of calsequestrinSepharose were added to 1.5-ml microfuge tubes and after 10 min at 22”C, the samples were centrifuged briefly in a microfuge. The optical densities of supernatants were measured and the amount of ruthenium red bound to calsequestrin was determined from the differences in absorbances of solutions before and after addition of resin. A linear standard absorption curve established the molar amounts of dye bound. Data analysis. Ruthenium red and 45Ca2+ binding to calsequestrin-Sepharose were analyzed with the Enzfitter nonlinear regression data analysis program developed for the IBM System 2 personal computer (written by Robin J. Leatherbarrow, Department of Chemistry, Imperial College of Science & Technology, South Kensington, London). Graphs were plotted using the Cricket program designed for use on the Macintosh personal computer. Dot blots of Optimization of ruthenium red binding. purified calsequestrin were used to visually estimate the optimal conditions required for ruthenium red staining. Aliquots (1 ~1) of a 2 mg/ml solution of calsequestrin were applied to l-cm2 pieces of nitrocellulose or Immobilon membranes. Unless otherwise specified, dot blots were incubated for 15 min at 22°C in 50 mM Tris-HCl (pH 8.0) and 25 mg/liter ruthenium red in the presence of different concentrations of various test compounds followed by destaining in the corresponding buffer without stain. Ruthenium red staining was also carried out at various pHs ranging from 2 to 11 using the following buffers at a concentration of 50 mM: glycine (pH 2-3); acetate (pH 4-5); Bis-tris2 (pH 6-7); Tris-HCl (pH 89); Caps (pH 10-11). Effects of ionic strength on ruthenium red staining were investigated using a range of * Abbreviations used: C&Es, octaethylene ether; Caps, 3-[cyclohexylaminol-1-propanesulfonic phate-buffered saline; SDS, sodium dodecyl hydroxyethyl)amino]tris(hydroxymethyl)methane.
glycol sulfate;
mono-n-dodecyl acid; PBS, phosBis-tris, [bis(Z-
RUTHENIUM
RED
STAINING
NaCl concentrations from 0.001 to 1.0 M. The time course required to reach maximal ruthenium red staining was monitored by incubating blots from 5 s to 24 h. The optimal dye concentration required for staining was investigated over a range of 0.6 rig/ml to 0.4 mg/ml ruthenium red. The sensitivity of staining was estimated by blotting 1 pg to 2 pugamounts of calsequestrin. Detection of Cazt-binding proteins following gel electrophoresis. Membranes or purified protein samples were electrophoresed on discontinuous, SDS-polyacrylamide minigels by the method of Laemmli (39) unless otherwise specified. Gels were either fixed in 10% isopropanol for 24 h or transferred to nitrocellulose paper electrophoretically (40). Gels were stained either with the carbocyanine dye Stains-all (41) or with 25 mg/liter ruthenium red in 60 mM KCl, 5 mM MgC&, 10 mM Tris-HCl (pH 7.5). Nitrocellulose transfers were first stained with Ponceau S, destained with PBS, and then overlaid with 1 &i/ml 45CaC12,60 mM KCl, 5 mM MgCI:!, 10 mM imidazole (pH 6.8) in the presence or absence of 50 mM CaCl*. Calcium-45 overlays were washed, air-dried, and autoradiographed as previously described (42). Nitrocellulose transfers were stained with 25 mg/liter ruthenium red in 60 mM KCl, 5 mM MgClz, 10 mM Tris-HCl (pH 7.5) in the presence or absence of 50 mM CaC&. Immunoblotting. Nitrocellulose transfers were immunoblotted by a modification of the method described by Olmsted (43). Transfers were blocked for 24 h in 0.25% gelatin, 10% ethanolamine, 0.1 M Tris-HCl (pH 9.0) and then incubated with a 1:lOOOdilution of aflinitypurified rabbit polyclonal antibody to human erythrocyte Band 3 protein in 0.25% gelatin, 0.05% NP-40, 5 mM EDTA, 0.15 M NaCl, 0.05 M Tris-HCl (pH 7.5) for 24 h. Bound antibodies were detected with affinity-purified, biotinylated second antibodies followed by reaction with Vectastain ABC. All incubations were conducted for 24-h periods at 22°C. Bound peroxidase was detected by incubating blots in 0.5 mg/ml 3’,3’-diaminobenzidene, 0.03% Hz02, 0.05 M Tris-HCI (pH 7.5). RESULTS
A relatively simple and rapid method for measuring Ca2+binding to calsequestrin was developed by coupling this protein to Sepharose 4B resin (44). Calsequestrin retained its ability to bind Ca” following coupling to Sepharose 4B and Scatchard analysis of the data revealed the presence of multiple binding sites with a range of affinities for Ca2+ (Fig. 1A). On the basis of a M, 42,435, determined from its cDNA sequence (45), 37 calcium ions were bound per immobilized calsequestrin molecule (B,,, = 882 nmol/mg protein; Kd = 0.5 mM), a similar number and average affinity of sites observed for the protein in solution (46). Although the number of Ca2+-binding sites on calsequestrin was similar in the presence of salt, the affinity of the protein for Ca2+ was
OF
Ca*+-BINDING
PROTEINS
125
considerably lower in the presence of 100 mM KC1 (Kd = 2.9 mM). Since sodium or choline chloride salts were equally as effective as KC1 in modulating the Ca2+-binding properties of calsequestrin, ionic strength appears to affect the binding affinity. In addition to the effect of salt, Ca2+ binding to calsequestrin was critically dependent on pH. Increased Ca2+ binding was observed at alkaline pH (1 pmol/mg protein at pH 9.0) while no Ca2+ bound at pH 4.0. Ruthenium red stained calsequestrin immobilized on Sepharose 4B a red color. Binding was measured by quantitating the decreased absorbance of samples at 533 nm (38) following incubation of the dye with aliquots of calsequestrin-Sepharose. Although the nonlinear Scatchard plot suggested various affinities of Ca2+-binding sites existed on calsequestrin, ruthenium red appeared to bind to a single class of high affinity sites (Fig. 1B). Only 10 binding sites for ruthenium red were detected on calsequestrin (B,,, = 218 nmol/mg protein; Kd = 0.7 PM), but the average affinity was 3 orders of magnitude greater for ruthenium red than Ca2+. The fewer number and higher affinity of ruthenium red binding sites on calsequestrin compared with Ca2+ could be attributed both to complexation of all three ruthenium cations in each dye molecule by the protein and recruitment of Ca2+-binding sites. Some recruitment of Ca2’binding sites by terbium has previously been noted for calsequestrin (47). No specific binding sites for either 45Ca2+or ruthenium red on uncoupled Sepharose 4B resin were observed in control experiments (not shown). The relationship between ruthenium red and Ca2+binding sites on calsequestrin was studied further. Table 1 shows that ruthenium red inhibited 45Ca2+binding to calsequestrin-Sepharose. Ca2+also inhibited ruthenium red binding to calsequestrin-Sepharose at similar concentrations required to saturate low affinity Ca2+binding sites (compare Table 1 with Fig. 1A). High concentrations of Mg2+ inhibited 45Ca2t binding to calsequestrin as previously reported (48). A similar concentration of Mg2’ also prevented the interaction of ruthenium red with calsequestrin (Table 1). Lanthanum ion, which can substitute for calcium ion (49), inhibited both the binding of 45Ca2+and ruthenium red to calsequestrin at similar, low concentrations (Table 1). Conditions for staining calsequestrin on either nitrocellulose or Immobilon membranes were optimized. Specific staining of protein dot blots occurred within a physiological pH range (pH 7.0-8.0). Low pH (~6.0) prevented staining of calsequestrin with ruthenium red while high pH (>lO.O) increased background binding of the dye to the membranes. At least 10 min was required to completely stain calsequestrin at 22°C and less than 1 pugof calsequestrin could be detected by concentrations of ruthenium red exceeding 5 hg/ml. The sensitivity of ruthenium red staining was critically dependent on the number of Ca2+-binding sites a particular protein pos-
126
CHARUK,
PIRRAGLIA,
'"""1 A
AND
REITHMEIER
b
Free [Ca],
mM
Free [Ruthenium
Red],pM
FIG. 1. Binding of Cazf and ruthenium red to calsequestrin. %a*+ (A) and ruthenium red (B) binding to calsequestrin immobilized on Sepharose 4B were measured as described under Methods. Scatchard analysis of data (insets) indicated that multiple binding sites for Ca2+ and ruthenium red existed. The curved nature of the Scatchard plot suggested that a range of affinities of Ca2+ for calsequestrin existed. Assuming linear relationships, 892 nmol Ca”/mg protein (Ka = 0.5 mM) and 218 nmol ruthenium red/mg protein (Kd = 0.7 jtM) could be maximally bound to calsequestrin-Sepharose.
sessed, however. Generally, 25 pg/ml of dye was used to achieve maximal staining of proteins in gels or on nitrocellulose. High ionic strength solutions (>lOO mM NaCl) inhibited staining of calsequestrin by ruthenium red. Concentrations of ATP or oxalate exceeding 1 mM diminished the staining of calsequestrin dramatically. ATP and oxalate anions likely prevented ruthenium red staining of calsequestrin by complexing with the dye as previously suggested (27). A solution that gave the greatest specificity for staining calsequestrin in gels or on ni-
TABLE
1
Inhibition of Cazf and Ruthenium Red Binding to Calsequestrin %a’+
binding
Ruthenium Mg2+ Las+
red
Ruthenium
I(, 72
ELM
12 mM 16
/.tM
red binding Ca’+ Mg2+ La3+
Ki
1.4 mM 30 mM 48
/.tM
Note. Binding of ‘%a’+ and ruthenium red to calsequestrin-sepharose was measured as described under Methods. Initial concentrations of %a*+ and ruthenium red used in the assays were 1 mM and 4 pM, respectively. Inhibition constants were determined by linear regression analysis of Dixon plots of binding data.
trocellulose was 25 PM ruthenium red, 60 mM KCl, 5 mM MgClz, 10 mM Tris-HCl (pH 7.5). The specificity of ruthenium red binding was investigated further by staining different types of Ca2+-binding proteins in SDS-polyacrylamide gels after reaction with Stains-all or on nitrocellulose transfers following calcium-45 autoradiography. Figure 2 shows the ruthenium red staining pattern of rabbit skeletal muscle sarcoplasmic reticulum proteins and a corresponding calcium-45 autoradiograph of the nitrocellulose transfer. Although no molecular weight standards were stained, both the lOO-kDa Ca2+-ATPase and 55-kDa calsequestrin proteins bound ruthenium red and 45Ca2+. The sensitivity of ruthenium red staining compared to calcium-45 autoradiography was greater because high background radioactivity detected with the overlay technique was difficult to reduce relative to specific 45Ca2+ binding. Lengthy or repeated washing of nitrocellulose transfers also reduced the amount of 45Ca2+ bound to Ca2’-ATPase and calsequestrin proteins. In addition to its greater sensitivity, the ruthenium red detection method was more rapid (10 min) than calcium-45 autoradiography which required 4 days of film exposure. The binding of ruthenium red or 45Ca2’ to either of these proteins was competed by 50 mM CaC12 (not shown). Similarly, nitrocellulose transfers or SDS-polyacrylamide gels of
RUTHENIUM
RED
STAINING 2+
-n-
PonS
RR
45Ca
‘2:: 66-
T-2459 362 L 2924-
2014-
FIG. 2. Ruthenium red staining of sarcoplasmic reticulum proteins. Samples of rabbit skeletal muscle sarcoplasmic reticulum membranes (SR) and purified calsequestrin (CS), corresponding to 20 and 2 pg of protein, respectively, were electrophoresed on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose paper as described under Methods. Nitrocellulose was first stained with Ponceau S (PonS) to reveal transferred proteins followed by calcium-45 autoradiography (45Ca2’) and then stained with ruthenium red (RR) to detect Ca2+-binding proteins as described under Methods. The arrowhead indicates the position of the lOO-kDa Ca2+-ATPase protein. An arrow shows the position of the 55kDa calsequestrin protein. MW represents the molecular weight standards, @-galactosidase (116 kDa), phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), and a-lactalbumin (14 kDa).
OF
Ca’+-BINDING
pared with calcium-45 autoradiography, ruthenium red stained these proteins on nitrocellulose transfers more sensitively. Although these protein bands were a blue color following reaction of polyacrylamide gels with Stains-all, as previously reported (41), considerable diffusion occurred during fixation due to their low AI,. Bovine brain calmodulin also was a blue color with Stains-all, but it failed to bind to nitrocellulose during electrotransfer as previously reported (52). Samples of highly purified calmodulin spotted directly onto nitrocellulose paper stained with ruthenium red. Less than 1 pugof calmodulin could be detected by ruthenium red and 50 mM CaCl, blocked the binding of the dye (not shown). Since ruthenium red is cationic and may simply interact with negatively charged groups on proteins the specificity of the dye for detecting only Ca’+-binding proteins was examined in another fashion. Proteins with various known isoelectric points were electrophoresed then either stained with ruthenium red or Stains-all or autoradiographed following calcium-45 overlay. As Fig. 4 shows, acidic proteins such as glucose oxidase and trypsin inhibitor (lanes b and c, respectively) were detected by ruthenium red better than basic proteins such as cytochrome c and trypsinogen (lanes a and e, respectively). However, these same acidic proteins also bound more calcium-45 than basic proteins. Conversely, Stains-all was less specific since it failed to detect trypsin inhibitor
RR sarcoplasmic reticulum proteins stained with ruthenium red could be completely destained in less than 30 min with 50 mM CaCl,, 10 mM Tris-HCl (pH 7.5). Although more Ca’+-ATPase was present in the sarcoplasmic reticulum preparation relative to calsequestrin, as judged by Ponceau S staining, the amount of ruthenium red bound to these protein bands was similar. This result could be accounted for by considering that 40-50 Ca2+ bind to 1 molecule of calsequestrin (46) whereas only 2 high affinity Ca2+-transport sites are associated with the Ca2+-ATPase enzyme (50). Although calsequestrin has previously been identified on SDS-polyacrylamide gels by staining blue with the cationic carbocyanine dye Stains-all, Ca2+-ATPase appears only as a light, pink band (41). It has therefore been difficult to unequivocally identify all types of Ca2+-binding proteins using the Stains-all detection method. A class of calcium-modulated proteins, which characteristically possesshigh-affinity Ca’+-binding sites that have a common EF hand structural conformation (51), were studied for their ability to bind ruthenium red. As Fig. 3 shows, ruthenium red stained both bovine brain S-100 protein and porcine skeletal muscle troponin C subunit on nitrocellulose transfers. Again, com-
127
PROTEINS
45d+,
I1
SA ,
MW SlOO Tn SlOO Tn SlOO
Tn
+ ^*
-&i
39-
2 V
27L
2
17-
FIG. 3. Ruthenium red staining of EF hand Ca’+-binding proteins. Samples (10 pg) of purified S-100 (SlOO) and troponin (Tn) proteins were electrophoresed on a 15% SDS-polyacrylamide gel and then either reacted with Stains-all or transferred to 0.05-pm pore size nitrocellulose paper as described under Methods. Nitrocellulose transfers were first calcium-45 autoradiographed and then stained with ruthenium red (RR). The arrowhead indicates the position of the 20-kDa troponin C subunit. An arrow shows the position of the lo-kDa S-100 protein. MW represents the prestained molecular weight standards, phosphorylase (135 kDa), bovine serum albumin (75 kDa), ovalbumin (50 kDa), carbonic anhydrase (39 kDa), soybean trypsin inhibitor (27 kDa), and lysozyme (17 kDa).
128
CHARUK,
PonS
45Ca*+
PIRRAGLIA,
SA
MWabcdeabcdeabcdeabCde’
FIG. 4. Ruthenium red staining of various isoelectric point proteins. Samples of cytochrome c (p/9.2), glucose oxidase (pl4.2), trypsin inhibitor (pZ4.6), carbonic anhydrase (pl6.6), and trypsinogen (pZ9.3), corresponding to 10 pg of protein each (lanes a-e, respectively), were electrophoresed on 16.5% Tricine-SDS polyacrylamide minigels (71). Proteins were either stained in the gel with Stains-all (SA) or transferred to nitrocellulose paper and stained with Ponceau S (PonS) followed by calcium-45 autoradiography (@‘Ca”) and reaction with ruthenium red (RR) as described under Methods.
(lane c) and also reacted with basic proteins such as trypsinogen (lane e). These results suggest that although ruthenium red may react with negatively charged groups on acidic proteins, these sites are also capable of Ca*+ binding. Ruthenium red was also used to detect Ca*+-binding proteins in membranes prepared from various tissues. The dye stained few proteins associated with dog kidney brush border membranes and only bound to some unresolved, high M, material on SDS-polyacrylamide gels of renal antiluminal membranes (not shown). Calcium-45 autoradiography and Stains-all also failed to detect any distinct Ca2+-binding proteins in these same membrane preparations. Ruthenium red detected two proteins in canine heart plasma membranes having an approximate M, of 100 and 130 kDa (not shown). These proteins corresponded to the two major Ca2+ and wheat germ agglutinin binding glycoproteins in bovine cardiac sarcolemma (53). These glycoproteins have recently been purified by lectin chromatography (54) and their reactivity with ruthenium red confirmed (M. Michalak, personal communication). Ruthenium red also reacted with at least three proteins in human red cell ghost membranes and binding was competed by Ca2+ (not shown). Similar to erythrocyte spectrins two high molecular weight proteins (>200 kDa) were removed from membranes by 1 mM EDTA and 1 M Kl and a corresponding loss of ruthenium red staining of these components on gels of extracted ghosts was observed (not shown). The amino acid sequence of cw-spectrin deduced from its nucleotide sequence has been shown to contain the equivalent of two EF hand structures (55), suggesting it may indeed bind Ca*+. Another intrinsic membrane protein, the red cell anion transporter, was previously reported to bind Ca2+ (56) and a protein having a M, similar to Band 3 also reacted with Stains-all to give a purple color (57). The interaction of Stains-all with components of the erythrocyte membrane was believed to be due to sialic acid
AND
REITHMEIER
residues on glycoproteins since samples treated with neuraminidase had reduced staining. A protein of 100 kDa in red cell ghost membranes also stained with ruthenium red (not shown). We investigated whether highly purified, deglycosylated, human Band 3 protein bound ruthenium red or Ca2+. As Fig. 5 shows, binding of ruthenium red and 45Ca2+to the lOO-kDa Band 3 protein was competed by 50 mM CaCl,. Ruthenium red detected Band 3 more sensitively than calcium-45 autoradiography of nitrocellulose transfers. Minor amounts of Band 3 proteolytic fragments detected by antibodies also bound ruthenium red and 45Ca2f. The smallest peptide fragment that remained both immunologically reactive and bound ruthenium red or 45Ca2+had a M, of only 23 kDa (Fig. 5). Since rabbit polyclonal antibodies prepared to the human erythrocyte anion transporter were directed solely toward the cytoplasmic, amino-terminal domain, as previously reported (58), the Ca*+-binding site must be localized within this region. Stains-all reacted only weakly with Band 3 regardless of whether the protein had been purified or deglycosylated prior to electrophoresis (not shown). Although Band 3 stained a pink color, another intrinsic membrane protein, which comigrated with the anion transporter when samples of red cell ghost membranes were electrophoresed, did stain a blue color on gels (not shown). This component presumably represented glycophorin, which has a similar mobility as Band 3 and contains a high concentration of sialic acid in its numerous carbohydrate structures (59). Interestingly, although glycophorin reacted strongly with Stains-all, it failed to bind noticeable
MW
-+-+
FIG. 5. Ruthenium red staining of erythrocyte Band 3 protein. A lo-rg sample of purified, deglycosylated human red blood cell membrane Band 3 protein was electrophoresed on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose paper as described under Methods. Nitrocellulose was first stained with Ponceau S (PonS) then calcium-45 autoradiographed (45CaZ+) and reacted with ruthenium red (RR) in the presence (+) or absence (-) of 50 mM CaCl,. An immunoblot (Ab) was formed by reacting the nitrocellulose transfer with a rabbit polyclonal antibody to human erythrocyte Band 3 protein. The arrowhead indicates the position of the lOO-kDa Band 3 protein. MW represents molecular weight standards as in Fig. 2.
RUTHENIUM
RED
STAINING
amounts of either 45Ca2f or ruthenium red (not shown). These results suggested Stains-all also reacts with proteins that do not necessarily possess Ca’+-binding sites. DISCUSSION
Two methods of identifying Ca2+-binding proteins following polyacrylamide gel electrophoresis have previously been described and each has been used extensively. The cationic carbocyanine dye, Stains-all was shown to bind to various known Ca2+-binding proteins turning them a blue color (41). However, in addition to its light sensitivity, its inability to detect several proteins known to contain Ca2+-binding sites such as the sarcoplasmic reticulum Ca2+-ATPase and its reactivity toward acidic sialoglycoproteins such as glycophorin, which is not known to bind Ca2+, limits the usefulness of Stains-all as a specific stain for Ca2+-binding proteins. Calcium-45 autoradiography of SDS-polyacrylamide gels (60) or more frequently, nitrocellulose transfers (42) have been popular techniques used to detect Ca2’-binding proteins. However, the low affinity of some proteins for Ca2+ (e.g., calsequestrin), the high background radioactivity, and the long exposure time required for autoradiographs, as well as the danger in handling this radioisotope, make this method unsuitable for routine detection of some Ca2+-binding proteins. These inherent problems with both techniques prompted the search for a relatively rapid, sensitive, safe, and inexpensive method for specifically detecting all classes of Ca2+binding proteins. The ability of ruthenium red to stain glycosaminoglycan-containing extracellular matrix (1) and some subcellular organelles (2-4), as well as to block Ca2+ channels (15-19) and inhibit various Ca2+-transport ATPases (27-30), suggested a possible interaction of this dye directly with Ca2+-binding sites. We found that ruthenium red not only reversibly bound with high affinity to the muscle protein calsequestrin, but also stained a wide assortment of proteins known to contain Ca2+-binding sites. Furthermore, ruthenium red detected Ca2+-binding proteins more sensitively than calcium-45 autoradiography and more reliably than Stainsall. Moreover, the interaction of ruthenium red with Ca2+-binding sites is extremely specific since binding was competed by Ca2+ and the dye failed to stain either kidney membrane proteins or protein molecular weight standards. Although ruthenium red stained acidic but not basic proteins the latter also failed to bind Ca2+. A good correlation between Ca2+-binding and ruthenium red staining of proteins was therefore observed. Glycosaminoglycans were originally thought to be involved in mitochondrial energy transduction based on the inhibitory effects of ruthenium red on Ca2+-transport (25) and its interaction with these components (1). Considering our present findings and their known extra-
OF
Ca’+-BINDING
PROTEINS
129
cellular localization, it is unlikely glycosaminoglycans are involved in mitochondrial function. Rather, mitochondrial Ca2+ fluxes mediated by specific ATPases or uniporters are more likely targets for the inhibitory action of ruthenium red (61). The effects of ruthenium red on Ca2+ fluxes in subcellular organelles is complex probably due to its multiple interactions with Ca2+-binding sites on ATPases, channels, and storage proteins such as calsequestrin. For example, Ca2+ transport by microsome fractions is considerably less sensitive to ruthenium red than mitochondria (26). The ruthenium redinsensitive Ca2+-transport activity is highest in heavy microsome fractions enriched in endoplasmic reticulum (62). The apparent resistance of endoplasmic reticulum Ca2+-ATPase to the inhibitory effects of ruthenium red may be due to several reasons. First, since Ca2+competes with dye-binding sites, excessive Ca2+ concentrations used in transport assays may prevent ruthenium red inhibition of Ca2+ uptake. Alternatively, blockade of Ca2+ channels by ruthenium red may increase Ca2+-loading rates into endoplasmic reticulum vesicles, thereby masking any effects of the dye on the Ca2’-ATPase. Finally, complexation of ruthenium red by anions such as oxalate and ATP, present in Ca2+-transport assays, may reduce its effective concentration, thereby preventing enzyme inhibition. The electrophysiological effects of ruthenium red on cells are also complex and have only been studied in detail for frog skeletal muscle fibers (63,64). The increased twitch potentiation caused by low amounts of dye (
130
CHARUK,
PIRRAGLIA,
binding domains. Purification and amino acid sequencing of these peptides may allow us to predict the structure of binding sites involved in Ca2+ translocation. These studies would help confirm the identity of residues involved in Ca2+ binding and transport predicted from site-directed mutagenesis experiments on proteins such as the Ca2+-ATPase of sarcoplasmic reticulum (70). We are currently taking advantage of our findings by attempting to immobilize ruthenium red on a resin suitable for protein chromatography. We are also using ruthenium red to identify new membrane Ca2+-binding proteins while continuing to investigate known proteins for their possible interaction with the dye. To date we have observed specific binding of ruthenium red to calreticulin and the 160-kDa glycoprotein of sarcoplasmic reticulum (sarcalumenin), and both the dihydropyridine- and ryanodine-sensitive Ca2’ channels of skeletal muscle (unpublished observations). Since ruthenium red has a characteristic absorption spectrum (38), we also hope to use the dye in spectrophotometric studies designed to investigate the mechanism of Ca2+ binding and translocation by membrane transporters and channels. We therefore feel that ruthenium red will be an extremely useful and versatile probe of the structure and function of Ca2+-binding sites in future protein biochemical studies. ACKNOWLEDGMENTS This work was funded by grants from the Medical Research Council of Canada and the Muscular Dystrophy Association of Canada to R.A.F.R. J.H.M.C. is a recipient of a postdoctoral fellowship from the Medical Research Council of Canada.
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188,123-131
Interaction Jeffrey
(1990)
of Ruthenium
H. M. Charuk,
Charles
Red with Ca*+-Binding
A. Pirraglia,
and Reinhart
MRC Group in Membrane Biology, Departments of Medicine University-of Toronto, Toronto, Ontario, Canada M5S IA8
Received
December
Academic
Press,
0003-2697/90 Copyright All rights
Clinical
Science Division,
4,1989
Inc.
The inorganic dye ruthenium red, [(NH&Ru-ORu(NH~)~-O-RU(NH~)~]C&. 4H20, was originally used as a histochemical stain for acidic glycosaminoglycans (1). The dye is relatively impermeable to intact cells but binds selectively to T-tubules and sarcoplasmic reticulum of skeletal and cardiac muscle (2-4). These membrane systems contain the dihydropyridineand ryanodine-sensitive calcium channels, respectively (5-9). Ruthenium red blocks Ca2+-induced Ca2+ release from both skinned slow or fast-twitch skeletal and cardiac muscle fibres (10-12) as well as sarcoplasmic reticulum vesicles isolated from either skeletal or cardiac muscle 1 To whom
A. F. Reithmeier’
and Biochemistry,
The interaction of ruthenium red, [(NH&Ru-ORu(NH~)~-O-RU(NH&,]C~~. 4Ha0, with various Ca’+binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (I& = 0.7 PM; B,, = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfatepolyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2+-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stainsall reaction. Ruthenium red bound to the same proteins detected by the 4sCa2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2+-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2+-binding proteins following electrophoresis. 0 1990
Proteins
correspondence
should
be addressed.
$3.00
0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
tissue (13,14). Furthermore, both the ruthenium red inhibition and ryanodine sensitivity (15-20) of Ca2+-induced Ca2+ release are found associated with the heavy vesicle fraction derived from the terminal cisternae of sarcoplasmic reticulum (21). The ryanodine receptor of sarcoplasmic reticulum has now been purified (22) and its identity with the Ca2+release channel and feet structures of the junctional terminal cisternae confirmed (23,24). In addition to its interaction with the Ca2+-channel protein of sarcoplasmic reticulum, ruthenium red was reported to block energy-dependent Ca2+ uptake into isolated mitochondria (2526) and sarcoplasmic reticulum (27). Inhibition of ATP hydrolysis by ruthenium red has also been observed for the Ca2+ + Mg’+-dependent ATPases of muscle sarcoplasmic reticulum (27,28) and sarcolemma (29). The effect of ruthenium red is specific for Ca2+-ATPases since neither Na+/K+-ATPase nor Mg2+-ATPase activities in red blood cell membranes were affected (30). The reduced rates of ATP hydrolysis and Ca2+uptake by sarcoplasmic reticulum treated with ruthenium red was associated with altered phosphorylation kinetics of the Ca2+-ATPase enzyme (31). In addition to its effects on Ca2+ channel and Ca2’ATPase functions, ruthenium red reduced the Ca2+ binding to lipid-free protein extracts of sarcoplasmic reticulum (28). Calsequestrin is the major Ca2+-binding protein in sarcoplasmic reticulum (32). It is localized within the heavy vesicle fractions (33) derived from terminal cisternae where the highest density of ruthenium red staining was previously observed (2-4). We were therefore prompted to investigate the possible interaction of ruthenium red with calsequestrin and its effect on Ca2+binding. We report that ruthenium red inhibits Ca2+binding to calsequestrin and specifically stains this and other proteins known to bind Ca2+. METHODS
Muterids. Rabbit skeletal muscle calsequestrin was prepared by ammonium sulfate fractionation (34). Rab123
124
CHARUK,
PIRRAGLIA,
bit skeletal muscle sarcoplasmic reticulum was isolated by the method of Campbell and MacLennan (35). Bovine brain S-100 protein was a gift from Dr. Alexander Marks (Department of Medical Research Banting and Best Institute, University of Toronto). Cyanogen bromide-activated Sepharose 4B was obtained from Pharmacia Chemicals. Ruthenium red, Ponceau S, 3’,3’-diaminobenzidine, protein molecular weight standards, bovine brain calmodulin, and porcine skeletal muscle troponin were purchased from Sigma Chemical Co. Prestained protein molecular weight standards were obtained from Bio-Rad. Stains-all dye was an Eastman Kodak product. Endoglycosidase F (catalogue No. 878740) was purchased from Boehringer-Mannheim. Nitrocellulose was from Schleicher and Schuell. Immobilon was purchased from Millipore. Vectastain ABC and biotinylated antibody to rabbit IgG were Vector Laboratory products purchased from Dimension Labs. Calcium-45 was obtained from New England Nuclear. Purification of erythrocyte Band 3 and antibody production. Human red blood cell Band 3 protein was purified from osmotically lysed and stripped ghost membranes by amino ethyl and parachloromercurobenzoic acid-Sepharose chromatography (36). Band 3 protein (l-2 mg/ml) was deglycosylated by incubation at 22°C for 24 h with endoglycosidase F (0.5 unit/mg protein) in 0.1% 2-mercaptoethanol, 0.1% C&Es, 5 mM sodium phosphate (pH 7.5). A polyclonal rabbit antibody to purified human Band 3 protein was prepared as previously described (37). Immobilization of calsequestrin on Sepharose 4B. A 25-mg sample of calsequestrin was dissolved in 15 ml of coupling buffer (0.1 M NaHC03, 0.5 M NaCl (pH 8.3)), added to 6 ml (bed volume) of swollen, washed, cyanogen bromide-activated Sepharose 4B, and then allowed to react for 21 h at 4°C with gentle shaking. The resin was washed on a sintered-glass filter funnel three times with 10 ml of coupling buffer, once with 10 ml of 0.5 M NaCl, 0.1 M sodium acetate (pH 4.0), three times with 10 ml of 0.5 M NaCl, 0.1 M Tris-HCl (pH 8.0), and once with 10 ml of 10 mM Tris-HCl (pH 7.5). Calsequestrin-sepharose was stored as a 1:l suspension in 10 mM Tris-HCl (pH 7.5) at 4°C for no longer than 1 week prior to use. The quantity of protein attached to the resin was determined by hydrolysis in 6 N HCl at 110°C for 24 h followed by amino acid analysis. With this method, 4 mg of calsequestrin were routinely coupled to 1 ml of Sepharose 4B. Ca’+-binding assays. Aliquots (200 ~1) of calsequestrin-Sepharose (1:l suspension) were incubated in the presence or absence of various concentrations of MgClz, LaCL, or ruthenium red. A stock solution of 100 mM 45CaC12 (lo6 cpm/nmol) in 10 mM Tris-HCl (pH 7.5) was added to tubes along with appropriate amounts of 10 mM Tris-HCl (pH 7.5) to give various or fixed concentra-
AND
REITHMEIER
tions of 45Ca2+ in a total volume of 0.5 ml. The tubes were shaken briefly by hand, and after 10 min at 22°C three 50-~1 aliquots of each suspension were withdrawn and the samples including pipet tips were counted for radioactivity using a Beckman LS 7800 counter. The samples were then centrifuged briefly in a microfuge and three 50-~1 aliquots of each supernatant were withdrawn and counted for radioactivity. The amounts of Ca2+ bound to calsequestrin-Sepharose were determined from the differences in total and free radioactivities of 45Ca2f in the samples. Ruthenium red binding assays. Optical densities at 533 nm (38) of l-ml samples of 10 mM Tris-HCl (pH 7.5) containing various or fixed concentrations of ruthenium red in the presence or absence of various concentrations of CaCl,, MgC12, or LaCl, were measured using a Gilford spectrophotometer. Aliquots (25 ~1) of calsequestrinSepharose were added to 1.5-ml microfuge tubes and after 10 min at 22”C, the samples were centrifuged briefly in a microfuge. The optical densities of supernatants were measured and the amount of ruthenium red bound to calsequestrin was determined from the differences in absorbances of solutions before and after addition of resin. A linear standard absorption curve established the molar amounts of dye bound. Data analysis. Ruthenium red and 45Ca2+ binding to calsequestrin-Sepharose were analyzed with the Enzfitter nonlinear regression data analysis program developed for the IBM System 2 personal computer (written by Robin J. Leatherbarrow, Department of Chemistry, Imperial College of Science & Technology, South Kensington, London). Graphs were plotted using the Cricket program designed for use on the Macintosh personal computer. Dot blots of Optimization of ruthenium red binding. purified calsequestrin were used to visually estimate the optimal conditions required for ruthenium red staining. Aliquots (1 ~1) of a 2 mg/ml solution of calsequestrin were applied to l-cm2 pieces of nitrocellulose or Immobilon membranes. Unless otherwise specified, dot blots were incubated for 15 min at 22°C in 50 mM Tris-HCl (pH 8.0) and 25 mg/liter ruthenium red in the presence of different concentrations of various test compounds followed by destaining in the corresponding buffer without stain. Ruthenium red staining was also carried out at various pHs ranging from 2 to 11 using the following buffers at a concentration of 50 mM: glycine (pH 2-3); acetate (pH 4-5); Bis-tris2 (pH 6-7); Tris-HCl (pH 89); Caps (pH 10-11). Effects of ionic strength on ruthenium red staining were investigated using a range of * Abbreviations used: C&Es, octaethylene ether; Caps, 3-[cyclohexylaminol-1-propanesulfonic phate-buffered saline; SDS, sodium dodecyl hydroxyethyl)amino]tris(hydroxymethyl)methane.
glycol sulfate;
mono-n-dodecyl acid; PBS, phosBis-tris, [bis(Z-
RUTHENIUM
RED
STAINING
NaCl concentrations from 0.001 to 1.0 M. The time course required to reach maximal ruthenium red staining was monitored by incubating blots from 5 s to 24 h. The optimal dye concentration required for staining was investigated over a range of 0.6 rig/ml to 0.4 mg/ml ruthenium red. The sensitivity of staining was estimated by blotting 1 pg to 2 pugamounts of calsequestrin. Detection of Cazt-binding proteins following gel electrophoresis. Membranes or purified protein samples were electrophoresed on discontinuous, SDS-polyacrylamide minigels by the method of Laemmli (39) unless otherwise specified. Gels were either fixed in 10% isopropanol for 24 h or transferred to nitrocellulose paper electrophoretically (40). Gels were stained either with the carbocyanine dye Stains-all (41) or with 25 mg/liter ruthenium red in 60 mM KCl, 5 mM MgC&, 10 mM Tris-HCl (pH 7.5). Nitrocellulose transfers were first stained with Ponceau S, destained with PBS, and then overlaid with 1 &i/ml 45CaC12,60 mM KCl, 5 mM MgCI:!, 10 mM imidazole (pH 6.8) in the presence or absence of 50 mM CaCl*. Calcium-45 overlays were washed, air-dried, and autoradiographed as previously described (42). Nitrocellulose transfers were stained with 25 mg/liter ruthenium red in 60 mM KCl, 5 mM MgClz, 10 mM Tris-HCl (pH 7.5) in the presence or absence of 50 mM CaC&. Immunoblotting. Nitrocellulose transfers were immunoblotted by a modification of the method described by Olmsted (43). Transfers were blocked for 24 h in 0.25% gelatin, 10% ethanolamine, 0.1 M Tris-HCl (pH 9.0) and then incubated with a 1:lOOOdilution of aflinitypurified rabbit polyclonal antibody to human erythrocyte Band 3 protein in 0.25% gelatin, 0.05% NP-40, 5 mM EDTA, 0.15 M NaCl, 0.05 M Tris-HCl (pH 7.5) for 24 h. Bound antibodies were detected with affinity-purified, biotinylated second antibodies followed by reaction with Vectastain ABC. All incubations were conducted for 24-h periods at 22°C. Bound peroxidase was detected by incubating blots in 0.5 mg/ml 3’,3’-diaminobenzidene, 0.03% Hz02, 0.05 M Tris-HCI (pH 7.5). RESULTS
A relatively simple and rapid method for measuring Ca2+binding to calsequestrin was developed by coupling this protein to Sepharose 4B resin (44). Calsequestrin retained its ability to bind Ca” following coupling to Sepharose 4B and Scatchard analysis of the data revealed the presence of multiple binding sites with a range of affinities for Ca2+ (Fig. 1A). On the basis of a M, 42,435, determined from its cDNA sequence (45), 37 calcium ions were bound per immobilized calsequestrin molecule (B,,, = 882 nmol/mg protein; Kd = 0.5 mM), a similar number and average affinity of sites observed for the protein in solution (46). Although the number of Ca2+-binding sites on calsequestrin was similar in the presence of salt, the affinity of the protein for Ca2+ was
OF
Ca*+-BINDING
PROTEINS
125
considerably lower in the presence of 100 mM KC1 (Kd = 2.9 mM). Since sodium or choline chloride salts were equally as effective as KC1 in modulating the Ca2+-binding properties of calsequestrin, ionic strength appears to affect the binding affinity. In addition to the effect of salt, Ca2+ binding to calsequestrin was critically dependent on pH. Increased Ca2+ binding was observed at alkaline pH (1 pmol/mg protein at pH 9.0) while no Ca2+ bound at pH 4.0. Ruthenium red stained calsequestrin immobilized on Sepharose 4B a red color. Binding was measured by quantitating the decreased absorbance of samples at 533 nm (38) following incubation of the dye with aliquots of calsequestrin-Sepharose. Although the nonlinear Scatchard plot suggested various affinities of Ca2+-binding sites existed on calsequestrin, ruthenium red appeared to bind to a single class of high affinity sites (Fig. 1B). Only 10 binding sites for ruthenium red were detected on calsequestrin (B,,, = 218 nmol/mg protein; Kd = 0.7 PM), but the average affinity was 3 orders of magnitude greater for ruthenium red than Ca2+. The fewer number and higher affinity of ruthenium red binding sites on calsequestrin compared with Ca2+ could be attributed both to complexation of all three ruthenium cations in each dye molecule by the protein and recruitment of Ca2+-binding sites. Some recruitment of Ca2’binding sites by terbium has previously been noted for calsequestrin (47). No specific binding sites for either 45Ca2+or ruthenium red on uncoupled Sepharose 4B resin were observed in control experiments (not shown). The relationship between ruthenium red and Ca2+binding sites on calsequestrin was studied further. Table 1 shows that ruthenium red inhibited 45Ca2+binding to calsequestrin-Sepharose. Ca2+also inhibited ruthenium red binding to calsequestrin-Sepharose at similar concentrations required to saturate low affinity Ca2+binding sites (compare Table 1 with Fig. 1A). High concentrations of Mg2+ inhibited 45Ca2t binding to calsequestrin as previously reported (48). A similar concentration of Mg2’ also prevented the interaction of ruthenium red with calsequestrin (Table 1). Lanthanum ion, which can substitute for calcium ion (49), inhibited both the binding of 45Ca2+and ruthenium red to calsequestrin at similar, low concentrations (Table 1). Conditions for staining calsequestrin on either nitrocellulose or Immobilon membranes were optimized. Specific staining of protein dot blots occurred within a physiological pH range (pH 7.0-8.0). Low pH (~6.0) prevented staining of calsequestrin with ruthenium red while high pH (>lO.O) increased background binding of the dye to the membranes. At least 10 min was required to completely stain calsequestrin at 22°C and less than 1 pugof calsequestrin could be detected by concentrations of ruthenium red exceeding 5 hg/ml. The sensitivity of ruthenium red staining was critically dependent on the number of Ca2+-binding sites a particular protein pos-
126
CHARUK,
PIRRAGLIA,
'"""1 A
AND
REITHMEIER
b
Free [Ca],
mM
Free [Ruthenium
Red],pM
FIG. 1. Binding of Cazf and ruthenium red to calsequestrin. %a*+ (A) and ruthenium red (B) binding to calsequestrin immobilized on Sepharose 4B were measured as described under Methods. Scatchard analysis of data (insets) indicated that multiple binding sites for Ca2+ and ruthenium red existed. The curved nature of the Scatchard plot suggested that a range of affinities of Ca2+ for calsequestrin existed. Assuming linear relationships, 892 nmol Ca”/mg protein (Ka = 0.5 mM) and 218 nmol ruthenium red/mg protein (Kd = 0.7 jtM) could be maximally bound to calsequestrin-Sepharose.
sessed, however. Generally, 25 pg/ml of dye was used to achieve maximal staining of proteins in gels or on nitrocellulose. High ionic strength solutions (>lOO mM NaCl) inhibited staining of calsequestrin by ruthenium red. Concentrations of ATP or oxalate exceeding 1 mM diminished the staining of calsequestrin dramatically. ATP and oxalate anions likely prevented ruthenium red staining of calsequestrin by complexing with the dye as previously suggested (27). A solution that gave the greatest specificity for staining calsequestrin in gels or on ni-
TABLE
1
Inhibition of Cazf and Ruthenium Red Binding to Calsequestrin %a’+
binding
Ruthenium Mg2+ Las+
red
Ruthenium
I(, 72
ELM
12 mM 16
/.tM
red binding Ca’+ Mg2+ La3+
Ki
1.4 mM 30 mM 48
/.tM
Note. Binding of ‘%a’+ and ruthenium red to calsequestrin-sepharose was measured as described under Methods. Initial concentrations of %a*+ and ruthenium red used in the assays were 1 mM and 4 pM, respectively. Inhibition constants were determined by linear regression analysis of Dixon plots of binding data.
trocellulose was 25 PM ruthenium red, 60 mM KCl, 5 mM MgClz, 10 mM Tris-HCl (pH 7.5). The specificity of ruthenium red binding was investigated further by staining different types of Ca2+-binding proteins in SDS-polyacrylamide gels after reaction with Stains-all or on nitrocellulose transfers following calcium-45 autoradiography. Figure 2 shows the ruthenium red staining pattern of rabbit skeletal muscle sarcoplasmic reticulum proteins and a corresponding calcium-45 autoradiograph of the nitrocellulose transfer. Although no molecular weight standards were stained, both the lOO-kDa Ca2+-ATPase and 55-kDa calsequestrin proteins bound ruthenium red and 45Ca2+. The sensitivity of ruthenium red staining compared to calcium-45 autoradiography was greater because high background radioactivity detected with the overlay technique was difficult to reduce relative to specific 45Ca2+ binding. Lengthy or repeated washing of nitrocellulose transfers also reduced the amount of 45Ca2+ bound to Ca2’-ATPase and calsequestrin proteins. In addition to its greater sensitivity, the ruthenium red detection method was more rapid (10 min) than calcium-45 autoradiography which required 4 days of film exposure. The binding of ruthenium red or 45Ca2’ to either of these proteins was competed by 50 mM CaC12 (not shown). Similarly, nitrocellulose transfers or SDS-polyacrylamide gels of
RUTHENIUM
RED
STAINING 2+
-n-
PonS
RR
45Ca
‘2:: 66-
T-2459 362 L 2924-
2014-
FIG. 2. Ruthenium red staining of sarcoplasmic reticulum proteins. Samples of rabbit skeletal muscle sarcoplasmic reticulum membranes (SR) and purified calsequestrin (CS), corresponding to 20 and 2 pg of protein, respectively, were electrophoresed on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose paper as described under Methods. Nitrocellulose was first stained with Ponceau S (PonS) to reveal transferred proteins followed by calcium-45 autoradiography (45Ca2’) and then stained with ruthenium red (RR) to detect Ca2+-binding proteins as described under Methods. The arrowhead indicates the position of the lOO-kDa Ca2+-ATPase protein. An arrow shows the position of the 55kDa calsequestrin protein. MW represents the molecular weight standards, @-galactosidase (116 kDa), phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), and a-lactalbumin (14 kDa).
OF
Ca’+-BINDING
pared with calcium-45 autoradiography, ruthenium red stained these proteins on nitrocellulose transfers more sensitively. Although these protein bands were a blue color following reaction of polyacrylamide gels with Stains-all, as previously reported (41), considerable diffusion occurred during fixation due to their low AI,. Bovine brain calmodulin also was a blue color with Stains-all, but it failed to bind to nitrocellulose during electrotransfer as previously reported (52). Samples of highly purified calmodulin spotted directly onto nitrocellulose paper stained with ruthenium red. Less than 1 pugof calmodulin could be detected by ruthenium red and 50 mM CaCl, blocked the binding of the dye (not shown). Since ruthenium red is cationic and may simply interact with negatively charged groups on proteins the specificity of the dye for detecting only Ca’+-binding proteins was examined in another fashion. Proteins with various known isoelectric points were electrophoresed then either stained with ruthenium red or Stains-all or autoradiographed following calcium-45 overlay. As Fig. 4 shows, acidic proteins such as glucose oxidase and trypsin inhibitor (lanes b and c, respectively) were detected by ruthenium red better than basic proteins such as cytochrome c and trypsinogen (lanes a and e, respectively). However, these same acidic proteins also bound more calcium-45 than basic proteins. Conversely, Stains-all was less specific since it failed to detect trypsin inhibitor
RR sarcoplasmic reticulum proteins stained with ruthenium red could be completely destained in less than 30 min with 50 mM CaCl,, 10 mM Tris-HCl (pH 7.5). Although more Ca’+-ATPase was present in the sarcoplasmic reticulum preparation relative to calsequestrin, as judged by Ponceau S staining, the amount of ruthenium red bound to these protein bands was similar. This result could be accounted for by considering that 40-50 Ca2+ bind to 1 molecule of calsequestrin (46) whereas only 2 high affinity Ca2+-transport sites are associated with the Ca2+-ATPase enzyme (50). Although calsequestrin has previously been identified on SDS-polyacrylamide gels by staining blue with the cationic carbocyanine dye Stains-all, Ca2+-ATPase appears only as a light, pink band (41). It has therefore been difficult to unequivocally identify all types of Ca2+-binding proteins using the Stains-all detection method. A class of calcium-modulated proteins, which characteristically possesshigh-affinity Ca’+-binding sites that have a common EF hand structural conformation (51), were studied for their ability to bind ruthenium red. As Fig. 3 shows, ruthenium red stained both bovine brain S-100 protein and porcine skeletal muscle troponin C subunit on nitrocellulose transfers. Again, com-
127
PROTEINS
45d+,
I1
SA ,
MW SlOO Tn SlOO Tn SlOO
Tn
+ ^*
-&i
39-
2 V
27L
2
17-
FIG. 3. Ruthenium red staining of EF hand Ca’+-binding proteins. Samples (10 pg) of purified S-100 (SlOO) and troponin (Tn) proteins were electrophoresed on a 15% SDS-polyacrylamide gel and then either reacted with Stains-all or transferred to 0.05-pm pore size nitrocellulose paper as described under Methods. Nitrocellulose transfers were first calcium-45 autoradiographed and then stained with ruthenium red (RR). The arrowhead indicates the position of the 20-kDa troponin C subunit. An arrow shows the position of the lo-kDa S-100 protein. MW represents the prestained molecular weight standards, phosphorylase (135 kDa), bovine serum albumin (75 kDa), ovalbumin (50 kDa), carbonic anhydrase (39 kDa), soybean trypsin inhibitor (27 kDa), and lysozyme (17 kDa).
128
CHARUK,
PonS
45Ca*+
PIRRAGLIA,
SA
MWabcdeabcdeabcdeabCde’
FIG. 4. Ruthenium red staining of various isoelectric point proteins. Samples of cytochrome c (p/9.2), glucose oxidase (pl4.2), trypsin inhibitor (pZ4.6), carbonic anhydrase (pl6.6), and trypsinogen (pZ9.3), corresponding to 10 pg of protein each (lanes a-e, respectively), were electrophoresed on 16.5% Tricine-SDS polyacrylamide minigels (71). Proteins were either stained in the gel with Stains-all (SA) or transferred to nitrocellulose paper and stained with Ponceau S (PonS) followed by calcium-45 autoradiography (@‘Ca”) and reaction with ruthenium red (RR) as described under Methods.
(lane c) and also reacted with basic proteins such as trypsinogen (lane e). These results suggest that although ruthenium red may react with negatively charged groups on acidic proteins, these sites are also capable of Ca*+ binding. Ruthenium red was also used to detect Ca*+-binding proteins in membranes prepared from various tissues. The dye stained few proteins associated with dog kidney brush border membranes and only bound to some unresolved, high M, material on SDS-polyacrylamide gels of renal antiluminal membranes (not shown). Calcium-45 autoradiography and Stains-all also failed to detect any distinct Ca2+-binding proteins in these same membrane preparations. Ruthenium red detected two proteins in canine heart plasma membranes having an approximate M, of 100 and 130 kDa (not shown). These proteins corresponded to the two major Ca2+ and wheat germ agglutinin binding glycoproteins in bovine cardiac sarcolemma (53). These glycoproteins have recently been purified by lectin chromatography (54) and their reactivity with ruthenium red confirmed (M. Michalak, personal communication). Ruthenium red also reacted with at least three proteins in human red cell ghost membranes and binding was competed by Ca2+ (not shown). Similar to erythrocyte spectrins two high molecular weight proteins (>200 kDa) were removed from membranes by 1 mM EDTA and 1 M Kl and a corresponding loss of ruthenium red staining of these components on gels of extracted ghosts was observed (not shown). The amino acid sequence of cw-spectrin deduced from its nucleotide sequence has been shown to contain the equivalent of two EF hand structures (55), suggesting it may indeed bind Ca*+. Another intrinsic membrane protein, the red cell anion transporter, was previously reported to bind Ca2+ (56) and a protein having a M, similar to Band 3 also reacted with Stains-all to give a purple color (57). The interaction of Stains-all with components of the erythrocyte membrane was believed to be due to sialic acid
AND
REITHMEIER
residues on glycoproteins since samples treated with neuraminidase had reduced staining. A protein of 100 kDa in red cell ghost membranes also stained with ruthenium red (not shown). We investigated whether highly purified, deglycosylated, human Band 3 protein bound ruthenium red or Ca2+. As Fig. 5 shows, binding of ruthenium red and 45Ca2+to the lOO-kDa Band 3 protein was competed by 50 mM CaCl,. Ruthenium red detected Band 3 more sensitively than calcium-45 autoradiography of nitrocellulose transfers. Minor amounts of Band 3 proteolytic fragments detected by antibodies also bound ruthenium red and 45Ca2f. The smallest peptide fragment that remained both immunologically reactive and bound ruthenium red or 45Ca2+had a M, of only 23 kDa (Fig. 5). Since rabbit polyclonal antibodies prepared to the human erythrocyte anion transporter were directed solely toward the cytoplasmic, amino-terminal domain, as previously reported (58), the Ca*+-binding site must be localized within this region. Stains-all reacted only weakly with Band 3 regardless of whether the protein had been purified or deglycosylated prior to electrophoresis (not shown). Although Band 3 stained a pink color, another intrinsic membrane protein, which comigrated with the anion transporter when samples of red cell ghost membranes were electrophoresed, did stain a blue color on gels (not shown). This component presumably represented glycophorin, which has a similar mobility as Band 3 and contains a high concentration of sialic acid in its numerous carbohydrate structures (59). Interestingly, although glycophorin reacted strongly with Stains-all, it failed to bind noticeable
MW
-+-+
FIG. 5. Ruthenium red staining of erythrocyte Band 3 protein. A lo-rg sample of purified, deglycosylated human red blood cell membrane Band 3 protein was electrophoresed on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose paper as described under Methods. Nitrocellulose was first stained with Ponceau S (PonS) then calcium-45 autoradiographed (45CaZ+) and reacted with ruthenium red (RR) in the presence (+) or absence (-) of 50 mM CaCl,. An immunoblot (Ab) was formed by reacting the nitrocellulose transfer with a rabbit polyclonal antibody to human erythrocyte Band 3 protein. The arrowhead indicates the position of the lOO-kDa Band 3 protein. MW represents molecular weight standards as in Fig. 2.
RUTHENIUM
RED
STAINING
amounts of either 45Ca2f or ruthenium red (not shown). These results suggested Stains-all also reacts with proteins that do not necessarily possess Ca’+-binding sites. DISCUSSION
Two methods of identifying Ca2+-binding proteins following polyacrylamide gel electrophoresis have previously been described and each has been used extensively. The cationic carbocyanine dye, Stains-all was shown to bind to various known Ca2+-binding proteins turning them a blue color (41). However, in addition to its light sensitivity, its inability to detect several proteins known to contain Ca2+-binding sites such as the sarcoplasmic reticulum Ca2+-ATPase and its reactivity toward acidic sialoglycoproteins such as glycophorin, which is not known to bind Ca2+, limits the usefulness of Stains-all as a specific stain for Ca2+-binding proteins. Calcium-45 autoradiography of SDS-polyacrylamide gels (60) or more frequently, nitrocellulose transfers (42) have been popular techniques used to detect Ca2’-binding proteins. However, the low affinity of some proteins for Ca2+ (e.g., calsequestrin), the high background radioactivity, and the long exposure time required for autoradiographs, as well as the danger in handling this radioisotope, make this method unsuitable for routine detection of some Ca2+-binding proteins. These inherent problems with both techniques prompted the search for a relatively rapid, sensitive, safe, and inexpensive method for specifically detecting all classes of Ca2+binding proteins. The ability of ruthenium red to stain glycosaminoglycan-containing extracellular matrix (1) and some subcellular organelles (2-4), as well as to block Ca2+ channels (15-19) and inhibit various Ca2+-transport ATPases (27-30), suggested a possible interaction of this dye directly with Ca2+-binding sites. We found that ruthenium red not only reversibly bound with high affinity to the muscle protein calsequestrin, but also stained a wide assortment of proteins known to contain Ca2+-binding sites. Furthermore, ruthenium red detected Ca2+-binding proteins more sensitively than calcium-45 autoradiography and more reliably than Stainsall. Moreover, the interaction of ruthenium red with Ca2+-binding sites is extremely specific since binding was competed by Ca2+ and the dye failed to stain either kidney membrane proteins or protein molecular weight standards. Although ruthenium red stained acidic but not basic proteins the latter also failed to bind Ca2+. A good correlation between Ca2+-binding and ruthenium red staining of proteins was therefore observed. Glycosaminoglycans were originally thought to be involved in mitochondrial energy transduction based on the inhibitory effects of ruthenium red on Ca2+-transport (25) and its interaction with these components (1). Considering our present findings and their known extra-
OF
Ca’+-BINDING
PROTEINS
129
cellular localization, it is unlikely glycosaminoglycans are involved in mitochondrial function. Rather, mitochondrial Ca2+ fluxes mediated by specific ATPases or uniporters are more likely targets for the inhibitory action of ruthenium red (61). The effects of ruthenium red on Ca2+ fluxes in subcellular organelles is complex probably due to its multiple interactions with Ca2+-binding sites on ATPases, channels, and storage proteins such as calsequestrin. For example, Ca2+ transport by microsome fractions is considerably less sensitive to ruthenium red than mitochondria (26). The ruthenium redinsensitive Ca2+-transport activity is highest in heavy microsome fractions enriched in endoplasmic reticulum (62). The apparent resistance of endoplasmic reticulum Ca2+-ATPase to the inhibitory effects of ruthenium red may be due to several reasons. First, since Ca2+competes with dye-binding sites, excessive Ca2+ concentrations used in transport assays may prevent ruthenium red inhibition of Ca2+ uptake. Alternatively, blockade of Ca2+ channels by ruthenium red may increase Ca2+-loading rates into endoplasmic reticulum vesicles, thereby masking any effects of the dye on the Ca2’-ATPase. Finally, complexation of ruthenium red by anions such as oxalate and ATP, present in Ca2+-transport assays, may reduce its effective concentration, thereby preventing enzyme inhibition. The electrophysiological effects of ruthenium red on cells are also complex and have only been studied in detail for frog skeletal muscle fibers (63,64). The increased twitch potentiation caused by low amounts of dye (
130
CHARUK,
PIRRAGLIA,
binding domains. Purification and amino acid sequencing of these peptides may allow us to predict the structure of binding sites involved in Ca2+ translocation. These studies would help confirm the identity of residues involved in Ca2+ binding and transport predicted from site-directed mutagenesis experiments on proteins such as the Ca2+-ATPase of sarcoplasmic reticulum (70). We are currently taking advantage of our findings by attempting to immobilize ruthenium red on a resin suitable for protein chromatography. We are also using ruthenium red to identify new membrane Ca2+-binding proteins while continuing to investigate known proteins for their possible interaction with the dye. To date we have observed specific binding of ruthenium red to calreticulin and the 160-kDa glycoprotein of sarcoplasmic reticulum (sarcalumenin), and both the dihydropyridine- and ryanodine-sensitive Ca2’ channels of skeletal muscle (unpublished observations). Since ruthenium red has a characteristic absorption spectrum (38), we also hope to use the dye in spectrophotometric studies designed to investigate the mechanism of Ca2+ binding and translocation by membrane transporters and channels. We therefore feel that ruthenium red will be an extremely useful and versatile probe of the structure and function of Ca2+-binding sites in future protein biochemical studies. ACKNOWLEDGMENTS This work was funded by grants from the Medical Research Council of Canada and the Muscular Dystrophy Association of Canada to R.A.F.R. J.H.M.C. is a recipient of a postdoctoral fellowship from the Medical Research Council of Canada.
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