ryanodine and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

Ryanodine receptor (RyR) type 1 (RyR1) exhibits a markedly lower gain of Ca(2+)-induced Ca(2+) release (CICR) activity than RyR type 3 (RyR3) in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle (selective stabilization of the RyR1 channel), and this reduction in the gain is largely eliminated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). We have investigated whether the hypothesized interdomain interactions within RyR1 are involved in the selective stabilization of the channel using [(3)H]ryanodine binding, single-channel recordings, and Ca(2+) release from the SR vesicles. Like CHAPS, domain peptide 4 (DP4, a synthetic peptide corresponding to the Leu(2442)-Pro(2477) region of RyR1), which seems to destabilize the interdomain interactions, markedly stimulated RyR1 but not RyR3. Their activating effects were saturable and nonadditive. Dantrolene, a potent inhibitor of RyR1 used to treat malignant hyperthermia, reversed the effects of DP4 or CHAPS in an identical manner. These findings indicate that RyR1 is activated by DP4 and CHAPS through a common mechanism that is probably mediated by the interdomain interactions. DP4 greatly increased [(3)H]ryanodine binding to RyR1 with only minor alterations in the sensitivity to endogenous CICR modulators (Ca(2+), Mg(2+), and adenine nucleotide). However, DP4 sensitized RyR1 four- to six-fold to caffeine in the caffeine-induced Ca(2+) release. Thus the gain of CICR activity critically determines the magnitude and threshold of Ca(2+) release by drugs such as caffeine. These findings suggest that the low CICR gain of RyR1 is important in normal Ca(2+) handling in skeletal muscle and that perturbation of this state may result in muscle diseases such as malignant hyperthermia.

Topics: Animals; Calcium; Cattle; Cholic Acids; Detergents; In Vitro Techniques; Protein Structure, Tertiary; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

We showed that frog alpha-ryanodine receptor (alpha-RyR) had a lower gain of Ca(2+)-induced Ca(2+) release (CICR) activity than beta-RyR in sarcoplasmic reticulum (SR) vesicles, indicating selective "stabilization" of the former isoform (Murayama T and Ogawa Y. J Biol Chem 276: 2953-2960, 2001). To know whether this is also the case with mammalian RyR1, we determined [(3)H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR vesicles. The value of [(3)H]ryanodine binding (B) was normalized by the number of maximal binding sites (B(max)), whereby the specific activity of each isoform was expressed. This B/B(max) expression demonstrated that ryanodine binding of individual channels for RyR1 was <15% that for RyR3. Responses to Ca(2+), Mg(2+), adenine nucleotides, and caffeine were not substantially different between in situ and purified isoforms. These results suggest that the gain of CICR activity of RyR1 is markedly lower than that of RyR3 in mammalian skeletal muscle, indicating selective stabilization of RyR1 as is true of frog alpha-RyR. The stabilization was partly eliminated by FK506 and partly by solubilization of the vesicles with CHAPS, each of which was additive to the other. In contrast, high salt, which greatly enhances [(3)H]ryanodine binding, caused only a minor effect on the stabilization of RyR1. None of the T-tubule components, coexisting RyR3, or calmodulin was the cause. The CHAPS-sensitive intra- and intermolecular interactions that are common between mammalian and frog skeletal muscles and the isoform-specific inhibition by FKBP12, which is characteristic of mammals, are likely to be the underlying mechanisms.

Topics: Adenine Nucleotides; Animals; Binding Sites; Caffeine; Calcium; Calmodulin; Cattle; Cholic Acids; Detergents; Diaphragm; Dose-Response Relationship, Drug; Magnesium; Phospholipids; Precipitin Tests; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sodium Chloride; Solubility; Tacrolimus Binding Protein 1A

Ryanodine receptors (RyRs) of skeletal muscle, as calcium release channels, have been found to form semicrystalline arrays in the membrane of sarcoplasmic reticulum. Recently, both experimental observations and theoretical simulations suggested cooperative coupling within interlocking RyRs. To better understand the interactions between RyRs and their modulation, the aggregation and dissociation of isolated RyRs in aqueous medium containing various Na(+) and K(+) concentrations were investigated using photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). RyRs aggregated readily at low salt concentrations. However, a different behavior was observed in the presence of Na(+) or K(+). Detectable aggregates were formed in 5 microg/mL RyR sample when the concentration of Na(+) and K(+) was reduced from 1 M to below 0.28 and 0.23 M, respectively. The dissociation of RyR aggregates was also examined when raising the salt concentration. While aggregates formed in 0.15 M NaCl medium could reverse almost completely, those formed in 0.15 M KCl medium only dissolved partly. When keeping the total salt concentration at 0.15 M, the aggregation and dissociation of RyRs were seen to evidently depend on the relative concentration of Na(+) and K(+). The interaction between RyRs was strengthened with increasing Na(+)/K(+) ratios in the mixed medium. Accompanying this, a decrease of [(3)H]ryanodine binding occurred. The results obtained with PCS and AFM provide further evidence for the interaction between RyRs and suggest the importance of Na(+), K(+), and their relative composition in modulating the interaction and cooperation between RyRs in vivo.

Topics: Absorptiometry, Photon; Animals; Calcium; Calcium Channels; Cholic Acids; In Vitro Techniques; Kinetics; Microscopy, Atomic Force; Muscle, Skeletal; Phosphatidylcholines; Potassium Chloride; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sodium Chloride

Optimal [3H]ryanodine binding to skeletal muscle sarcoplasmic reticulum membranes is dependent on a number of factors such as Ca2+ concentration, ionic strength, and the presence of modulators of the Ca2+ release channel. The rate of association of [3H]-ryanodine with its binding site is slower than a diffusion limited process, and often the binding reaches a peak value which is followed by a slow decline. This phenomenon makes it extremely difficult to determine kinetic constants for [3H]ryanodine binding. The inclusion of bovine serum albumin (BSA) or the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) in the incubation buffer prevents the decrease in [3H]ryanodine binding observed in association studies. BSA or Chaps slows this decline in binding partially by preventing a conversion to a more rapidly dissociating component. Pretreatment of the membranes with Chaps does not prevent the decrease in [3H]ryanodine binding, suggesting that Chaps is not exerting its effect by extracting a lipid or peripheral membrane protein. The decrease in affinity observed in the absence of BSA and Chaps appears to require the occupation of the high-affinity ryanodine binding site. Incubation for extended times in the absence of [3H]ryanodine prior to the initiation of the association produced similar curves to those obtained without preincubation. These combined results suggest that Chaps and BSA stabilize the ryanodine-modified Ca2+ release channel by preventing an alteration in the ryanodine binding site which leads to decreased affinity, thus allowing for a more quantitative interpretation of binding data.

Topics: Animals; Calcium; Calcium Channels; Cholic Acids; Detergents; Hydrogen-Ion Concentration; Kinetics; Muscle, Skeletal; Osmolar Concentration; Rabbits; Radioligand Assay; Ryanodine; Sarcoplasmic Reticulum; Serum Albumin, Bovine; Tritium

We have devised a novel procedure, employing Chaps rather than Triton [Costello B., Chadwick C., Saito A., Chu A., Maurer A., Fleischer S. J Cell Biol 1986; 103: 741-753], for obtaining vesiculated derivatives of the junctional face membrane (JFM) domain of isolated terminal cisternae (TC) from fast skeletal muscle of the rabbit. Enriched JFM is minimally contaminated with junctional transverse tubules. The characteristic ultrastructural features and the most essential features of TC function relating to this membrane domain-i.e. both the Ca(2+)-release system and the Ca2+ and calmodulin (CaM)-dependent protein kinase (CaM I PK) system-appear to be retained in enriched JFM. We show that our isolation procedure, yielding up to a 2.5-fold enrichment in ryanodine receptor (RyR) protein and in the maximum number of high affinity [3H]-ryanodine binding sites, does not alter the assembly for integral proteins associated with the receptor in its native membrane environment, i.e. FKBP-12, triadin and the structurally related protein junction [Jones L.R., Zhang L., Sanborn K., Jorgensen A., Kelley J. J Biol Chem 1995; 270: 30787-30796] having, in common, the property to bind calsequestrin (CS) in overlays in the presence of EGTA. The substrate specificity of endogenous CaM I PK is also the same as that of parent TC vesicles. Phosphorylation of mainly triadin and of a high M(r) polypeptide, and not of the RyR, is the most remarkable common property. Retention of peripheral proteins, like CS and histidine-rich Ca(2+)-binding protein, although not that endogenous CaM, and of a unique set of CaM-binding proteins, unlike that of junctional SR-specific integral proteins, is shown to be influenced by the concentration of Ca2+ during incubation of TC vesicles with Chaps. Characterization of RyR functional behaviour with [3H]-ryanodine has indicated extensive similarities between the enriched JFM and parent TC vessicles, as far as the characteristic bell shaped Ca(2+)-dependence of [3H]-ryanodine binding and the dose-dependent sensitization to Ca2+ by caffeine, reflecting the inherent properties of SR Ca(2+)-release channel, as well as concerning the stimulation of [3H]-ryanodine binding by increasing concentrations of KCl. Stabilizing the RyR in a maximally active state by optimizing concentrations of KCl (1 M), at also optimal concentrations of Ca2+ (pCa 4), rendered the receptor less sensitive to inhibition by 1 microM CaM, to a greater extent in the case of enriched JF

Topics: Animals; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinase Type 1; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cholic Acids; Digoxigenin; Electrophoresis, Polyacrylamide Gel; Immune Sera; Immunoblotting; Immunoglobulin G; Intracellular Membranes; Isradipine; Muscle Fibers, Fast-Twitch; Phosphorylation; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Trypsin

To understand the functions of the two ryanodine receptor isoforms (alpha- and beta-RyRs) in nonmammalian skeletal muscles, we determined [3H]ryanodine binding to these isoforms purified from bullfrog skeletal muscle. In 0.17 M-NaCl medium both isoforms demonstrated similar Ca2+ dependent ryanodine binding activities, while the Ca2+ sensitivity for activation of beta-RyR was increased in 1 M-NaCl medium. This enhancement in Ca2+ sensitivity depended on the kinds of salts used. These results imply that alpha- and beta-RyRs may have similar properties as Ca2+-induced Ca2+ release channels in bullfrog skeletal muscle.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channels; Cholic Acids; Muscle Proteins; Muscle, Skeletal; Phospholipids; Rana catesbeiana; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sodium Chloride; Tritium

[3H]9-Methyl-7-bromoeudistomin D ([3H]MBED), the most powerful Ca2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [3H]MBED binding. [3H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine-5'-(beta,gamma-methylene)triphosphate. The Bmax value was 170 times more than that of [3H]ryanodine binding. The profile of sucrose-density gradient centrifugation of solubilized microsomes indicated that [3H]MBED binding protein was different from [3H]ryanodine binding protein. These results suggest that there are MBED/caffeine-binding sites in brain that are distinct from the ryanodine receptor and that MBED becomes an essential molecular probe for characterizing caffeine-binding protein in the central nervous system.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Binding, Competitive; Brain; Caffeine; Calcium; Calcium Channels; Carbolines; Cholic Acids; Cyclic GMP; Detergents; Guinea Pigs; Inositol 1,4,5-Trisphosphate; Kinetics; Microsomes; Muscle Proteins; Procaine; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel

1. An intracellular anion channel, known to be co-localized in brain endoplasmic reticulum membranes with ryanodine-sensitive calcium-release channels, was incorporated into voltage-clamped planar lipid bilayers from sheep brain microsomal membrane vesicles. 2. Single channels, which displayed a main open-state conductance of 80-100 pS in symmetric 450 mM choline Cl, reduced to approximately 20 pS in symmetric 225 mM (choline)2SO4 (the solutions also contained 10 mM Tris-HCl, pH 7.4), discriminated poorly between Cl- and choline+ (relative permeability ratio, PCl-/Pcholine+, 2.5). 3. Sheep brain microsomal membrane proteins were solubilized in the zwitterionic detergent CHAPS, and subjected to sequential anion-exchange and size-exclusion chromatography; the solubilizate, and partially-purified protein fractions, were then incorporated into large unilamellar liposomes by freeze-thaw sonication. 4. Reconstituted passive anion (Cl-)-transport, which was reduced by approximately 60% in the presence of SO4(2-), was assayed by measuring the efflux of entrapped 36Cl- (compared to the efflux of [3H]inulin), and also by monitoring the fluorescence quenching of entrapped SPQ by Cl(-)-influx. 5. Cl(-)-transporting activity was enriched up to 200-fold after two stages of purification, and the partially-purified channel protein was incorporated from reconstituted proteoliposomes into planar lipid bilayers, where its permeation behaviour remained very similar to that observed for the native channel.

Topics: Animals; Cerebral Cortex; Chloride Channels; Chlorides; Cholic Acids; Choline; Chromatography, Gel; Chromatography, Ion Exchange; Detergents; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Ion Transport; Lipid Bilayers; Membrane Proteins; Oxidation-Reduction; Patch-Clamp Techniques; Phosphatidylethanolamines; Phosphatidylserines; Ryanodine; Sheep; Solubility; Spectrometry, Fluorescence; Sulfates

A full-length cDNA encoding the ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was transiently expressed in COS-1 cells. Immunoblotting studies showed that the expressed ryanodine receptor and the native ryanodine receptor of rabbit skeletal muscle were indistinguishable in molecular size and immunoreactivity. Scatchard analysis of [3H]ryanodine binding to transfected COS-1 cell microsomes resulted in a Bmax of 0.22 pmol/mg of protein and a Kd of 16.2 nM. Expressed ryanodine receptors were solubilized in CHAPS and were shown to cosediment with native ryanodine receptors in a sucrose density gradient. Thus, the expressed receptor, like the native receptor, is assembled as a large oligomeric complex. Single-channel recordings in planar lipid bilayers were used to investigate the functional properties of the sucrose gradient-purified complex. The expressed ryanodine receptor formed a large conductance channel activated by ATP and Ca2+ and inhibited by Mg2+ and ruthenium red. Ryanodine reduced the conductance and increased the mean open time in a manner consistent with that of native channels. These results demonstrated that functional binding sites for the physiological ligands (Ca2+, Mg2+, and ATP) and pharmacological ligands (ruthenium red and ryanodine) controlling gating of the Ca2+ release channel are encoded in the ryanodine receptor cDNA and are faithfully expressed in COS-1 cells. Ryanodine receptors expressed in COS-1 cells displayed several conductance states > or = 1 nS not present in native channels. Such anomalous conductance states of the expressed channel might be referable to lack of muscle-specific posttranslational processing or to the need for components not present in COS-1 cells, which may be required to stabilize the channel structure.

Topics: Adenosine Triphosphate; Animals; Calcium Channels; Cell Line; Centrifugation, Density Gradient; Cholic Acids; DNA; DNA Restriction Enzymes; Electric Conductivity; Gene Expression; Immunohistochemistry; Muscle Proteins; Muscles; Plasmids; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Solubility; Transfection

In this study, the effects of Ca(2+)-activated neutral protease (CANP) upon skeletal muscle heavy sarcoplasmic reticulum (HSR) structure and function were investigated. CANP was immunolocalized to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent-insoluble fraction of purified HSR membranes. Ca2+ activation of the endogenous membrane-bound CANP produced a characteristic partial fragmentation of the HSR 565-kDa Ca2+ release channel. Similarly, the major substrate for both micromolar and millimolar Ca(2+)-sensitive isoforms of exogenous CANP was the Ca2+ release channel with proteolysis of a 88-kDa HSR protein also observed. Ca2+ release channel proteolysis was initiated at a single cleavage site with coincidental production of 410- and 150-kDa peptide fragments. Appearance of 160- and 137-kDa limiting peptides accompanied secondary proteolysis of the primary 410- and 150-kDa fragments, respectively. Despite extensive proteolysis of the Ca2+ release channel, CANP did not dramatically alter the Ca2+ handling and ryanodine binding properties of HSR membranes. The association of CANP with isolated HSR membranes suggests that, in vivo, this protease may modify an additional property of the Ca2+ release channel. This may be related to the CANP-susceptible structural association of the Ca2+ release channel with dihydropyridine receptors at T-tubule/sarcoplasmic reticulum junctions.

Topics: Adenosine Triphosphate; Animals; Calcium; Calpain; Cell Fractionation; Cholic Acids; Electrophoresis, Polyacrylamide Gel; Kinetics; Membrane Proteins; Muscles; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Spectrophotometry

Sheep cardiac muscle sarcoplasmic reticulum ryanodine receptors have been isolated by density-gradient centrifugation following solubilisation with the zwitterionic detergent, CHAPS. The functional state of the receptor complex has been assessed by quantification of [3H]ryanodine binding and by characterisation of single-channel conductance and gating properties following reconstitution into unilamellar proteo-liposomes and incorporation into planar phospholipid bilayers. A method of solubilisation is described which yields a receptor displaying high-affinity [3H]ryanodine binding (Kd 2.8 nM, Bmax 352 pmol/mg protein) and which functions as a cation-selective, ligand-regulated channel under voltage clamp conditions. Previous reports of channel activity of purified rabbit skeletal and canine cardiac muscle ryanodine receptors describe a range of sub- or variable-conductance events. In contrast, the sheep cardiac ryanodine receptor-channels isolated using the optimal conditions described in this report consistently display a single open state conductance with either Ca2+ or K+ as the charge carrying species.

Topics: Animals; Calcium; Cholic Acids; Electric Conductivity; Lipid Bilayers; Liposomes; Muscle Proteins; Myocardium; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sheep

Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.

Topics: Animals; Cholic Acids; Chromatography, Gel; Dogs; Kinetics; Microsomes; Molecular Weight; Muscles; Myocardium; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

The ryanodine receptor has been purified from junctional terminal cisternae of fast skeletal muscle sarcoplasmic reticulum (SR). The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and stabilized by addition of phospholipids. The solubilized receptor showed the same [3H]ryanodine binding properties as the original SR vesicles in terms of affinity, Ca2+ dependence, and salt dependence. Purification of the ryanodine receptor was performed by sequential column chromatography on heparin-agarose and hydroxylapatite in the presence of CHAPS. The purified receptor bound 393 +/- 65 pmol of ryanodine/mg of protein (mean +/- S.E., n = 5). The purified receptor showed three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr of 360,000, 330,000, and 175,000. Densitometry indicates that these are present in the ratio of 2/1/1, suggesting a monomer Mr of 1.225 X 10(6) and supported by gel exclusion chromatography in CHAPS. Electron microscopy of the purified preparation showed the square shape of 210 A characteristic of and comparable in size and shape to the feet structures of junctional terminal cisternae of SR, indicating that ryanodine binds directly to the feet structures. From the ryanodine binding data, the stoichiometry between ryanodine binding sites to the number of feet structures is estimated to be about 2. Since the ryanodine receptor is coupled to Ca2+ gating, the present finding suggests that the ryanodine receptor and Ca2+ release channel represent a functional unit, the structural unit being the foot structure which, in situ, is junctionally associated with the transverse tubules. It is across this triad junction that the signal for Ca2+ release is expressed. Thus, the foot structure appears to directly respond to the signal from transverse tubules, causing the release of Ca2+ from the junctional face membrane of the terminal cisternae of SR.

Topics: Animals; Calcium; Cholic Acids; Microscopy, Electron; Molecular Weight; Muscles; Osmolar Concentration; Phospholipids; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

The Ca2+-dependent ryanodine binding site of rabbit cardiac sarcoplasmic reticulum is solubilized by treatment with 20 mM CHAPS detergent and 1 M NaCl for 30 min at 0 degrees C. Ca2+ added at 5 microM enhances binding, at 0.5 mM increases both the affinity and number of [3H]ryanodine binding sites, while at 10 mM only the number of binding sites is increased. Mg2+ up to 1 mM does not significantly affect [3H]ryanodine binding. Radioligand binding is strongly enhanced by all alkali metal chlorides except LiCl. NaCl increases the rate of association of the ligand and the affinity of the binding site but does not influence the dissociation. NaCl and CaCl2 enhance the thermal stability of the [3H]ryanodine-binding protein. Thiol groups are essential for [3H]ryanodine binding. Ruthenium red and Cd2+ inhibit binding, while theophylline is stimulatory at low (micromolar) Ca2+ concentrations by a mechanism other than phosphodiesterase inhibition. Gel permeation chromatography establishes that the ryanodine binding protein is localized only in the high molecular mass fraction (greater than 669 kDa). Polyacrylamide gel electrophoresis of the proteins following treatment with SDS and 2-mercaptoethanol indicates that more than 90% are of low molecular mass (34-70 kDa) and that two stain blue with Stains-all as expected of Ca2+-binding proteins.

Topics: Animals; Calcium; Cholic Acids; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Hot Temperature; Molecular Weight; Myocardium; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sodium Chloride; Solubility; Tritium