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S Fleischer

Publications and source records attributed to S Fleischer.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cryoelectron microscopy and image analysis of the cardiac ryanodine receptor.

The three-dimensional structure of the cardiac muscle ryanodine receptor (RyR2) is described and compared with its skeletal muscle isoform (RyR1). Previously, structural studies of RyR2 have not been as informative as those for RyR1 because optimal conditions for electron microscopy, which require low levels of phospholipid, are destabilizing for RyR2. A simple procedure was devised for diluting RyR2 (in phospholipid-containing buffer) into a lipid-free buffer directly on the electron microscope grid, followed by freezing within a few seconds. Cryoelectron microscopy of RyR2 so prepared yielded images of sufficient quality for analysis by single particle image processing. Averaged projection images for RyR2, as well as for RyR1, prepared under the same conditions, were found to be nearly identical in overall dimensions and appearance at the resolution attained, approximately 30 A. An initial three-dimensional reconstruction of RyR2 was determined (resolution approximately 41 A) and compared with previously reported reconstructions of RyR1. Although they looked similar, which is consistent with the similarity found for the projection images, and with expectations based on the 66% amino acid sequence identity of the two isoforms, structural differences near the corners of the cytoplasmic assembly were observed in both two- and three-dimensional studies.

Animals↗
PubMed1998

Purification and characterization of ryanodine receptor 3 from mammalian tissue.

The ryanodine receptors are intracellular Ca2+ release channels that play a key role in cell signaling via Ca2+. There are three isoforms. Isoform 1 from skeletal muscle and isoform 2 from heart have been characterized. Isoform 3 is widely distributed in many mammalian tissues although in minuscule amounts. Its low abundance has hampered its study. We now describe methodology to isolate mammalian isoform 3 in amounts sufficient for biochemical and biophysical characterization. Bovine diaphragm sarcoplasmic reticulum fractions enriched in terminal cisternae containing both isoforms 1 (>95%) and 3 (<5% of the ryanodine binding) served as starting source. Isoform 3 was selectively immunoprecipitated from the 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized fraction and eluted with peptide epitope. Isoform 3 thus prepared is highly purified as characterized by SDS-polyacryamide gel electrophoresis, Coomassie Blue staining, and by high affinity ryanodine binding. The purified isoform 3 was incorporated into planar lipid bilayers, and its channel properties were studied. Channel characteristics in common with the other two isoforms are slope conductance, higher selectivity to Ca2+ versus K+ (PCa/K approximately 6), and response to drugs and ligands. In its response to Ca2+ and ATP, it more closely resembles isoform 2. The first two-dimensional structure of isoform 3 was obtained by cryoelectron microscopy and image enhancement techniques.

Animals↗
PubMed1998

Specific interaction of Golgi coatomer protein alpha-COP with phosphatidylinositol 3,4,5-trisphosphate.

The phosphoinositide binding selectivity of Golgi coatomer COPI polypeptides was examined using photoaffinity analogs of the soluble inositol polyphosphates Ins(1,4,5)P3, Ins(1,3,4,5)P4, and InsP6, and of the polyphosphoinositides PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2. Highly selective Ins(1,3,4,5)P4-displaceable photocovalent modification of the alpha-COP subunit was observed with a p-benzoyldihydrocinnamide (BZDC)-containing probe, [3H]BZDC-Ins(1,3,4,5)P4. A more highly phosphorylated probe, [3H]BZDC-InsP6 probe labeled six of the seven subunits, with only beta, beta', delta, and epsilon-COP showing competitive displacement by excess InsP6. Importantly, [3H]BZDC-triester-PtdIns(3,4,5)P3, the lipid with the same phosphorylation pattern as Ins(1,3,4,5)P4, showed specific, PtdIns(3,4,5)P3-displaceable labeling of only alpha-COP. Labeling by the PtdIns(4,5)P2 and PtdIns(3,4)P2 photoaffinity probes was less intense and showed no discrimination based on PtdInsPn ligand. Thus, both the D-3 and D-5 phosphates are critical for the alpha-COP-PtdIns(3,4,5)P3 interaction, suggesting an important role for this polyphosphoinositide in vesicular trafficking.

Affinity Labels↗
PubMed1998

Localization of ryanodine receptors in smooth muscle.

The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide-stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR-Ca2+-release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca2+ content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca2+-release channels and origins of Ca2+ sparks, Ca2+ oscillations, and Ca2+ waves.

Animals↗
PubMed1997

Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor.

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large ( approximately 80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.

Animals↗
PubMed1997

Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors.

The single-channel activity of rabbit skeletal muscle ryanodine receptor (skeletal RyR) and dog cardiac RyR was studied as a function of cytosolic [Ca2+]. The studies reveal that for both skeletal and cardiac RyRs, heterogeneous populations of channels exist, rather than a uniform behavior. Skeletal muscle RyRs displayed two extremes of behavior: 1) low-activity RyRs (LA skeletal RyRs, approximately 35% of the channels) had very low open probability (Po < 0.1) at all [Ca2+] and remained closed in the presence of Mg2+ (2 mM) and ATP (1 mM); 2) high-activity RyRs (HA skeletal RyRs) had much higher activity and displayed further heterogeneity in their Po values at low [Ca2+] (< 50 nM), and in their patterns of activation by [Ca2+]. Hill coefficients for activation (nHa) varied from 0.8 to 5.2. Cardiac RyRs, in comparison, behaved more homogeneously. Most cardiac RyRs were closed at 100 nM [Ca2+] and activated in a cooperative manner (nHa ranged from 1.6 to 5.0), reaching a high Po (> 0.6) in the presence and absence of Mg2+ and ATP. Heart RyRs were much less sensitive (10x) to inhibition by [Ca2+] than skeletal RyRs. The differential heterogeneity of heart versus skeletal muscle RyRs may reflect the modulation required for calcium-induced calcium release versus depolarization-induced Ca2+ release.

Adenosine Triphosphate↗
PubMed1997

Gating of the skeletal calcium release channel by ATP is inhibited by protein phosphatase 1 but not by Mg2+.

We have previously found that dephosphorylation/phosphorylation of the calcium release channel (CRC) of skeletal muscle confers channel sensitivity/insensitivity to the block by physiological [Mg2+] (approximately 1 mM). These studies have now been extended to modulation by ATP. Terminal cisternae vesicles of sarcoplasmic reticulum were incorporated into planar lipid bilayers. CRC gating by ATP (0.67 mM), in the absence of Ca2+ (< 1nM), was studied by treatment with protein kinase A (PKA) or phosphatase 1 (PPT1) and assayed in the presence and absence of free Mg2+ (1 mM). PPT1, PKA, and Mg2+ were directly applied to the bilayer using the microsyringe method, which controls the environment of the CRC in the bilayer for phosphorylation/ dephosphorylation cycles and for assays. PKA treated channels were activated by ATP to high open probabilities, while PPT1 treated channels were not activatable by ATP. Opening and closing of channels during cycles of PKA and PPT1 applications, respectively, provided evidence that the change of CRC activity is due to cyclic phosphorylation/dephosphorylation. Free Mg2+ (1 mM) did not block channels activated by ATP. The new finding is that channel gating by ATP can be controlled by the state of phosphorylation without inhibition by free Mg2+.

Adenosine Triphosphate↗
PubMed1997

Impaired cortico-bulbar tract function in dysarthria due to hemispheric stroke. Functional testing using transcranial magnetic stimulation.

We investigated cortico-lingual and cortico-orofacial tract function utilizing transcranial magnetic stimulation in 18 consecutive patients with dysarthria due to hemispheric stroke. Delayed responses (conduction time > mean + 2.5 SD of that of 43 controls) or absent responses were considered abnormal. In all patients, motor-cortex stimulation of the lesion side demonstrated absent (13 patients) or delayed (five patients) responses to the tongue bilaterally (17 patients) or unilaterally (one patient). In 14 patients the contralateral orofacial responses were either absent (13 patients) or delayed (one patient). According to the electrophysiological findings, all lesions revealed by CT or MRI, were located within the pyramidal tract at the lower motor cortex (n = 4), the corona radiata (n = 7), and the genu of the internal capsule (n = 3) or its posterior limb (n = 4). We conclude that interruption of the cortico-bulbar tract fibres to muscles involved in articulation is a frequent cause of dysarthria in hemispheric stroke.

Aged↗
PubMed1997

Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms.

In the present study, we compare functional consequences of dissociation and reconstitution of binding proteins FKBP12 and FKBP12.6 with ryanodine receptors from cardiac (RyR2) and skeletal muscle (RyR1). The skeletal muscle RyR1 channel became activated on removal of endogenously bound FKBP12, consistent with previous reports. Both FKBP12 and FKBP12.6 rebind to FKBP-depleted RyR1 and restore its quiescent channel behavior by altering ligand sensitivity, as studied by single-channel recordings in planar lipid bilayers, and macroscopic behavior of the channels (ryanodine binding and net energized Ca2- uptake). By contrast, removal of FKBP12.6 from the cardiac RyR2 did not modulate the function of the channel using the same types of assays as for RyR1. FKBP12 or FKBP12.6 had no effect on channel activity of FKBP12.6-depleted cardiac RyR2, although FKBP12.6 rebinds. Our studies reveal important differences between the two ryanodine receptor isoforms with respect to their functional interaction with FKBP12 and FKBP12.6.

Adenosine Triphosphate↗
PubMed1996

Selective binding of FKBP12.6 by the cardiac ryanodine receptor.

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer)4(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35S-labeled FKBP12 and 35S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12. 6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBP-stripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.

Animals↗
PubMed1996

Wild type and mutant human heart (R)-3-hydroxybutyrate dehydrogenase expressed in insect cells.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. PC is an allosteric activator that enhances NAD(H) binding to BDH. The enzyme serves as a paradigm to study specific lipid-protein interactions in membranes. Analysis of the primary sequence of BDH, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the C-terminal third of the protein [Marks, A. R., McIntyre, J. O., Duncan, T. M., Erdjument-Bromage, H., Tempst, P., & Fleischer, S. (1992) J. Biol. Chem. 267, 15459-15463]. The mature form of human heart BDH has now been expressed in catalytically active form in insect cells (Sf9, Spodoptera frugiperda) transfected with BDH-cDNA in baculovirus. Endogenous PC in the insect cells fulfills the lipid requirement for the expressed BDH since enzymatic activity is lost upon digestion with phospholipase A2 and restored selectively by reconstitution with PC vesicles. The K(m)s for NAD+ and (R)-3-hydroxybutyrate (R-HOB) of expressed BDH are similar to those for bovine heart or rat liver BDH in mitochondria. Replacing Cys242 (the only cysteine in the C-terminal domain) with serine by site-directed mutagenesis resulted in a 10-fold increase in K(m) for R-HOB with no change in the K(m) for NAD+, indicating a role for Cys242 in substrate binding. Carboxypeptidase cleavage studies had indicated a requirement of the C-terminal for catalysis and a role in lipid binding [Adami, P., Duncan, T. M., McIntyre, J. O., Carter, C. E., Fu, C., Melin, M., Latruffe, N., & Fleischer, S. (1993) Biochem J. 292, 863-872]. We now show that deletion of twelve C-terminal amino acids to form a truncated BDH mutant results in loss of enzymic function. The expression in Sf 9 cells of the constitutively active full-length mature form of human heart BDH and the first expression and characterization of BDH mutants validate this system for structure-function studies of BDH.

3-Hydroxybutyric Acid↗
PubMed1996

Specific interaction of (R)-3-hydroxybutyrate dehydrogenase with membrane phosphatidylcholine as studied by ESR spectroscopy in oriented phospholipid multibilayers: coenzyme binding enhances the interaction with phosphatidylcholine.

The interaction of phospholipid with (R)-3-hydroxybutyrate dehydrogenase, a phosphatidylcholine-requiring membrane enzyme, has been studied using ESR spectroscopy of spin-labeled lipids, both as ordered multibilayers and in lipid vesicle suspensions (liposomes). Partially oriented phospholipid multibilayers were prepared from lipid vesicles composed of a 1:1 mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Vesicles containing (R)-3-hydroxybutyrate dehydrogenase yielded active preparations of the enzyme in such multibilayers. With increasing protein/lipid ratio, the order of the multibilayers was disrupted as monitored by ESR spectroscopy with a spin-labeled analogue of PC, 5-doxyl-PC (5 mol %, 10% of total PC) as a probe. The outer peak separation of 5-doxyl-PC varied with the lipid/protein ratio. The lower the ratio, the larger was the separation, with higher activity enzyme being more effective in exerting this effect. When 5-doxylstearic acid was substituted for 5-doxyl-PC or when the enzyme was inactive, the 2A(zz) value stayed practically constant at its lower limit (about 54 G). Multilayers composed of 81% PE, 11% diphosphatidylglycerol (DPG), and 8% 5-doxyl-PC (no unlabeled PC present) gave similar results. With this lipid mixture, the maximal 2A(zz) value (about 61 G) was reached at lower protein/lipid ratios, although the enzymic activity of (R)-3-hydroxybutyrate dehydrogenase is reduced to 40% in this system. The outer peak separation also depended on the presence of the coenzyme, NAD+, and 2-methylmalonate. The latter enhances binding of NAD+ about 100-fold by forming a ternary complex. With this ternary complex, the 2A(zz) values were increased unless the maximal values had been reached already in the absence of coenzyme. In all these experiments only a single ESR spectral component was observed. Similar results were obtained for the enzyme in liposomes, although the effect was less pronounced apparently due to the higher mobility of the probe. It is concluded that PC is motionally restricted by (R)-3-hydroxybutyrate dehydrogenase and yet is in rapid exchange with the bulk lipid on the ESR time scale. PC is required for formation of tight and functional complexes with NAD [Rudy et al. (1989) Biochemistry 28, 5354-5366], and such complexes strengthen the interaction of the enzyme with PC.

Animals↗
PubMed1996

Cryoelectron microscopy resolves FK506-binding protein sites on the skeletal muscle ryanodine receptor.

A 12-kDa immunophilin (FKBP12) is an integral component of the skeletal muscle ryanodine receptor (RyR). The RyR is a hetero-oligomeric complex with structural formula (FKBP)4(Ryr1)4, where Ryr1 is the 565-kDa product of the Ryr1 gene. To aid in the detection of the immunophilin's location in the receptor, we exchanged the FKBP12 present in RyR-enriched vesicles derived from sarcoplasmic reticulum with an engineered construct of FKBP12 fused to glutathione S-transferase and then isolated the complexes. Cryoelectron microscopy and image averaging of the complexes (in an orientation displaying the RyR's fourfold symmetry) revealed four symmetrically distributed, diffuse density regions that were located just outside the boundary defining the cytoplasmic assembly of the RyR. These regions are attributed to the glutathione transferase portion of the fusion protein because they are absent from receptors lacking the fusion protein. To more precisely define the location of FKBP12, we similarly analyzed complexes of RyR containing FKBP12 itself. Apparently some FKBP is lost during the purification or storage of the RyR because, to detect the receptor-bound immunophilin, it was necessary to add FKBP12 to the purified receptor before electron microscopy. Averaged images of these complexes showed a region of density that had not been observed previously in images of isolated receptors, and its position, along the edges of the transmembrane assembly, agreed with the position of the FKBP12 deduced from the experiments with the fusion protein. The proposed locations for FKBP12 are about 10 nm from the transmembrane baseplate assembly that contains the ion channel of the RyR.

Animals↗
PubMed1996

Different vagal modulation of the sinoatrial node and AV node in patients with congestive heart failure.

1. We have previously shown that in healthy young men autonomic control of the sinoatrial (SA) and AV node may be independent during sleep. It is conceivable, that this independence is lost in patients with high sympathetic activity. This would be in analogy to exercise in normal subjects, where an increase in sinus rate is associated with a shortening of the PR interval. 2. The aim of this study was to investigate whether this independence of SA and AV nodal autonomic modulation is maintained in patients with congestive heart failure. 3. For analysis of heart rate variability (HRV) the ECG was online digitized from 10 pm to 6 am in six patients with congestive heart failure (EF < 40%). The onset of P-waves and QRS-complexes was recognized by a computer algorithm with an accuracy of +/-1 ms. Power spectra of PR intervals and PP intervals were calculated for consecutive 256 second segments. The power in the high frequency component. (HF, 0.15 - 0.4 Hz) of PP intervals was used as an index of vagal drive to the SA node. The vagal input to the AV node was determined by the spectral power of the corresponding PR intervals. 4. All patients showed the typical spectral peak in the HF band, both in PP and PR. The power spectral density of HF varied over time with different patterns for PP and PR. The ratio of the HF power derived from PP and PR was calculated for each segment. This ratio was not constant, but showed a distinct time course. 5. Congestive heart failure did not abolish the independence of vagal modulation of SA and AV node, as assessed by the HF power derived from PP and PR intervals. Thus, the difference in vagal traffic to the SA and AV node was maintained even in the setting of high background sympathetic activity. Further investigation is needed to analyze potential factors responsible for this difference in patterns and the clinical relevance of this finding.

Adult↗
PubMed1996

Dysarthria and lacunar stroke: pathophysiologic aspects.

We investigated the supranuclear tracts involved in speech production in 8 patients with dysarthria associated with a single lacunar stroke. Magnetic resonance imaging revealed the lesion site in 7 out of 8 patients. We tested corticobulbar tract function using transcranial magnetic stimulation and demonstrated impairment of the corticolingual projections in all the patients and in 5 patients impairment of the corticofacial projections. Sensory function in the oral cavity was impaired in 1 out of 8 patients. 99mTc hexamethyl-propylene amine oxime-single-photon emission computerized tomography (HMPAO-SPECT) imaging, performed in 5 patients, showed cerebellar diaschisis in 1, suggesting additional cerebropontocerebellar tract impairment. Dysarthria observed in the 2 patients with impaired sensation in the oral cavity or with cerebellar diaschisis did not differ from the dysarthria found in the other patients. We conclude that interruption of the corticolingual pathways to the tongue is crucial in the pathogenesis of dysarthria following extracerebellar lacunar stroke.

Aged↗
PubMed1995

A novel FK506 binding protein can mediate the immunosuppressive effects of FK506 and is associated with the cardiac ryanodine receptor.

FK506, an immunosuppressant that prolongs allograft survival, is a co-drug with its intracellular receptor, FKBP12. The FKBP12.FK506 complex inhibits calcineurin, a critical signaling molecule during T-cell activation. FKBP12 was, until recently, the sole FKBP known to mediate calcineurin inhibition at clinically relevant FK506 concentrations. The best characterized cellular function of FKBP12 is the modulation of ryanodine receptor isoform-1, a component of the calcium release channel of skeletal muscle sarcoplasmic reticulum. Recently, a novel protein, FKBP12.6, was found to inhibit calcineurin at clinically relevant FK506 concentrations. We have cloned the cDNA encoding human FKBP12.6 and characterized the protein. In transfected Jurkat cells, FKBP12.6 is equivalent to FKBP12 at mediating the inhibitory effects of FK506. Upon binding rapamycin, FKBP12.6 complexes with the 288-kDa mammalian target of rapamycin. In contrast to FKBP12, FKBP12.6 is not associated with ryanodine receptor isoform-1 but with the distinct ryanodine receptor isoform-2 in cardiac muscle sarcoplasmic reticulum. Our results suggest that FKBP12.6 has both a unique physiological role in excitation-contraction coupling in cardiac muscle and the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin.

Amino Acid Sequence↗
PubMed1995

Affinity purification of the ryanodine receptor/calcium release channel from fast twitch skeletal muscle based on its tight association with FKBP12.

The ryanodine receptor (RyR)/calcium release channel isolated from skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) is tightly associated with FK506 binding protein of 12.0 kDa (FKBP12) (Jayaraman et al., (1992) J.Biol.Chem. 267, 9474-9477). In this study, we describe a new method of affinity chromatography for purifying the RyR from skeletal muscle SR based on: 1) its tight association with FKBP12; and 2) the finding that bound FKBP on the RyR can be exchanged with soluble FKBP12 (Timerman et al., (1995) J.Biol.Chem. 270, 2451-2459). Soluble glutathione S-transferase/FKBP12 (GST/FKBP12) fusion protein was first exchanged with bound FKBP12 on the RyR of TC. The TC were then solubilized with CHAPS and the complex of RyR.GST/FKBP12 was specifically adsorbed by glutathione Sepharose 4B and then eluted with glutathione. The RyR, purified by this method, has similar characteristics by SDS-PAGE, radioligand binding and immuno-reactivity as the RyR purified by multiple sequential column chromatography.

Animals↗