Search PubMed⌕ Search

Biomedical subjects

A Margreth

Publications and source records attributed to A Margreth.

At least 19 recordsLinked to original sources
PubMed2001

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.

Animals↗
PubMed2001

Clues to calcineurin function in mammalian fast-twitch muscle.

It is believed that brief, high amplitude Ca2+ transients, as found in fast-twitch muscles, are not sufficient to activate the calcineurin (Cn)-dependent signaling pathway involved in regulation of slow myosin and slow sarcoplasmic reticulum Ca2+-ATPase genes (Olson and Williams, Cell 101: 689-692, 2000). The results reported here try to fill the gap between this molecular knowledge, and the still fragmentary pieces of information on a possible different role of calcineurin in the same type of muscles. In the present work calcineurin was determined immunocytochemically by labeling fast- and slow-twitch fibers of representative rabbit muscles with anti-CnB antibodies, and was assessed by western blotting of isolated subcellular fractions. Calcineurin was found to be largely soluble and to be constitutively overexpressed in fast muscle as CnAalpha and CnAbeta isoforms, the latter appearing to be predominant. Particulate calcineurin was not only associated with myofibrils but also with membranes of various origins. Fluorescence microscopy showed that calcineurin was distributed in the same pattern with respect to sarcomeres in both types of fibers, and formed punctate dots spanning the I-Z-I region, rather than being exclusively located at the Z-line, a disposition described for cardiomyocytes (Frey et al., Proc Natl Acad Sci USA 97: 14,632-14,637, 2000). From knowledge that, in mammalian skeletal muscle fibers, junctional triads are located at the A-I band boundary, we explored the distribution of calcineurin between triadic components, after having verified that it was present in very low amounts in dystrophin-enriched sarcolemmal membranes. Our results demonstrate that a small but significant proportion of calcineurin coenriched with transverse tubules (TT), and copurified with the DHPR and with DHPR-associated PKA-AKAP15/18, thus suggesting that it is assembled as a multiprotein complex in the junctional membrane domain of TT. The membrane specificity of this association is further corroborated by the negative evidence for the presence of calcineurin in SR terminal cisternae. Calcineurin was separated from the DHPR and isolated as a AKAP15/18 subcomplex, including beta2 adrenergic receptor, in addition to PKA and calcineurin, following equilibrium centrifugation of detergent extracts on a linear sucrose gradient. We show that the alpha1 subunit skeletal isoform of the DHPR, is a substrate for calcineurin dephosphorylation, after previous phosphorylation by endogenous PKA.

Animals↗
PubMed2001

Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum.

Skeletal muscle triadin is a sarcoplasmic reticulum (SR) membrane protein that had been shown to interact structurally and functionally at the cytoplasmic domain (amino acid residues 1-47) with the ryanodine receptor (RyR1), and to undergo phosphorylation by endogenous calmodulin protein kinase (CaM K II) in isolated terminal cisternae from rabbit fast-twitch muscle. Here we show that triadin cytoplasmic domain expressed as glutathione-S-transferase fusion protein, is a substrate of the protein kinase. This finding is corroborated by identification of a specific consensus sequence in the deduced amino sequence between residue 34 and 37 of triadin. Confirming the regulatory features of CaM K II, we show the phosphorylation of triadin cytoplasmic segment by the kinase, when converted to the autonomous form. We propose that triadin modulates RyR1 in a phosphorylation-dependent manner.

Animals↗
PubMed2000

Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle.

Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR.

Animals↗
PubMed2000

Coordinate expression of Ca2+-ATPase slow-twitch isoform and of beta calmodulin-dependent protein kinase in phospholamban-deficient sarcoplasmic reticulum of rabbit masseter muscle.

Modulation of sarcoplasmic reticulum (SR) Ca(2+) transport by endogenous calmodulin-dependent protein kinase II (CaM K II) involves covalent changes of regulatory protein phospholamban (PLB), as a common, but not the only mechanism, in limb slow-twitch muscles of certain mammalian species, such as the rabbit. Here, using immunofluorescent techniques in situ, and biochemical and immunological methods on the isolated SR, we have demonstrated that rabbit masseter, a muscle with a distinct embryological origin, lacks PLB. Accommodating embryological heterogeneity in the paradigm of neural-dependent expression of specific isogenes in skeletal muscle fibers, our results provide novel evidence for the differential expression in the SR of 72 kDa beta components of CaM K II, together with the expression of a slow-twitch sarcoendoplasmic reticulum Ca(2+)-ATPase isoform, both in limb muscle and in the masseter.

Animals↗
PubMed2000

Variation of phospholamban in slow-twitch muscle sarcoplasmic reticulum between mammalian species and a link to the substrate specificity of endogenous Ca(2+)-calmodulin-dependent protein kinase.

Systematic immunological and biochemical studies indicate that the level of expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase regulatory protein phospholamban (PLB) in mammalian slow-twitch fibers varies from zero, in the rat, to significant levels in the rabbit, and even higher in humans. The lack of PLB expression in the rat, at the mRNA level, is shown to be exclusive to slow-twitch skeletal muscle, and not to be shared by the heart, thus suggesting a tissue-specific, in addition to a species-specific regulation of PLB. A comparison of sucrose density-purified SR of rat and rabbit slow-twitch muscle, with regard to protein compositional and phosphorylation properties, demonstrates that the biodiversity is two-fold, i.e. (a) in PLB membrane density; and (b) in the ability of membrane-bound Ca(2+)-calmodulin (CaM)-dependent protein kinase II to phosphorylate both PLB and SERCA2a (slow-twitch isoform of Ca(2+)-ATPase). The basal phosphorylation state of PLB at Thr-17 in isolated SR vesicles from rabbit slow-twitch muscle, colocalization of CaM K II with PLB and SERCA2a at the same membrane domain, and the divergent subcellular distribution of PKA, taken together, seem to argue for a differential heterogeneity in the regulation of Ca(2+) transport between such muscle and heart muscle.

Animals↗
PubMed2000

Pharmacological clues to calmodulin-mediated activation of skeletal ryanodine receptor using [3H]-ryanodine binding.

The hypothesis that calmodulin (CaM) may act as a positive modulator of junctional SR Ca2+-release channel/ ryanodine receptor (RyRl) rests largerly on the demonstrated capacity of CaM to interact structurally and functionally with RyRl at pCa > 8 (Tripathy et al., 1995). The goal of the present [3H]-ryanodine binding study was to produce, in isolated terminal cisternae (TC) and in purified junctional face membrane (JFM), CaM-mediated activation of RyRl at less extreme pCa values, i.e. closer to resting myoplasmic pCa, and to analyze more accurately the corresponding changes in binding affinity for ryanodine of the receptor. We were able to monitor these changes at an optimum pCa of 6.5, following pre-activation of native RyRl by mM concentrations of caffeine or microM concentrations of antraquinone compound doxorubicin, and at various doses of these triggers. CaM increased the affinity of ryanodine binding to isolated TC in the presence of 1 mM AMP-PCP as an activator of RyRl; the Kd for ryanodine binding was reduced from 21.8 nM to 13.2 nM by 1microM CaM. Similar effects of CaM were seen when AMP-PCP was replaced by either caffeine or doxorubicin. In order to discount the involvement of SR extrajunctional proteins in this effect, the experiments were repeated on purified JFM. Again, CaM increased the affinity of ryanodine binding; the Kd was reduced from 11.1 nM to 7.0 nM by 1 microM CaM (in the presence of doxorubicin). Pharmacological triggers of CaM-activatory action on native RyRl, like caffeine and doxorubicin, have long been characterized for their ability to activate RyRl by increasing the Ca2+-sensitivity of the receptor. We speculate that the triggering effect of these agents on the CaM-mediated mechanism in vitro might mimick one of the early effects of the activation of RyRl in skeletal muscle, during E-C coupling.

Adenosine Triphosphate↗
PubMed1999

Interaction of triadin with histidine-rich Ca(2+)-binding protein at the triadic junction in skeletal muscle fibers.

The present study documents the binding interaction of skeletal muscle sarcoplasmic reticulum (SR) transmembrane protein triadin with peripheral histidine-rich, Ca(2+)-binding protein (HCP). In addition to providing further evidence that HCP coenriches with RyR1, FKBP-12, triadin and calsequestrin (CS) in sucrose-density-purified TC vesicles, using specific polyclonal antibody, we show it to be expressed as a single protein species, both in fast-twitch and slow-twitch fibers, and to identically localize to the I-band. Colocalization of HCP and triadin at junctional triads is supported by the overlapping staining pattern using monoclonal antibodies to triadin. We show a specific binding interaction between digoxigenin-HCP and triadin, using ligand blot techniques. The importance of this finding is strengthened by the similarities in binding affinity and in Ca2+ dependence, (0.1-1 mM Ca2+) of the interaction of digoxigenin-HCP with immobilized TC vesicles. Suggesting that triadin dually interacts with HCP and with CS, at distinct sites, we have found that triadin-CS interaction in overlays does not require the presence of Ca2+. Consistent with the binding of CS to triadin luminal domain (Guo and Campbell, 1995), we show that binding sites for digoxigenin-CS, although not binding sites for digoxigenin-HCP, can be recovered in the 92 kDa triadin fragment, after chymotryptic cleavage of the NH2-terminal end of the folded molecule in intact TC vesicles. These differential effects form the basis for the hypothesis that HCP anchors to the junctional membrane domain of the SR, through binding to triadin short cytoplasmic domain at the NH2 terminus. Although the function of this interaction, as such, is not well understood, it seems of potential biological interest within the more general context of the structural-functional role of triadin at the triadic junction in skeletal muscle.

Animals↗
PubMed1999

Sarcoplasmic reticulum in aged skeletal muscle.

A decrease in muscle mass and strength and a slowing of muscle contraction are common features of the ageing process. Recent advances in basic biochemical knowledge have provided new insights into pathogenetic mechanisms underlying age-related changes in the excitation-contraction coupling process, Ca2+-transients and isometric twitch-contraction time. Sarcoplasmic reticulum (SR) Ca2+-pumps are not basically altered in physiological ageing, but several aspects of the Ca2+-transport system remain controversial, regarding phosphorylation-dependent regulation in slow-twitch muscles, in particular. It seems that conflicting reports and divergent interpretations concerning the effect of ageing on SR Ca2+-release arise from the type of muscle, the stage of the ageing process and the animal species. A cause-effect relationship between the decrease in dihydropyridine receptors and in muscle strength is strongly suggested by studies in transgenic mice, but is unsupported by our studies with fast-twitch and slow-twitch muscles of old rats. Our experimental evidence also seems to exclude the occurrence of age-related changes in the number and in the functional behaviour of Ca2+-release channels/ryanodine receptors (RyR1), based on ¿3H-ryanodine binding studies. There is emerging, although only suggestive evidence, so far, that modulation of RyR1 by SR luminal protein calsequestrin, or the functional coupling of RyRs by FKBP-12, may be altered in ageing skeletal muscle.

Aging↗
PubMed1997

Functional behaviour of the ryanodine receptor/Ca(2+)-release channel in vesiculated derivatives of the junctional membrane of terminal cisternae of rabbit fast muscle sarcoplasmic reticulum.

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 JFM. That was not accounted for by any significant difference in the IC50 concentrations of CaM varying between 40 nM to approximately 80 nM, at low-intermediate and at high KCl concentrations, respectively. Additional results with enriched JFM using doxorubicin, a pharmacological Ca2+ channel allosteric modifier, strengthen the hypothesis that the conformational state at which RyR is stabilized, according to the experimental assay conditions for [3H]-ryanodine binding, directly influences CaM-sensitivity.

Animals↗
PubMed1996

Colocalization of the dihydropyridine receptor, the plasma-membrane calcium ATPase isoform 1 and the sodium/calcium exchanger to the junctional-membrane domain of transverse tubules of rabbit skeletal muscle.

The subcellular distribution of the calmodulin-stimulated plasma-membrane Ca(2+)-ATPase (PMCA) has been studied in rat and rabbit skeletal muscle cells by indirect (calmodulin gel overlays) and direct (Western blotting with specific antibodies) methods. It has also been studied in situ in immunocytochemistry experiments. The distribution of PMCA has been compared with that of the NA+/Ca2+ exchanger and of the dihydropyridine receptor, which has been studied by Western blotting with specific antibodies. Both PMCA and the Na+/Ca2+ exchanger had a dual localization, i.e., they were found in the plasma membrane and in the transverse-tubule fractions of the two main types of skeletal muscles studied. The pump and the exchanger were not diffusely distributed in the transverse-tubule-membrane system, but specifically confined to the membrane domain where the dihydropyridine receptor was also localized, i.e., the junctional membrane. Experiments with isoform-specific antibodies have shown that the pump isoform expressed in skeletal muscle is PMCA 1.

Animals↗
PubMed1996

Skeletal muscle sarcoplasmic reticulum phenotype in myotonic dystrophy.

In this study we investigated the sarcoplasmic reticulum (SR), alongside myofibrillar phenotype, in muscle samples from five Myotonic Dystrophy (DM) patients and five control individuals. DM muscles exhibited as a common feature, a decrease in the slow isoform of myosin heavy chain (MHC) and of troponin C in myofibrils. We observed a match between myofibrillar changes and changes in SR membrane markers specific to fiber type, i.e. the fast (SERCA1) Ca(2+)-ATPase isoform increased concomitantly with a decrease of protein phospholamban (PLB), which in native SR membranes colocalizes with the slow (SERCA2a) SR Ca(2+)-ATPase, and regulates its activity depending on phosphorylation by protein kinases. Our results outline a cellular process selectively affecting slow-twitch fibers, and non-degenerative in nature, since neither the total number of Ca(2+)-pumps or of ryanodine receptor/Ca(2+)-release channels, or their ratio to the dihydropyridine receptor/voltage sensor in junctional transverse tubules, were found to be significantly changed in DM muscle. The only documented, apparently specific molecular changes associated with this process in the SR of DM muscle, are the defective expression of the slow/cardiac isoform of Ca(2+)-binding protein calsequestrin, together with an increased phosphorylation activity of membrane-bound 60 kDa Ca(2+)-calmodulin (CaM) dependent protein kinase. Enhanced phosphorylation of PLB by membrane-bound Ca(2+)-CaM protein kinase also appeared to be most pronounced in biopsy from a patient with a very high CTG expansion, as was the overall 'slow-to-fast' transformation of the same muscle biopsy. Animal studies showed that endogenous Ca(2+)-CaM protein kinase exerts a dual activatory role on SERCA2a SR Ca(2+)-ATPase, i.e. either by direct phosphorylation of the Ca(2+)-ATPase protein, or mediated by phosphorylation of PLB. Our results seem to be consistent with a maturational-related abnormality and/or with altered modulatory mechanisms of SR Ca(2+)-transport in DM slow-twitch muscle fibers.

Adolescent↗
PubMed1996

Age-related abnormalities in regulation of the ryanodine receptor in rat fast-twitch muscle.

The tibialis anterior (TA) muscles of 6-month-old and 24-month-old male Wistar rats, after being characterized, at the fast motor unit level, for twitch properties, were dissected and processed by a procedure [Margreth A., Damiani E., Tobaldin G. Biochem Biophys Res Commun 1993; 197: 1303-1311] aimed at obtaining a representative total membrane fraction comprising 70-80% of the total muscle content of sarcoplasmic reticulum (SR) and transverse tubule (TT) membranes (about 20 mg protein/g). Skeletal muscle membranes were analyzed for protein composition, and the content and functional properties of specific components of the free and junctional subcompartments of the SR and of junctional TT. Our results, while confirming a twitch prolongation in TA of old rats, do not demonstrate any associated age-related change concerning: (a) the overall number and functional properties of Ca2+ pumps, as characterized by kinetic parameters, Ca(2+)-dependency, and the protein isoform specificity of SR Ca(2+)-ATPase; (b) the number of functional junctional SR Ca(2+)-release channels, on the basis of Bmax values for high-affinity binding of [3H]-ryanodine to skeletal muscle membranes at optimal Ca2+; (c) the overall muscle dihydropyridine receptor/ryanodine receptor (RyR) ratio. We conclude from these findings, and the additional negative evidence for changes in membrane density of specific components of junctional SR, including 60 kDa Ca(2+)-calmodulin protein kinase, that this membrane domain, like the Ca(2+)-pump domain of the SR, are in no way basically altered at early stages of the aging process, as investigated here. Because of that, we allege particular significance to the occurrence of age-related, specific abnormalities in regulation of RyR in rat TA. The main supportive evidence is as follows: (a) an increased sensitivity to Ca2+ of the RyR of old muscle, and, more importantly; (b) an increased sensitivity to caffeine of [3H]ryanodine binding to the RyR at optimal Ca2+ and also optimal for the activity of the Ca(2+)-release channel. The results reported here also demonstrate that there are two classes of caffeine sites in rat TA muscle, as defined by differences in EC50 values at resting (pCa 7) and at high Ca2+ (pCa 4-5), that sites involved in stimulation of [3H]-ryanodine binding to the RyR are distinguished by a higher affinity (caffeine below mM), and that only these sites undergo age-related changes. Thus, although the underlying age-related abnormality of the RyR remains to be elucidated, it appears to satisfy the requirement for being regarded as a specific change, which in itself might argue for its being fundamentally related to the twitch prolongation of the muscle.

Aging↗
PubMed1995

Identification of triadin and of histidine-rich Ca(2+)-binding protein as substrates of 60 kDa calmodulin-dependent protein kinase in junctional terminal cisternae of sarcoplasmic reticulum of rabbit fast muscle.

The endogenous calmodulin-protein kinase system of sarcoplasmic reticulum terminal cisternae of rabbit fast-twitch muscle was studied. Investigation of a single Ca(2+)-channel in terminal cisternae fused to planar lipid bilayers demonstrated that the endogenous kinase inhibits the channel, although it remained unclear whether the phosphorylation sites are on the channel protein or on other junctional sarcoplasmic reticulum specific proteins [Hain et al., (1994) Biophys. J. 67, 1823-1833]. Our results, which show that two junctional sarcoplasmic reticulum specific proteins, i.e., triadin and histidine-rich Ca(2+)-binding protein, but not the ryanodine receptor/Ca(2+)-channel protein, are phosphorylated by membrane-bound 60 kDa protein kinase, seem to be able to resolve this ambiguity. Furthermore, such a probably specific protein isoform of calmodulin-protein kinase, by its substrate specificity and exposure to the cytoplasmic side of terminal cisternae at the junctional membrane domain and based on protease sensitivity, also seems to possess some of the potential requirements for a regulatory role in the functional state of the Ca(2+)-channel.

Animals↗
PubMed1995

Evidence that spinach leaves express calreticulin but not calsequestrin.

The presence of either calreticulin (CR) or calsequestrin (CS-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca(2+)-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn(2+) (i.e., under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca(2+). Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca(2+)-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.

Amino Acid Sequence↗
PubMed1994

Identification of two ryanodine receptor transcripts in neonatal, slow-, and fast-twitch rabbit skeletal muscles.

Analysis of the primary structure of the rabbit skeletal muscle ryanodine receptor led to the identification of two molecules of 5032 and 5037 residues, respectively. Such a sequence discrepancy is likely to be due to the alternative splicing of a 15 bp exon (1) encoding a 5 amino acid insertion (Ala-Gly-Asp-Ala-Gln) after residue 3479. By using PCR on first strand cDNA, we searched for the 15 base pair insertion in the ryanodine receptor mRNA from adult slow- and fast-twitch skeletal muscle, as well as from fast-muscles, at various stages of post-natal development. All rabbit skeletal muscle mRNAs, regardless of their developmental stage and twitch properties, contain two RYR transcripts, suggesting the coexistence of two RYR isoforms in mammalian skeletal muscle.

Aging↗
PubMed1994

Characterization study of the ryanodine receptor and of calsequestrin isoforms of mammalian skeletal muscles in relation to fibre types.

We have investigated high-affinity ryanodine-binding sites in membrane preparations from representative fast-twitch and slow-twitch muscles of the rabbit and rat, as well as from human mixed muscle. Our results, obtained in high-ionic strength binding buffer, demonstrate extensive similarities in binding affinity for [3H]ryanodine (Kd: about 10 nM) and a two-fold to four-fold difference in membrane density of the ryanodine receptor between fast-twitch and slow-twitch muscle of the rat and rabbit, respectively. The [3H]ryanodine-pCa relationship for the Ca(2+)-activation curve of ryanodine binding was found to be similar for all mammalian muscles, as tested at 20 nM ryanodine. With 10 mM caffeine or 50 microM doxorubicin the pCa for half-maximal activation of [3H]ryanodine binding invariably shifted from an average pCa value of 6.5 to pCa 7.1-7.3. IC50 values for the inhibition of [3H]ryanodine binding by Ruthenium Red, a Ca(2+)-release channel blocker, did not differ significantly (range 0.3-1.0 microM). The Ca(2+)-dependence curve (range 1 nM-10 mM free Ca2+) that we have observed at 5 nM ryanodine, for [3H]ryanodine binding to terminal cisternae from rabbit fast-twitch, as well as slow-twitch muscle, is bell-shaped and differs from that obtained with cardiac terminal cisternae from the same species. Cardiac ryanodine receptor is also clearly distinguishable for electrophoretic mobility, Cleveland's peptide maps, and, most strikingly, for total lack of cross-reactivity with polyclonal antibody to fast skeletal RyR. By the same properties, the ryanodine receptor of fast- and slow-twitch muscle appear to be the same or a similar protein. On investigating the composition of calsequestrin in rat and human skeletal muscles, both in membrane-bound form and after purification by phenyl-Sepharose chromatography, we have been able to show that, independent of the animal species, the cardiac isoform, as characterized by the identical amino-terminal amino-acid sequence, pattern of immunoreactivity, and lack of Ca(2+)-dependent shift in mobility on SDS-PAGE, is exclusively expressed in slow-twitch fibres, together with the main fast-skeletal calsequestrin isoform. While our experimental findings strongly argue for the presence of only one population of skeletal-specific Ca(2+)-release channels in junctional terminal cisternae of mammalian fast-twitch and slow-twitch muscle, they at the same time suggest the existence of differences in calsequestrin modulation of Ca(2+)-release, depending on its isoform composition.

Amino Acid Sequence↗