Polymorphism of sarcoplasmic-reticulum adenosine triphosphatase of rabbit skeletal muscle.
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ABSTRACT:
SUBMITTER: Damiani E
PROVIDER: S-EPMC1163077 | biostudies-other | 1981 Jul
REPOSITORIES: biostudies-other
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Project description:DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.
| S-EPMC1609923 | biostudies-literature
Project description:The isolation and the determination of the amino-acid sequences of the soluble tryptic peptides, derived by cleavage at arginine residues, of the succinylated (3-carboxypropionylated) S-carboxymethylated adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum are described. Treatment of the protein with succinic anhydride gave a derivative that was readily digested with trypsin, yielding two distinct sets of peptides. One set comprises large, relatively hydrophobic, peptides that are highly aggregated (or insoluble) in aqueous solution and that have been identified, by several criteria, with the portion of the protein embedded in the lipid bilayer in the sarcoplasmic reticulum. The second set, which is described here, comprises peptides that have properties typical of those derived from soluble globular proteins and that constitute that part of the protein external to the lipid bilayer. The sequences of these soluble tryptic peptides contain 586 unique residues. Details of the isolation of the peptides and the determination of the sequences are contained in Supplementary Publication SUP 50102 (88 pages) which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
| S-EPMC1162440 | biostudies-other
Project description:The soluble peptides from the peptic digest of the reduced S-carboxymethylated 3-carboxypropionylated adenosine triphosphatase protein have been isolated and most of their structures have been determined. About 397 residues of the protein were represented in these peptides. The reduced S-carboxymethylated protein was digested with thermolysin, and peptides containing arginine or carboxymethylcysteine were isolated and characterized. Some peptides isolated from tryptic and staphylococcal-proteinase digests of the protein are described. The information contained within the structures of these peptides has been used to reconstruct long stretches of the sequence of the ATPase protein that constitute most of the protein structure external to the lipid bilayer (Allen, Trinnaman and Green (1980) Biochem. J. 187, 591-616). The details of some of the chromatographic steps used in the isolation of the peptides and the properties of the peptides are contained in Supplementary Publication SUP 50104 (45 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
| S-EPMC1162442 | biostudies-other
Project description:The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.
| S-EPMC1164174 | biostudies-other
Project description:The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.
| S-EPMC1186603 | biostudies-other
Project description:A preliminary investigation of the primary structure of the Ca(2+-transporting ATPase (adenosine triphosphatase) protein of rabbit skeletal-muscle sarcoplasmic reticulum is reported. The preparation of derivatives of delipidated protein in a form suitable for sequence analysis is described. Tryptic peptides containing S-carboxymethylcysteine residues were isolated from the reduced carboxymethylated protein, and their sequences were partially determined. The results are consistent with mol.wt. about 105000 for the polypeptide, and the absence of extended repeated lengths of sequence. The distribution of tryptophan and cysteine residues between large, aggregated peptides and soluble tryptic peptides shows that these residues are concentrated in different regions of the primary structure. This observation agrees with other evidence that these residues are, on the whole, widely separated in the native protein. The details of the procedures used to isolate the peptides, and the evidence for the determination of their sequences, are given Supplementary Publication SUP 50085 (30 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem.J. (1978) 169, 5.
| S-EPMC1185791 | biostudies-other
Project description:TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca(2+) channel, it contributes to intracellular calcium concentration ([Ca(2+)]i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca(2+) imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca(2+)]i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca(2+) elevation suggesting that TRPV1 may participate to a secondary SR Ca(2+) liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca(2+) leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.
| S-EPMC3594164 | biostudies-literature
Project description:Skeletal muscle stores Ca²(+) in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²(+) in the SR ([Ca²(+)](SR)) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²(+)](SR) in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²(+)](SR, free) is 390 ?M. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²(+)](SR, free) to ?8 ?M, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²(+) content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²(+) and for future studies of the role of SR Ca²(+) in healthy and diseased mammalian muscle.
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Project description:Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca(2+) channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca(2+) were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.
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Project description:Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309-314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 μm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.
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