FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2018485485> ?p ?o ?g. }

• W2018485485 endingPage "24037" @default.
• W2018485485 startingPage "24030" @default.
• W2018485485 abstract "We investigated type 3 isoform (RyR3) of ryanodine receptor in rabbit skeletal muscles using an antibody specific for RyR3. By Western blot analysis and by immunoprecipitation, a single polypeptide for RyR3 was detected in sarcoplasmic reticulum vesicles from rabbit diaphragm but not in those from back muscle. The molecular mass was slightly smaller than that of RyR1, the major isoform in skeletal muscles. Each of RyR1 and RyR3 formed a homotetramer in rabbit diaphragm. RyR3 had a single class of [3H]ryanodine binding sites of high affinity (K D = 1.6 nm). From theB max of the binding, the content of RyR3 was estimated to be only 0.6% of RyR1 in rabbit diaphragm. [3H]Ryanodine binding to RyR3 was biphasically dependent on Ca2+, as is true of RyR1, and was stimulated further by adenine nucleotide, caffeine, or high salt concentration. Procaine and ruthenium red inhibited the binding. RyR3 was more resistant to Mg2+ inhibition than RyR1. Interestingly, RyR3 showed about a 7-fold lower Ca2+ sensitivity for activation than RyR1. Comparison with the counterparts in bullfrog skeletal muscles indicates that the Ca2+ sensitivities of RyR3 homologs are similar to each other, whereas those of RyR1 homologs are species-specific. We investigated type 3 isoform (RyR3) of ryanodine receptor in rabbit skeletal muscles using an antibody specific for RyR3. By Western blot analysis and by immunoprecipitation, a single polypeptide for RyR3 was detected in sarcoplasmic reticulum vesicles from rabbit diaphragm but not in those from back muscle. The molecular mass was slightly smaller than that of RyR1, the major isoform in skeletal muscles. Each of RyR1 and RyR3 formed a homotetramer in rabbit diaphragm. RyR3 had a single class of [3H]ryanodine binding sites of high affinity (K D = 1.6 nm). From theB max of the binding, the content of RyR3 was estimated to be only 0.6% of RyR1 in rabbit diaphragm. [3H]Ryanodine binding to RyR3 was biphasically dependent on Ca2+, as is true of RyR1, and was stimulated further by adenine nucleotide, caffeine, or high salt concentration. Procaine and ruthenium red inhibited the binding. RyR3 was more resistant to Mg2+ inhibition than RyR1. Interestingly, RyR3 showed about a 7-fold lower Ca2+ sensitivity for activation than RyR1. Comparison with the counterparts in bullfrog skeletal muscles indicates that the Ca2+ sensitivities of RyR3 homologs are similar to each other, whereas those of RyR1 homologs are species-specific. Ryanodine receptor (RyR) 1The abbreviations used are: RyR, ryanodine receptor; CICR, Ca2+-induced Ca2+ release; SR, sarcoplasmic reticulum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPSO, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; AMP-PCP, adenosine 5′-(β,γ-methylenetriphosphate). 1The abbreviations used are: RyR, ryanodine receptor; CICR, Ca2+-induced Ca2+ release; SR, sarcoplasmic reticulum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPSO, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; AMP-PCP, adenosine 5′-(β,γ-methylenetriphosphate). is one of the Ca2+ release channels of intracellular Ca2+stores and may play important roles not only in muscles but also in various other cells (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (834) Google Scholar, 3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (227) Google Scholar, 4Sorrentino V. Adv. Pharmacol. 1995; 33: 67-90Crossref PubMed Scopus (71) Google Scholar, 5Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (363) Google Scholar). Molecular cloning of cDNAs encoding mammalian RyRs has shown that there are three distinct isoforms of RyR (RyR1–3) encoded by different genes (6Takeshima H. Nishimura S. Matsumoto T. Ishida H. Kangawa K. Minamino N. Matsuo H. Ueda M. Hanaoka M. Hirose T. Numa S. Nature. 1989; 339: 439-445Crossref PubMed Scopus (856) Google Scholar, 7Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 8Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (287) Google Scholar, 9Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 10Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (350) Google Scholar, 11Marziali G. Rossi D. Giannini G. Charlesworth A. Sorrentino V. FEBS Lett. 1996; 394: 76-82Crossref PubMed Scopus (40) Google Scholar). Although recent studies by RNase protection analysis and by reverse transcription-polymerase chain reaction analysis revealed that mRNA for all RyR isoforms could be widely detected in various tissues (12Furuichi T. Furutama D. Hakamata Y. Nakai J. Takeshima H. Mikoshiba K. J. Neurosci. 1994; 14: 4794-4805Crossref PubMed Google Scholar, 13Ledbetter M.W. Preiner J.K. Louis C.F. Mickelson J.R. J. Biol. Chem. 1994; 269: 31544-31551Abstract Full Text PDF PubMed Google Scholar, 14Giannini G. Conti A. Mammarella S. Scrobogna M. Sorrentino V. J. Cell Biol. 1995; 128: 893-904Crossref PubMed Scopus (476) Google Scholar), these isoforms are highly tissue-specific: RyR1 is expressed primarily in skeletal muscles and cerebellar Purkinje cells, RyR2 in cardiac muscles and ubiquitous regions of brain, and RyR3 in specific regions of brain and various peripheral tissues. Whereas RyR1 and RyR2 were obtained as purified proteins and their functional properties have been extensively examined (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (834) Google Scholar, 3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (227) Google Scholar, 4Sorrentino V. Adv. Pharmacol. 1995; 33: 67-90Crossref PubMed Scopus (71) Google Scholar, 5Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (363) Google Scholar), molecular characterization of RyR3 remains to be elucidated because of its more miniscule amount. Several studies have been done on the function of RyR3 in tissues where mRNA for RyR3 is expressed exclusively (15Giannini G. Clementi E. Ceci R. Marziali G. Sorrentino V. Science. 1992; 257: 91-94Crossref PubMed Scopus (211) Google Scholar, 16Hakamata Y. Nishimura S. Nakai J. Nakashima Y. Kita T. Imoto K. FEBS Lett. 1994; 352: 206-210Crossref PubMed Scopus (70) Google Scholar, 17Takeshima H. Yamazawa T. Ikemoto T. Takekura H. Nishi M. Noda T. Iino M. EMBO J. 1995; 14: 2999-3006Crossref PubMed Scopus (128) Google Scholar), but there have been few investigations on RyR3 protein and its functions. We purified α- and β-RyR from bullfrog skeletal muscles where the two isoforms coexist in almost equal amounts (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar) and found them to be homologs of RyR1 and RyR3, respectively, from similarities in the amino acid sequences deduced from their cDNAs (19Oyamada H. Murayama T. Takagi T. Iino M. Iwabe N. Miyata T. Ogawa Y. Endo M. J. Biol. Chem. 1994; 269: 17206-17214Abstract Full Text PDF PubMed Google Scholar). Taking advantage of this, we recently produced an antibody (anti-RyR3) that reacts specifically with RyR3 among the three RyR isoforms, and we revealed by immunoprecipitation with anti-RyR3 that a homotetramer of RyR3 protein is expressed in some specific regions of mammalian brain as a caffeine-sensitive Ca2+-induced Ca2+ release (CICR) channel (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Analysis of its mRNA showed that there were several tissue-specific alternative splicing variants of RyR3, especially between brain and peripheral tissues (11Marziali G. Rossi D. Giannini G. Charlesworth A. Sorrentino V. FEBS Lett. 1996; 394: 76-82Crossref PubMed Scopus (40) Google Scholar, 21Miyatake R. Furukawa A. Matsushita M. Iwahashi K. Nakamura K. Ichikawa Y. Suwaki H. FEBS Lett. 1996; 395: 123-126Crossref PubMed Scopus (32) Google Scholar). These alternatively spliced variants might generate potential heterogeneity in the function of RyR3 among tissues. mRNA for RyR3 was also found in mammalian skeletal muscles that express primarily RyR1 (14Giannini G. Conti A. Mammarella S. Scrobogna M. Sorrentino V. J. Cell Biol. 1995; 128: 893-904Crossref PubMed Scopus (476) Google Scholar, 15Giannini G. Clementi E. Ceci R. Marziali G. Sorrentino V. Science. 1992; 257: 91-94Crossref PubMed Scopus (211) Google Scholar, 17Takeshima H. Yamazawa T. Ikemoto T. Takekura H. Nishi M. Noda T. Iino M. EMBO J. 1995; 14: 2999-3006Crossref PubMed Scopus (128) Google Scholar). Conti et al. (22Conti A. Gorza L. Sorrentino V. Biochem. J. 1996; 316: 19-23Crossref PubMed Scopus (97) Google Scholar) recently demonstrated minor amounts of RyR3 protein in mammalian skeletal muscles by Western blot analysis. Interestingly, the content of RyR3 varied among different muscles in rat: higher levels in diaphragm and soleus, lower levels in abdominal muscles and tibialis anterior, and no detectable amounts in the extensor digitorum longus. A particularly high content of RyR3 in the diaphragm was observed in several mammals examined (rat, mouse, rabbit, and cow). To learn whether there are functional differences in RyR3 between brain and skeletal muscles, we identified and characterized here RyR3 expressed in rabbit skeletal muscles using the anti-RyR3 antibody (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The results of this study show that a homotetramer of RyR3 is expressed in rabbit diaphragm but is undetectable in back muscle. It may function as a CICR channel that is similar to RyR3 in rabbit brain. Further unique properties of RyR3 are also revealed. The peptide corresponding to the amino acid sequence 4375–4387 of the rabbit RyR3 (RyR3-peptide) was synthesized at the Central Laboratory of Medical Sciences, Division of Biochemical Analysis, Juntendo University School of Medicine (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). [3H]Ryanodine (60–90 Ci/mmol) was purchased from NEN Life Science Products. Goat anti-rabbit IgG-agarose was from Sigma. Egg lecithin (egg total phosphatide extract) was from Avanti Polar Lipids. All other reagents were of analytical grade. Heavy fraction of SR vesicles was prepared from rabbit diaphragm or back muscle according to Murayama and Ogawa (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar) in the presence of a mixture of protease inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml antipain, 2 μg/ml pepstatin A, and 2 μg/ml chymostatin). The isolated membranes were quickly frozen in liquid N2 and stored at −80 °C until use. SR vesicles (2–4 mg/ml at the final concentration) were incubated for 15 min on ice with 2% CHAPS and 1% egg lecithin in a buffer containing 0.5 m NaCl, 20 mm Tris-HCl, pH 7.4, 2 mm dithiothreitol, and a mixture of protease inhibitors. The supernatant after centrifugation at 100,000 × g for 30 min was collected and used for detection and characterization of RyR3. Sucrose gradient ultracentrifugation with 5–20% linear gradients was performed as described (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar). SDS-polyacrylamide gel electrophoresis was performed with 2–12% linear gradient gels (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar, 20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The molecular mass (in kDa) of standards used here was 205 (myosin heavy chain), 116 (β-galactosidase), 97.4 (phosphorylaseb), 66 (bovine serum albumin), 45 (ovalbumin), and 29 (carbonic anhydrase). Gels were stained with Coomassie Brilliant Blue. For Western blotting, the separated proteins were transferred electrophoretically onto polyvinylidene difluoride membranes at 40 V overnight in the presence of 0.02% SDS to facilitate the transfer of high molecular weight proteins (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar, 20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Anti-RyR3 antibody was produced in rabbits against the synthetic peptide corresponding to the amino acid sequence 4375–4387 of rabbit RyR3, and was purified with protein-bound polyvinylidene difluoride membranes of β-RyR from bullfrog skeletal muscle as described in Murayama and Ogawa (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The antibody reacted with RyR3 of mammalian brain and β-RyR of frog or chicken skeletal muscle, but no cross-reactivity was observed with mammalian RyR1, RyR2, or α-RyR of non-mammalian vertebrate skeletal muscles (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Western blotting was carried out colorimetrically as described previously (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar) using peroxidaseconjugated goat anti-rabbit IgG as the secondary antibody and 3,3′-diaminobenzidine as the substrate. Immunoprecipitation of RyR3 was performed using the purified anti-RyR3 antibody and goat anti-rabbit IgG-agarose beads (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Solubilized SR vesicles (1–2 mg of protein) were incubated overnight at 4 °C with 100 μl of anti-RyR3 antibody and 30 μl of anti-rabbit IgG-agarose beads. For detection of the polypeptide band for RyR3, the beads were washed five times with a buffer containing 0.5 m NaCl, 50 mm Tris-HCl, pH 7.5, 0.05% Tween 20, 0.1% CHAPS, and 2 mmdithiothreitol and were resuspended in 40 μl of 2 × Laemmli sample buffer (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) containing 0.1 m dithiothreitol. Aliquots of 15–20 μl were subjected to SDS-polyacrylamide gel electrophoresis. Assays were carried out as described previously (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). SR vesicles (0.2 mg) were incubated with [3H]ryanodine (2–21 nm) for 4 h at 25 °C in 0.5 ml of a binding buffer containing 0.3 or 1m NaCl, 10 mm MOPSO/NaOH, pH 6.8, 2 mm dithiothreitol, 1% CHAPS, 0.5% egg lecithin, the mixture of protease inhibitors, and a specified concentration of free Ca2+ buffered with 10 mm EGTA. Then RyR3 was immunoprecipitated with 100 μl of anti-RyR3 and 30 μl of anti-rabbit IgG-agarose beads. The radioactivity of the beads after five washings with a buffer (1 m NaCl, 10 mmMOPSO/NaOH, pH 6.8, 1% CHAPS, 0.5% egg lecithin, 2 mmdithiothreitol, and 0.1 mm Ca2+) was determined as the activity for RyR3. The nonspecific radioactivity was determined in the absence of anti-RyR3 in each experiment. The value was similar to that determined with the addition of 30 μmRyR3-peptide (see Fig. 5). The nonspecific activity was decreased as the salt concentration of the medium was increased and approached a value similar to the result on the addition of excess unlabeled ryanodine (10–50 μm) at 0.3 m NaCl. These findings indicate that it may be caused probably by direct binding of [3H]ryanodine and RyR1 to the agarose beads (see Fig. 2) rather than weak binding to anti-RyR3. By compensating for the nonspecific radioactivity thus determined, [3H]ryanodine binding specific to RyR3 can be obtained. In this study, the [3H]ryanodine binding to RyR3 could not be determined accurately in an isotonic medium containing 0.17 m NaCl because of a high nonspecific radioactivity. Instead, assays were carried out in a medium containing 0.3 m NaCl where the properties are assumed to be more physiological than those in 1m NaCl (see Figs. 8 and 9, Tables I and II). The binding for RyR1 was measured by filtering an aliquot of the remaining supernatants after immunoprecipitation through polyethyleneimine-treated Whatman GF/B glass filters (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar). Free Ca2+ was calculated using values of 8.79 × 105m−1 and 1.82 × 103m−1 as the apparent binding constants for Ca2+ of EGTA (24Harafuji H. Ogawa Y. J. Biochem. 1980; 87: 1305-1312Crossref PubMed Scopus (345) Google Scholar) and of AMP-PCP (25Ogawa Y. Kurebayashi N. Harafuji H. J. Biochem. 1986; 100: 1305-1318Crossref PubMed Scopus (17) Google Scholar), respectively.Figure 2Immunoprecipitation of RyR3 in rabbit skeletal muscle SR vesicles. 5 mg of solubilized SR vesicles from rabbit diaphragm (panel A) or back muscle (panel B) was incubated with 100 μl of anti-RyR3 antibody and 30 μl of goat anti-rabbit IgG-agarose beads as described under “Experimental Procedures” in the absence (left lane) and presence (right lane) of 30 μm RyR3-peptide. The immunoprecipitated products were resolved on a 2–12% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. The band for RyR3 which selectively disappeared in the presence of the peptide is found only in diaphragm muscle SR (arrowhead).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Ca2+ concentration dependence of [3H]ryanodine binding to RyR3. Assays were carried out as in Table I in the presence of 1 mm AMP-PCP and various concentrations of free Ca2+. Panel A, [3H]ryanodine binding to RyR3 in the medium containing 0.3 m (open circles) or 1 m NaCl (closed circles). Panel B, Ca2+dependences of the normalized [3H]ryanodine binding activity of RyR1 (open squares) and RyR3 (open circles) in 0.3 m NaCl medium. The values for 100% denote 4.3 and 0.023 pmol/mg of protein for RyR1 and RyR3, respectively. The data are the mean ± S.E. of three to six determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Dose-dependent inhibition of [3H]ryanodine binding to RyR1 and RyR3 by Mg2+. Assays were carried out as in Table I in the presence of 0.1 mm free Ca2+ with various concentrations of Mg2+. The data are mean ± S.E. of four determinations. The values for 100% denote 3.22 and 0.021 pmol/mg of protein for RyR1 and RyR3, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IEffect of CICR activators on [3H]ryanodine binding to RyR3Ligands added[3H]Ryanodine boundStimulationfmol/mg protein-foldNone (10 mmEGTA)0.3 ± 0.2—+10 μm Ca2+6.3 ± 0.71+10 μm Ca2+ + 1 mm AMP-PCP18.1 ± 2.13.0+10 μm Ca2+ + 10 mm caffeine22.4 ± 1.63.7+10 μm Ca2+, 1 m NaCl34.1 ± 1.45.70.2 mg of rabbit diaphragm SR was incubated with 8.5 nm[3H]ryanodine in the medium containing 0.3 mNaCl. 10 mm MOPSO/NaOH, pH 6.8, 1% CHAPS, 0.5% egg lecithin, 2 mm dithiothreitol for 4 h at 25 °C. Immunoprecipitation of RyR3 was carried out as described under “Experimental Procedures.” The data were the mean ± S.E. of four determinations. Open table in a new tab Table IIEffect of CICR inhibitors on [3H]ryanodine binding to RyR3Ligands added[3H]Ryanodine boundInhibitionfmol/mg protein% of controlNone (0.1 mmCa2+)17.1 ± 0.4100+1 μm ruthenium red4.5 ± 0.124+10 mm procaine3.2 ± 0.318+10 mmMg2+11.0 ± 0.665Assays were carried out as in Table I. The data were the mean ± S.E. of four determinations. Open table in a new tab 0.2 mg of rabbit diaphragm SR was incubated with 8.5 nm[3H]ryanodine in the medium containing 0.3 mNaCl. 10 mm MOPSO/NaOH, pH 6.8, 1% CHAPS, 0.5% egg lecithin, 2 mm dithiothreitol for 4 h at 25 °C. Immunoprecipitation of RyR3 was carried out as described under “Experimental Procedures.” The data were the mean ± S.E. of four determinations. Assays were carried out as in Table I. The data were the mean ± S.E. of four determinations. To identify RyR3 in skeletal muscles, we prepared terminal cisternae-rich fractions of SR vesicles from rabbit skeletal muscles where RyR3 is reported to be localized (22Conti A. Gorza L. Sorrentino V. Biochem. J. 1996; 316: 19-23Crossref PubMed Scopus (97) Google Scholar). Because the content of RyR3 varied among different muscles in rat (22Conti A. Gorza L. Sorrentino V. Biochem. J. 1996; 316: 19-23Crossref PubMed Scopus (97) Google Scholar), we used diaphragm (which was reported to express the highest content of RyR3 among skeletal muscles examined) and back muscle (material commonly used for SR vesicles) as materials. Fig.1 A shows a Coomassie Brilliant Blue-stained SDS-polyacrylamide gel of SR vesicles from diaphragm and back muscle. The two specimens showed very similar patterns of protein composition. Mammalian skeletal muscles express primarily RyR1, in contrast to skeletal muscles of non-mammalian vertebrates which have nearly equal amounts of two RyR isoforms (α- and β-RyR). Consistently, single bands for RyR1 of nearly equal density were clearly detected at the low mobility range of the gel (arrowhead) in both SR preparations, and the band for RyR3 could not be identified in diaphragm or back muscle SR on the Coomassie-stained gel. When the transferred membrane was probed with anti-RyR3, the antibody raised against a synthetic peptide (RyR3-peptide) corresponding to 4375–4387 of rabbit RyR3 which reacts highly specifically with mammalian RyR3 among the three isoforms (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), a single band for RyR3 was faintly but significantly reacted just below the RyR1 band in diaphragm SR, but not in back muscle SR (Fig. 1 B). Thus, a minute amount of RyR3 in addition to dominant RyR1 was expressed in rabbit diaphragm, whereas no RyR3 was detected in back muscle SR. The expression of RyR3 was also examined by immunoprecipitation using antibody-conjugated agarose beads (see “Experimental Procedures”). Fig. 2 shows Coomassie Brilliant Blue-stained gels of the proteins immunoprecipitated with the anti-RyR3 from CHAPS/egg lecithin-solubilized SR. In diaphragm SR, a single high molecular weight band (arrowhead) was observed, which specifically disappeared by the addition of 30 μmRyR3-peptide during immunoprecipitation (panel A). This band was reacted with anti-RyR3 on Western blot analysis (data not shown). These results indicate that RyR3 is definitely expressed in rabbit diaphragm. A band seen above RyR3 band was a minute contamination of RyR1 because of its positive reaction with anti-RyR1 antibody (data not shown). Because the band did not disappear by the RyR3-peptide, the precipitation of RyR1 may be the result of nonspecific binding to the agarose beads rather than weak binding to anti-RyR3. No specific bands, in contrast, were immunoprecipitated from back muscle SR (panel B), suggesting that there are no detectable amounts of RyR3 in back muscle, the same conclusion as shown in Fig. 1 B. The varied contents of RyR3 in contrast to similar amounts of RyR1 between diaphragm and back muscle corresponded well to the results with rat skeletal muscles (22Conti A. Gorza L. Sorrentino V. Biochem. J. 1996; 316: 19-23Crossref PubMed Scopus (97) Google Scholar). The following experiments to characterize RyR3 in skeletal muscles were therefore carried out with diaphragm SR vesicles. The molecular mass of rabbit RyR3 protein is estimated to be 552 kDa from its predicted amino acid sequence (10Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (350) Google Scholar), which is slightly smaller than that of rabbit RyR1 (565 kDa) (6Takeshima H. Nishimura S. Matsumoto T. Ishida H. Kangawa K. Minamino N. Matsuo H. Ueda M. Hanaoka M. Hirose T. Numa S. Nature. 1989; 339: 439-445Crossref PubMed Scopus (856) Google Scholar, 7Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar). As shown in Fig.3, the mobility of immunoprecipitated RyR3 was slightly but significantly larger than that of RyR1 on the Coomassie Brilliant Blue-stained SDS-polyacrylamide gel. The mobilities of RyR1 and RyR3 were similar to those of bullfrog α- and β-RyR, the homologs of RyR1 and RyR3, respectively, in non-mammalian vertebrates (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar, 19Oyamada H. Murayama T. Takagi T. Iino M. Iwabe N. Miyata T. Ogawa Y. Endo M. J. Biol. Chem. 1994; 269: 17206-17214Abstract Full Text PDF PubMed Google Scholar). Formation of tetramer is one of the typical characteristics of RyRs. As described above, rabbit diaphragm expresses considerable amounts of RyR1 and minor levels of RyR3 (see Fig. 1). It is therefore important to determine whether RyR3 forms a homotetramer of its subunit or heterotetramer with RyR1. The tetramer formation of RyR is detected easily by the sedimentation pattern of sucrose density gradients: a tetrameric RyR sediments to higher density fractions of the gradient because of its large sedimentation coefficient of about 30 S, whereas monomeric RyR subunits remain in lower density fractions as is true with the other proteins (26Lai F.A. Erickson H.P. Rousseau E. Liu Q.-Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (65) Google Scholar, 27Lai F.A. Misra M. Xu L. Smith H.A. Meissner G. J. Biol. Chem. 1989; 264: 16776-16785Abstract Full Text PDF PubMed Google Scholar). Fig.4 A shows the low mobility range of Coomassie Brilliant Blue-stained gels of total proteins (30 μl of each fraction) of fractions that were divided after centrifugation through 5–20% sucrose density gradient of solubilized rabbit diaphragm SR. The band for RyR1 was detected in higher density fractions (fractions 6 and 7) of the gradient. This pattern corresponds well with our previous results with α- and β-RyR of bullfrog skeletal muscle (18Murayama T. Ogawa Y. J. Biochem. 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar) and RyR2 and RyR3 of rabbit brain microsomes (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) under identical conditions, as is true of RyR of rabbit skeletal muscle SR (26Lai F.A. Erickson H.P. Rousseau E. Liu Q.-Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (65) Google Scholar, 27Lai F.A. Misra M. Xu L. Smith H.A. Meissner G. J. Biol. Chem. 1989; 264: 16776-16785Abstract Full Text PDF PubMed Google Scholar). The sedimentation pattern of RyR3 through the sucrose gradient was similarly examined on the Coomassie Brilliant Blue-stained gel after immunoprecipitation of a large volume of the gradient fractions (4 ml of each fraction) with anti-RyR3. As shown in Fig.4 B, the bands for RyR3 were detected in the identical fractions of nos. 6 and 7. It should be noted that only a single RyR3 band was detected in the immunoprecipitated products on each lane of the gel. If RyR3 forms a heterotetramer with RyR1, the two should be coprecipitated, resulting in the detection of dual bands on the Coomassie Brilliant Blue-stained gel because of their different mobilities (see Fig. 3). The absence of an RyR1 band clearly excludes the possibility of heterotetramer formation of RyR3 with RyR1. These results indicate that each of RyR1 and RyR3 coexpressed in rabbit diaphragm exclusively forms a homotetramer. Homotetramer formation of RyR3 is also observed in rabbit brain, which expresses a high content of RyR2 (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The functional properties of RyR3 in rabbit skeletal muscles were determined through the [3H]ryanodine binding. Solubilized SR was incubated with [3H]ryanodine to achieve ryanodine binding, and then RyR3 was specifically immunoprecipitated with anti-RyR3 as described in Murayama and Ogawa (20Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Fig. 5 shows the radioactivity immunoprecipitated with anti-RyR3 from SR of rabbit diaphragm or back muscle. In diaphragm SR, significant radioactivity was precipitated with the antibody. Thirty μmRyR3-peptide, which completely prevented RyR3 from being immunoprecipitated (see Fig. 2), reduced the radioactivity to the background level. A similar reduction in the radioactivity was also observed in determination without anti-RyR3 (data not shown). Therefore, the radioactivity is caused by RyR3 itself, but not by minute amounts of contaminating RyR1. The addition of excess concentrations (10–50 μm) of unlabeled ryanodine caused almost total loss of the radioactivity (data not shown). These results suggest that RyR3" @default.
• W2018485485 created "2016-06-24" @default.
• W2018485485 creator A5002974385 @default.
• W2018485485 creator A5048262927 @default.
• W2018485485 date "1997-09-01" @default.
• W2018485485 modified "2023-09-26" @default.
• W2018485485 title "Characterization of Type 3 Ryanodine Receptor (RyR3) of Sarcoplasmic Reticulum from Rabbit Skeletal Muscles" @default.
• W2018485485 cites W113046133 @default.
• W2018485485 cites W12635643 @default.
• W2018485485 cites W1499385605 @default.
• W2018485485 cites W1525891657 @default.
• W2018485485 cites W1529262908 @default.
• W2018485485 cites W1547730299 @default.
• W2018485485 cites W1566622078 @default.
• W2018485485 cites W1601185542 @default.
• W2018485485 cites W1641967096 @default.
• W2018485485 cites W1740533879 @default.
• W2018485485 cites W1809317608 @default.
• W2018485485 cites W1840022026 @default.
• W2018485485 cites W1852991130 @default.
• W2018485485 cites W1914131776 @default.
• W2018485485 cites W1938317606 @default.
• W2018485485 cites W1970311357 @default.
• W2018485485 cites W1972823724 @default.
• W2018485485 cites W1976257073 @default.
• W2018485485 cites W1984843607 @default.
• W2018485485 cites W1986154065 @default.
• W2018485485 cites W1986897813 @default.
• W2018485485 cites W1989256274 @default.
• W2018485485 cites W2011809437 @default.
• W2018485485 cites W2037744654 @default.
• W2018485485 cites W2039547987 @default.
• W2018485485 cites W2051392031 @default.
• W2018485485 cites W2055779520 @default.
• W2018485485 cites W2060055554 @default.
• W2018485485 cites W2060571726 @default.
• W2018485485 cites W2063081959 @default.
• W2018485485 cites W2067297047 @default.
• W2018485485 cites W2080901566 @default.
• W2018485485 cites W2085326980 @default.
• W2018485485 cites W2096827547 @default.
• W2018485485 cites W2100837269 @default.
• W2018485485 cites W2102972209 @default.
• W2018485485 cites W2113007237 @default.
• W2018485485 cites W2128113643 @default.
• W2018485485 cites W2138450406 @default.
• W2018485485 cites W2358927500 @default.
• W2018485485 cites W2418748206 @default.
• W2018485485 cites W252963246 @default.
• W2018485485 cites W3013605051 @default.
• W2018485485 cites W36028778 @default.
• W2018485485 doi "https://doi.org/10.1074/jbc.272.38.24030" @default.
• W2018485485 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9295356" @default.
• W2018485485 hasPublicationYear "1997" @default.
• W2018485485 type Work @default.
• W2018485485 sameAs 2018485485 @default.
• W2018485485 citedByCount "71" @default.
• W2018485485 countsByYear W20184854852013 @default.
• W2018485485 countsByYear W20184854852014 @default.
• W2018485485 countsByYear W20184854852018 @default.
• W2018485485 countsByYear W20184854852019 @default.
• W2018485485 countsByYear W20184854852020 @default.
• W2018485485 countsByYear W20184854852021 @default.
• W2018485485 crossrefType "journal-article" @default.
• W2018485485 hasAuthorship W2018485485A5002974385 @default.
• W2018485485 hasAuthorship W2018485485A5048262927 @default.
• W2018485485 hasConcept C105702510 @default.
• W2018485485 hasConcept C105795698 @default.
• W2018485485 hasConcept C113217602 @default.
• W2018485485 hasConcept C12554922 @default.
• W2018485485 hasConcept C126322002 @default.
• W2018485485 hasConcept C134018914 @default.
• W2018485485 hasConcept C158617107 @default.
• W2018485485 hasConcept C170493617 @default.
• W2018485485 hasConcept C185592680 @default.
• W2018485485 hasConcept C190712762 @default.
• W2018485485 hasConcept C2779884254 @default.
• W2018485485 hasConcept C2779959927 @default.
• W2018485485 hasConcept C33923547 @default.
• W2018485485 hasConcept C55493867 @default.
• W2018485485 hasConcept C71924100 @default.
• W2018485485 hasConcept C86803240 @default.
• W2018485485 hasConcept C95444343 @default.
• W2018485485 hasConceptScore W2018485485C105702510 @default.
• W2018485485 hasConceptScore W2018485485C105795698 @default.
• W2018485485 hasConceptScore W2018485485C113217602 @default.
• W2018485485 hasConceptScore W2018485485C12554922 @default.
• W2018485485 hasConceptScore W2018485485C126322002 @default.
• W2018485485 hasConceptScore W2018485485C134018914 @default.
• W2018485485 hasConceptScore W2018485485C158617107 @default.
• W2018485485 hasConceptScore W2018485485C170493617 @default.
• W2018485485 hasConceptScore W2018485485C185592680 @default.
• W2018485485 hasConceptScore W2018485485C190712762 @default.
• W2018485485 hasConceptScore W2018485485C2779884254 @default.
• W2018485485 hasConceptScore W2018485485C2779959927 @default.
• W2018485485 hasConceptScore W2018485485C33923547 @default.
• W2018485485 hasConceptScore W2018485485C55493867 @default.
• W2018485485 hasConceptScore W2018485485C71924100 @default.

• next