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• W2071234680 abstract "Previously we identified a protein of apparent Mr = 26,000 as the major calsequestrin binding protein in junctional sarcoplasmic reticulum vesicles isolated from cardiac and skeletal muscle (Mitchell, R. D., Simmerman, H. K. B., and Jones, L. R.(1988) J. Biol. Chem. 263, 1376-1381). Here we describe the purification and primary structure of the 26-kDa calsequestrin binding protein. The protein was purified 164-fold from cardiac microsomes and shown by immunoblotting to be highly enriched in junctional membrane subfractions. It ran as a closely spaced doublet on SDS-polyacrylamide gel electrophoresis and bound 125I-calsequestrin intensely. Cloning of the cDNA predicted a protein of 210 amino acids containing a single transmembrane domain. The protein has a short N-terminal region located in the cytoplasm, and the bulk of the molecule, which is highly charged and basic, projects into the sarcoplasmic reticulum lumen. Significant homologies were found with triadin and aspartyl β-hydroxylase, suggesting that all three proteins are members of a family of single membrane-spanning endoplasmic reticulum proteins. Immunocytochemical labeling localized the 26-kDa protein to junctional sarcoplasmic reticulum in cardiac and skeletal muscle. The same gene product was expressed in these two tissues. The calsequestrin binding activity of the 26-kDa protein combined with its codistribution with calsequestrin and ryanodine receptors strongly suggests that the protein plays an important role in the organization and/or function of the Ca2+ release complex. Because the 26-kDa calsequestrin binding protein is an integral component of the junctional sarcoplasmic reticulum membrane in cardiac and skeletal muscle, we have named it Junctin. Previously we identified a protein of apparent Mr = 26,000 as the major calsequestrin binding protein in junctional sarcoplasmic reticulum vesicles isolated from cardiac and skeletal muscle (Mitchell, R. D., Simmerman, H. K. B., and Jones, L. R.(1988) J. Biol. Chem. 263, 1376-1381). Here we describe the purification and primary structure of the 26-kDa calsequestrin binding protein. The protein was purified 164-fold from cardiac microsomes and shown by immunoblotting to be highly enriched in junctional membrane subfractions. It ran as a closely spaced doublet on SDS-polyacrylamide gel electrophoresis and bound 125I-calsequestrin intensely. Cloning of the cDNA predicted a protein of 210 amino acids containing a single transmembrane domain. The protein has a short N-terminal region located in the cytoplasm, and the bulk of the molecule, which is highly charged and basic, projects into the sarcoplasmic reticulum lumen. Significant homologies were found with triadin and aspartyl β-hydroxylase, suggesting that all three proteins are members of a family of single membrane-spanning endoplasmic reticulum proteins. Immunocytochemical labeling localized the 26-kDa protein to junctional sarcoplasmic reticulum in cardiac and skeletal muscle. The same gene product was expressed in these two tissues. The calsequestrin binding activity of the 26-kDa protein combined with its codistribution with calsequestrin and ryanodine receptors strongly suggests that the protein plays an important role in the organization and/or function of the Ca2+ release complex. Because the 26-kDa calsequestrin binding protein is an integral component of the junctional sarcoplasmic reticulum membrane in cardiac and skeletal muscle, we have named it Junctin. INTRODUCTIONThe technique of membrane subcellular fractionation has proven to be invaluable for biochemical dissection of the molecular components of the SR 1The abbreviations used are: SRsarcoplasmic reticulumbpbase pair(s)MOPS3-(N-morpholino)propanesulfonic acidCSQcalsequestrinERendoplasmic reticulumPAGEpolyacrylamide gel electrophoresis. responsible for Ca2+ uptake and Ca2+ release in striated muscle(1.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 2.Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (836) Google Scholar). Utilizing this approach it is possible to isolate biochemically subspecialized regions of SR in the form of sealed membrane vesicles from both cardiac (3.Jones L.R. Cala S.E. J. Biol. Chem. 1981; 256: 11809-11818Abstract Full Text PDF PubMed Google Scholar) and skeletal muscle(4.Meissner G. Biochim. Biophys. Acta. 1975; 389: 51-68Crossref PubMed Scopus (290) Google Scholar). Free SR vesicles originate from the region of the SR surrounding the myofibrils. Ca2+ is actively transported here into the SR lumen(5.Jorgensen A.O. Shen A.C.-Y. Daly P. MacLennan D.H. J. Cell Biol. 1982; 93: 883-892Crossref PubMed Scopus (42) Google Scholar, 6.Sommer J.R. Jennings R.B. Fozzard H.A. Jennings R.B. Haber E. Katz A.M. Morgan H.E. The Heart and Cardiovascular System. 2nd Ed. Raven Press, Ltd., New York1992: 3-50Google Scholar). Junctional SR vesicles arise from the regions of the SR making contact with the sarcolemma where Ca2+ is released to initiate muscle contraction (1.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 2.Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (836) Google Scholar, 6.Sommer J.R. Jennings R.B. Fozzard H.A. Jennings R.B. Haber E. Katz A.M. Morgan H.E. The Heart and Cardiovascular System. 2nd Ed. Raven Press, Ltd., New York1992: 3-50Google Scholar, 7.Franzini-Armstrong C. Jorgensen A.O. Annu. Rev. Physiol. 1994; 56: 509-534Crossref PubMed Scopus (342) Google Scholar). A few of the junctional SR proteins participating in Ca2+ release have been purified and cloned(1.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 2.Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (836) Google Scholar). Ryanodine receptors, large tetrameric proteins of subunit molecular weight approximately one-half million, are the Ca2+ release channels. Ryanodine receptors constitute the SR feet, structures that join the junctional SR membrane to transverse tubules and regions of surface sarcolemma in intact muscle. CSQ, a peripheral membrane protein, is a high capacity Ca2+ binding protein located in the lumen of the junctional SR(1.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 2.Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (836) Google Scholar). CSQ is visible as an electron-dense matrix, which appears to be associated with the inner surface of the junctional SR membrane(1.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 6.Sommer J.R. Jennings R.B. Fozzard H.A. Jennings R.B. Haber E. Katz A.M. Morgan H.E. The Heart and Cardiovascular System. 2nd Ed. Raven Press, Ltd., New York1992: 3-50Google Scholar). CSQ acts as a Ca2+ reservoir, providing a ready source for Ca2+ when ryanodine receptors open and Ca2+ is released into the cytoplasm. Although both ryanodine receptors (9.Otsu 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, 10.Takeshima 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 (857) Google Scholar) and CSQ(11.Scott B.T. Simmerman H.K.B. Collins J.H. Nadal-Ginard B. Jones L.R. J. Biol. Chem. 1988; 263: 8958-8964Abstract Full Text PDF PubMed Google Scholar, 12.Fliegel L. Ohnishi M. Carpenter M.R. Khanna V.K. Reithmeier R.A.F. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1167-1171Crossref PubMed Scopus (187) Google Scholar) are the products of separate genes in cardiac and skeletal muscle, the molecular architecture at the junctional region in each tissue is similar(1.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 6.Sommer J.R. Jennings R.B. Fozzard H.A. Jennings R.B. Haber E. Katz A.M. Morgan H.E. The Heart and Cardiovascular System. 2nd Ed. Raven Press, Ltd., New York1992: 3-50Google Scholar). The close proximity of CSQ to ryanodine receptors in striated muscle suggests that specific protein interactions are required for stabilization and efficient operation of the Ca2+ release process. The membrane surface where these protein interactions occur is called the junctional face membrane (8.Costello B. Chadwick C. Saito A. Chu A. Maurer A. Fleischer S. J. Cell Biol. 1986; 103: 741-753Crossref PubMed Scopus (82) Google Scholar).Identification of all of the molecular components of the junctional face membrane important for Ca2+ release is an area of intense study(1.Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (442) Google Scholar, 2.Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (836) Google Scholar, 7.Franzini-Armstrong C. Jorgensen A.O. Annu. Rev. Physiol. 1994; 56: 509-534Crossref PubMed Scopus (342) Google Scholar). “Rope-like fibers” (8.Costello B. Chadwick C. Saito A. Chu A. Maurer A. Fleischer S. J. Cell Biol. 1986; 103: 741-753Crossref PubMed Scopus (82) Google Scholar) or “joining strands” (13.Franzini-Armstrong C. Kenney L.J. Varriano-Marston E. J. Cell Biol. 1987; 105: 49-56Crossref PubMed Scopus (167) Google Scholar) have been visualized by electron microscopy, which appear to anchor CSQ to the junctional membrane at sites apposed to ryanodine receptors. Recently, it has been proposed that triadin, a 95-kDa junctional SR glycoprotein(14.Caswell A.H. Brandt N.R. Brunschwig J.-P. Purkerson S. Biochemistry. 1991; 30: 7507-7513Crossref PubMed Scopus (134) Google Scholar, 15.Brandt N.R. Caswell A.H. Lewis Carl S.A. Ferguson D.A. Brandt T. Brunschwig J.P. Basset A.L. J. Membr. Biol. 1993; 131: 219-228Crossref PubMed Scopus (20) Google Scholar, 16.Knudson C.M. Stang K.K. Moomaw C.R. Slaughter C.A. Campbell K.P. J. Biol. Chem. 1993; 268: 12646-12654Abstract Full Text PDF PubMed Google Scholar, 17.Peng M. Fan H. Kirley T.L. Caswell A.H. Schwartz A. FEBS Lett. 1994; 348: 17-20Crossref PubMed Scopus (24) Google Scholar), may be a component of these anchoring strands by virtue of its ability to bind both CSQ and ryanodine receptors(18.Guo W. Campbell K.P. J. Biol. Chem. 1995; 270: 9027-9030Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In an earlier study, we described a different CSQ binding protein localized to both canine cardiac and rabbit fast skeletal muscle junctional SR vesicles(19.Mitchell R.D. Simmerman H.K.B. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar). This protein was detected by binding 125I-labeled CSQ to junctional SR proteins transferred to nitrocellulose. It was the major CSQ binding protein identified and migrated as a closely spaced doublet of apparent molecular weight 26,000. Subsequently, using the same approach, Damiani and Margreth (20.Damiani E. Margreth A. Biochem. Biophys. Res. Commun. 1990; 172: 1253-1259Crossref PubMed Scopus (24) Google Scholar) confirmed the presence of the 26-kDa CSQ binding protein in junctional face membranes prepared from rabbit fast skeletal muscle. A similar 26-kDa protein doublet had been described earlier as a major constituent of this membrane(8.Costello B. Chadwick C. Saito A. Chu A. Maurer A. Fleischer S. J. Cell Biol. 1986; 103: 741-753Crossref PubMed Scopus (82) Google Scholar). A protein doublet of approximately the same molecular weight, which appeared to interact functionally with CSQ and regulate the amount of Ca2+ released from junctional SR vesicles by caffeine, was also recently reported by Ikemoto et al.(21.Ikemoto N. Ronjat M. Meszaros L.G. Koshita M. Biochemistry. 1989; 28: 6764-6771Crossref PubMed Scopus (187) Google Scholar).Here we have purified the 26-kDa CSQ binding protein and deduced its complete primary structure by cDNA cloning. We show that the same protein is expressed in heart and skeletal muscle and with use of specific antibodies localize the protein to the junctional SR in these two tissues. This newly characterized protein of the junctional face membrane shares many similarities with triadin and is likely to play an important role in Ca2+ release in striated muscle. A preliminary report on purification of the 26-kDa CSQ binding protein appeared in abstract form(23.Jones L.R. Kelley J.S. Sanborn K.L. Biophys. J. 1994; 66 (abstr.): 315Google Scholar).EXPERIMENTAL PROCEDURESIsolation of Membrane SubfractionsProcedure I microsomes were isolated from canine left ventricle(22.Jones L.R. Besch Jr., H.R. Fleming J.W. McConnaughey M.M. Watanabe A.M. J. Biol. Chem. 1979; 254: 530-539Abstract Full Text PDF PubMed Google Scholar). Subfractionation of these microsomes was by Ca2+ oxalate loading followed by sucrose density gradient centrifugation(3.Jones L.R. Cala S.E. J. Biol. Chem. 1981; 256: 11809-11818Abstract Full Text PDF PubMed Google Scholar, 22.Jones L.R. Besch Jr., H.R. Fleming J.W. McConnaughey M.M. Watanabe A.M. J. Biol. Chem. 1979; 254: 530-539Abstract Full Text PDF PubMed Google Scholar). Subfractions A, B, C, and D were collected at the 0.6, 0.8, 1.0, and 1.5 M sucrose interfaces, respectively. Subfraction E was obtained as the pellet below 1.5 M sucrose. Subfractions A, D, and E are enriched in sarcolemmal vesicles, and junctional and free SR vesicles, respectively(3.Jones L.R. Cala S.E. J. Biol. Chem. 1981; 256: 11809-11818Abstract Full Text PDF PubMed Google Scholar, 22.Jones L.R. Besch Jr., H.R. Fleming J.W. McConnaughey M.M. Watanabe A.M. J. Biol. Chem. 1979; 254: 530-539Abstract Full Text PDF PubMed Google Scholar, 24.Seiler S. Wegener A.D. Whang D.D. Hathaway D.R. Jones L.R. J. Biol. Chem. 1984; 259: 8550-8557Abstract Full Text PDF PubMed Google Scholar). Further purification of junctional SR vesicles (subfraction D membranes) was achieved by a second round of Ca2+ oxalate loading, this time in the presence of 300 μM ryanodine, to effect a ryanodine-mediated density shift(25.Cala S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Abstract Full Text PDF PubMed Google Scholar). Following Ca2+ oxalate loading of subfraction D microsomes in the presence of ryanodine, the membranes were centrifuged through a 1.3 M sucrose cushion. P, T, and B designate the starting parent (P) membranes (or subfraction D microsomes), the membranes collected on top of the 1.3 M sucrose cushion (T), and the membranes pelleted at the bottom (B) of the sucrose layer. As a control, subfraction D microsomes were also loaded in the absence of ryanodine. Procedure I microsomes were also prepared from canine fast twitch (tensor fasciae latae) and slow twitch (vastus intermedius) skeletal muscle. Canine liver microsomes were obtained as the membranes sedimenting between 10,000 and 100,000 × gmax(25.Cala S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Abstract Full Text PDF PubMed Google Scholar). All membranes were stored frozen at −40°C in small aliquots in 0.25 M sucrose, 30 mM histidine (pH 7.2).Solubilization of the 26-kDa CSQ Binding Protein32 μl (166 μg of protein) of subfraction D membranes in 0.25 M sucrose, 30 mM histidine (pH 7.2) were added to a Beckman TL-100 centrifuge tube, followed by addition of 8 μl of 10% Triton X-100. 13 μl of sample was saved for analysis, and the remainder was centrifuged at 100,000 rpm for 5 min. The supernatant (S1) was collected, and the pellet (P1) was resuspended in 27 μl of 20 mM MOPS, 0.5 M NaCl, and 2% Triton X-100. 13 μl was saved, and the remainder was spun at 100,000 rpm for 10 min to yield supernatant (S2) and pellet (P2) fractions. P2 was resuspended in 13 μl of 0.25 M sucrose. 13 μl from all fractions were analyzed for 125I-CSQ binding as described below.Purification of the 26-kDa CSQ Binding ProteinFor a typical purification, 214 mg of procedure I canine cardiac microsomal protein at a concentration of 7.74 mg/ml was solubilized at room temperature for 10 min in 0.22 M sucrose, 26 mM histidine, 0.5 M NaCl, and 2% Triton X-100. The sample was sedimented at 45,000 rpm for 30 min in a Beckman Ti 70 rotor. The supernatant was diluted with an equal volume (27.7 ml) of 20 mM MOPS, 0.1% Triton X-100 (pH 7.2) (Buffer A) and loaded over a 20-ml phosphocellulose column preequilibrated with Buffer A. The flow-through fraction was collected, and the column was eluted with 20-ml washes of Buffer A containing 0.2-0.9 M NaCl applied in 0.1 M concentration steps. Aliquots of the column fractions were electrophoresed and transferred to nitrocellulose to localize 125I-CSQ binding proteins, as described below. The 0.5-0.8 M NaCl wash fractions containing the partially purified 26-kDa CSQ binding protein were pooled and concentrated, and the sample was subjected to preparative SDS-PAGE using a Bio-Rad 491 Prep Cell(25.Cala S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Abstract Full Text PDF PubMed Google Scholar). The PAGE fractions containing the purified 26-kDa protein were identified by 125I-CSQ binding assay and combined. In the typical preparation described 150 μg of purified 26-kDa CSQ binding protein was isolated. Protein concentration was determined by the Amido Black method(27.Schaffner W. Weissman C. Anal. Biochem. 1973; 56: 502-514Crossref PubMed Scopus (1946) Google Scholar).SDS-PAGE and 125I-CSQ BindingSDS-PAGE was conducted with a 7% polyacrylamide gel according to Porzio and Pearson (28.Porzio M.A. Pearson A.M. Biochim. Biophys. Acta. 1977; 490: 27-34Crossref PubMed Scopus (507) Google Scholar) (Fig. 1 only) or by the method of Laemmli (26.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar) with 9 or 10% separating gels and a 3% stacking gel. Gels were stained with Coomassie Blue or, for detection of 125I-CSQ binding proteins, proteins were transferred to nitrocellulose(19.Mitchell R.D. Simmerman H.K.B. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar). Nitrocellulose sheets were stained with Amido Black (29.Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5868) Google Scholar) to localize proteins and then blocked two times for 30 min in 20 mM MOPS, 150 mM KCl (pH 7.2) (Buffer B) containing 1% horse hemoglobin. Blots were washed three times for 5 min in 30 ml of Buffer B containing 1 mM EGTA, and then 5-10 μg of 125I-CSQ in 50 μl (approximately 8 × 106 cpm) were added, and the blot was incubated for 90 min at room temperature. After incubation with radioactive CSQ, blots were washed three times for 15 min in Buffer B containing 1 mM EGTA, and then autoradiography was performed. Canine cardiac CSQ was prepared by phenyl-Sepharose chromatography (30.Cala S.E. Jones L.R. J. Biol. Chem. 1983; 258: 11932-11936Abstract Full Text PDF PubMed Google Scholar) and iodinated using Enzymobeads(19.Mitchell R.D. Simmerman H.K.B. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar).Protein and Peptide Sequencing50-100 μg of purified 26-kDa CSQ binding protein was precipitated (31.Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3128) Google Scholar) and resuspended in 60% n-propyl alcohol, 0.1% trifluoroacetic acid for direct sequence analysis or in 200 μl of 20 mM Tris, 0.1% hydrogenated Triton X-100 (pH 8.5) (32.Fernandez J. DeMott M. Atherton D. Mische S.M. Anal. Biochem. 1992; 201: 255-264Crossref PubMed Scopus (247) Google Scholar) for digestion with proteases. Trypsin, endoproteinase Lys-C, or endoproteinase Asp-N was added at 1/20 weight ratios, and digestions were conducted overnight at 37°C. Peptides were separated by C18 reverse-phase chromatography using a Pharmacia Biotech Inc. PepRPC column and sequenced by Edman degradation with an Applied Biosystems model 477A automated sequencer(25.Cala S.E. Ulbright C. Kelley J.S. Jones L.R. J. Biol. Chem. 1993; 268: 2969-2975Abstract Full Text PDF PubMed Google Scholar).Antibody Production and ImmunoblottingAntibodies were produced to the purified 26-kDa CSQ binding protein and to synthetic peptides made to residues 77-91 (Peptide 1) (EGPGGVAKRKTKAKV) and to residues 94-108 (Peptide 2) (LTKEELKKEKEKTES). Peptides were synthesized with N-terminal cysteine residues and coupled to thyroglobulin and bovine serum albumin(33.Green N. Alexander H. Olson A. Alexander S. Shinnick T.M. Sutcliffe J.G. Lerner R.A. Cell. 1982; 28: 477-487Abstract Full Text PDF PubMed Scopus (511) Google Scholar). Affinity purification of antibodies was by the method of Olmsted(34.Olmsted J.B. J. Biol. Chem. 1981; 256: 11955-11957Abstract Full Text PDF PubMed Google Scholar). Immunoblotting was done as described previously(35.Jones L.R. Simmerman H.K.B. Wilson W.W. Gurd F.R.N. Wegener A.D. J. Biol. Chem. 1985; 260: 7721-7730Abstract Full Text PDF PubMed Google Scholar, 36.Xiao R.-P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar). Monoclonal antibody 2A7-A1 was produced to the Ca2+-ATPase in canine cardiac SR vesicles(37.Moversusesian M.A. Karimi M. Green K. Jones L.R. Circulation. 1994; 90: 653-657Crossref PubMed Scopus (212) Google Scholar).Immunofluorescence LabelingImmunofluorescence labeling with affinity-purified rabbit antibodies to the intact 26-kDa CSQ binding protein was performed as described previously(38.Jorgensen A.O. Jones L.R. J. Cell Biol. 1987; 104: 1343-1352Crossref PubMed Scopus (32) Google Scholar, 39.Jorgensen A.O. Kalnins V. MacLennan D.H. J. Cell Biol. 1979; 80: 372-384Crossref PubMed Scopus (78) Google Scholar). To localize the fast (type II) isoform of the SR Ca2+-ATPase, mouse monoclonal antibody IIH11 was used(40.Jorgensen A.O. Arnold W. Pepper D.R. Kahl S.D. Campbell K.P. Cell Motil. Cytoskel. 1988; 9: 164-174Crossref PubMed Scopus (76) Google Scholar). Confocal microscopy was carried out as recently described. 2Guo, W., Jorgensen, A. O., Jones, L. R., and Campbell, K. P.(1996) J. Biol. Chem., in press. Trypsin Digestion of Junctional SR Vesicles10 μg of procedure I microsomes from dog heart were incubated at room temperature for 30 min in 50 mM MOPS (pH 7.0), 3 mM MgCl2, 100 mM KCl, 0.1 mM CaCl2, and 0.5 μg of trypsin in the presence and absence of 0.2% Triton X-100. Control samples were incubated without trypsin and Triton X-100. Reactions were quenched by boiling in Laemmli solubilization buffer containing 10% SDS. Samples were analyzed by immunoblotting.cDNA Cloning and SequencingA canine cardiac λgt11 expression library was screened with affinity-purified antisera against the 26-kDa CSQ binding protein and peptide 1 as described previously (11.Scott B.T. Simmerman H.K.B. Collins J.H. Nadal-Ginard B. Jones L.R. J. Biol. Chem. 1988; 263: 8958-8964Abstract Full Text PDF PubMed Google Scholar). Positive phage clones were plaque-purified, and DNA was prepared by the plate lysate method(41.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). EcoRI inserts were subcloned into pBluescript. Clones from a canine skeletal muscle λZAPII cDNA library (Stratagene) were identified with affinity-purified antibodies to the intact protein. A canine cardiac λgt10 cDNA library (42.Palmer C.J. Scott B.T. Jones L.R. J. Biol. Chem. 1991; 266: 11126-11130Abstract Full Text PDF PubMed Google Scholar) was screened with a 32P-labeled synthetic oligonucleotide probe corresponding to the 5′-end of clone 40 (bp −80 to −51 (see Fig. 8)). Double-stranded sequencing of all clones was performed by the dideoxy method(43.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52363) Google Scholar), using universal plasmid primers and a series of internal synthetic primers(41.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Sequence analyses and data base searches were conducted with software from MacVector.Fig. 8Nucleotide and deduced amino acid sequence of the 26-kDa CSQ binding protein. The start (ATG) and stop (TGA) codons and the first methionine residue (M) are in boldface. Polyadenylation signals are boldface and underlined. Underlined amino acids were confirmed by N-terminal sequencing the intact protein or proteolytic peptides purified by reverse-phase chromatography. Dots denote four amino acid residues not confirmed. The double underline marks the predicted transmembrane domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Northern Blot Analysis30 μg of total RNA prepared from canine heart and skeletal muscle were electrophoresed in a 0.8% agarose gel containing 2.2 M formaldehyde and transferred to a GeneScreen membrane. Hybridization was with a 32P-labeled probe made to the EcoRI insert in clone 8 (see Fig. 7). Autoradiography was performed at −80°C for 3 days with an intensifying screen.Fig. 7Restriction map of 26-kDa CSQ binding protein cDNA. Solid black bars denote open reading frames with start (ATG) and stop (TGA) codons indicated for full-length Clone 2. The striped boxes and A(n) indicate polyadenylation signals and tails, respectively. All clones were isolated from canine libraries, either cardiac or skeletal muscle, as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)[3H]Ryanodine Binding[3H]Ryanodine binding to 40 μg of membrane subfractions was performed by filtration assay in 0.6 M NaCl, 1 mM CaCl2, 20 mM MOPS, and 20 nM radioactive ligand (pH 7.1)(44.Rardon D.P. Cefali D.C. Mitchell R.D. Seiler S.M. Jones L.R. Circ. Res. 1989; 64: 779-789Crossref PubMed Scopus (51) Google Scholar). Nonspecific binding was determined in the same medium containing 10 μM non-radioactive ryanodine.RESULTSSolubilization and Purification of the 26-kDa CSQ Binding Protein from Canine Cardiac SRConsistent with our earlier study(19.Mitchell R.D. Simmerman H.K.B. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar), a major radioactive protein band of apparent Mr = 26,000 was observed when canine cardiac junctional SR vesicles were transferred to nitrocellulose and incubated with 125I-CSQ in Ca2+-free medium (Fig. 1, arrow). Several other minor 125I-labeled bands were also detected. The two radioactive bands running just below the 45-kDa protein standard (arrowheads) are cardiac isoforms of triadin.2 (These two 125I-CSQ binding proteins were designated bands 4 and 5 in our earlier study (Fig. 11 of (19.Mitchell R.D. Simmerman H.K.B. Jones L.R. J. Biol. Chem. 1988; 263: 1376-1381Abstract Full Text PDF PubMed Google Scholar)).) Another minor labeled band was detected migrating just above the 31-kDa protein standard (asterisk). This minor band may represent a protein isoform of the 26-kDa CSQ binding protein but is not considered further in this work. 2% Triton X-100 was insufficient to solubilize a significant amount of the 26-kDa CSQ binding protein from junctional SR vesicles (Fig. 1, S1). With addition of 0.5 M NaCl, however, most of the protein became soluble (Fig. 1, S2). The requirement of a relatively high concentration of detergent plus a high ionic strength medium for efficient solubilization of the 26-kDa CSQ binding protein suggested that it was an integral membrane protein. Consistent with this, 100 mM sodium carbonate at pH 11.4 also failed to solubilize the protein.Fig. 11Sequence alignment of aspartyl β-hydroxylase (OHase), Junctin (Junct), and triadin (Triad). Identical residues in all three proteins are shaded. Different residues are marked by vertical ticks. Dashes in the Junctin sequence denote a gap for proper alignment with OHase. Residue numbers for the three proteins are given at the left and right margins. For triadin, only residues spanning the region of homology are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For purification of the 26-kDa CSQ binding protein, we used procedure I cardiac membrane vesicles as the starting material, which is a heterogeneous microsomal preparation consisting mostly of sarcolemmal and free and junctional SR vesicles(3.Jones L.R. Cala S.E. J. Biol. Chem. 1981; 256: 11809-11818Abstract Full Text PDF PubMed Google Scholar, 22.Jones L.R. Besch Jr., H.R. Fleming J.W. McConnaughey M.M. Watanabe A.M. J. Biol. Chem. 1979; 254: 530-539Abstract Full Text PDF PubMed Google Scholar, 24.Seiler S. Wegener A.D. Whang D.D. Hathaway D.R. Jones L.R. J. Biol. Chem. 1984; 259: 8550-8557Abstract Full Text PDF PubMed Google Scholar). The detergent extract from these membranes was loaded over a phosphocellulose column (Fig. 2). All of the 26-kDa CSQ binding protein was retained by the column and was subsequently eluted in the 0.5-0.7 M NaCl wash fractions. A closely spaced doublet corresponding to the 26-kDa CSQ binding protein was visible in these fractions by Coomassie Blue staining (Fig. 2A) and also by 125I-CSQ binding (Fig. 2B). (Both bands of the 26-kDa doublet reacted equally with labeled CSQ but are" @default.
• W2071234680 created "2016-06-24" @default.
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• W2071234680 title "Purification, Primary Structure, and Immunological Characterization of the 26-kDa Calsequestrin Binding Protein (Junctin) from Cardiac Junctional Sarcoplasmic Reticulum" @default.
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