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• W1986247804 abstract "Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na+-Ca2+exchanger. In the presence of 1 mm Ca2+, the major product was a peptide of ∼37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, ∼16- and ∼19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105–110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca2+. Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [γ-32P]ATP led to incorporation of 32P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca2+ regulatory domain of a scallop muscle Na+-Ca2+ exchanger that mediates direct modulation of secondary Ca2+ regulation by cAMP. Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na+-Ca2+exchanger. In the presence of 1 mm Ca2+, the major product was a peptide of ∼37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, ∼16- and ∼19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105–110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca2+. Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [γ-32P]ATP led to incorporation of 32P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca2+ regulatory domain of a scallop muscle Na+-Ca2+ exchanger that mediates direct modulation of secondary Ca2+ regulation by cAMP. Sarco(endo)plasmic veticulum calcium ATPase sarcoplasmic reticulum nonaethylene glycol dodecyl ether deoxycholate protein kinase A (cAMP-dependent kinase) sarcolemma 3-(N-morpholino)propanesulfonic acid polyacrylamide gel electrophoresis 3-(cyclohexylamino)propanesulfonic acid N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine phosphate-buffered saline The Na+-Ca2+ exchangers of the plasma membrane catalyze a secondary active transport process dependent on the Na+ electrochemical gradient generated by the Na+,K+-ATPase and play a major role in cellular Ca2+ homeostasis in many tissues (1Blaustein M. Goldman W.F. Fontana G. Kreuger B.K. Santiago E.M. Steele T.D. Weiss D.N. Yarowsky P.J. Ann. N. Y. Acad. Sci. 1991; 639: 254-274Crossref PubMed Scopus (109) Google Scholar). Three Na+-Ca2+ exchanger proteins (NCX1, NCX2, and NCX3) have been described in vertebrates (2Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-564Crossref PubMed Scopus (627) Google Scholar, 3Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar, 4Nicoll D.A. Quednau B.D. Qui Z. Xia Y.-R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 5Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar). The molecular biology of the Na+-Ca2+ exchanger (Calx) from Drosophila has been described (6Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (180) Google Scholar, 7Ruknudin A. Valdiva C. Kofuji P. Lederer W.J. Schulze D.H. Am. J. Physiol. 1997; 273: C257-C265Crossref PubMed Google Scholar), and an exchanger from squid has been reported (8He Z. Tong Q. Quednau B. Philipson K.D. Hilgemann D.W. J. Gen. Physiol. 1998; 111: 857-873Crossref PubMed Scopus (45) Google Scholar). An electroneutral Na+-Ca2+ antiporter has also been found in mitochondria, where it may be involved in modulating matrix Ca2+ in response to changes in cytoplasmic Ca2+concentration (9Cox D.A. Matlib M.A. Trends Pharmacol. Sci. 1993; 14: 408-413Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 10Li W. Shariat-Madar Z. Powers M. Sun X. Lane R.D. Garlid K.D. J. Biol. Chem. 1992; 267: 17983-17989Abstract Full Text PDF PubMed Google Scholar).The protein moieties of the plasma membrane Na+-Ca2+ exchanger proteins are close in overall size (∼103–106 kDa) (4Nicoll D.A. Quednau B.D. Qui Z. Xia Y.-R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 11Philipson K.D. Longoni S. Ward R. Biochim. Biophys. Acta. 1988; 945: 298-306Crossref PubMed Scopus (159) Google Scholar) to the SERCA1-type Ca2+-ATPase pumps, which have a molecular mass of ∼110 kDa (12Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (655) Google Scholar). In the case of NCX1, a signal sequence of 32 amino acid residues at the N terminus of the protein is removed post-translationally (13Durkin J.T. Ahrens D.C. Pan Y.-C. Reeves J.P. Arch. Biochem. Biophys. 1991; 290: 369-375Crossref PubMed Scopus (64) Google Scholar, 14Hryshko L. Nicoll D.A Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google Scholar, 15Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar). From the N terminus, structure prediction algorithms suggest that five transmembrane segments lead to a large cytoplasmic, followed by four C-terminal transmembrane helices (14Hryshko L. Nicoll D.A Weiss J.N. Philipson K.D. Biochim. Biophys. Acta. 1993; 1151: 35-42Crossref PubMed Scopus (62) Google Scholar, 15Cook O. Low W. Rahamimoff H. Biochim. Biophys. Acta. 1998; 1371: 40-52Crossref PubMed Scopus (34) Google Scholar, 16Nicoll D.A. Ottolia L. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Two internal stretches of the f loop, Phe407(375)–Asp478(443) and Thr538(506)–Tyr613(581), 2Residue positions are given first for the coded protein sequence and then in parentheses for the processed polypeptide. 2Residue positions are given first for the coded protein sequence and then in parentheses for the processed polypeptide.show similarity to one another, and are termed the β-1 and β-2 repeats (6Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (180) Google Scholar). A high affinity Ca2+ binding region (Kd, 0.1–3 μm) is present in the large cytoplasmic domain (17Matsuoka S. Nicoll D.A. Reilly R.F. Hilgemann D.W. Philipson K.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3870-3874Crossref PubMed Scopus (200) Google Scholar, 18Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar, 19Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 404-420Crossref Scopus (204) Google Scholar, 20Philipson K.D. Nicoll D.A. Matsuoka S. Hryshko L.V. Levitsky D.O. Weiss J.N. Ann. N. Y. Acad. Sci. 1996; 779: 20-28Crossref PubMed Scopus (40) Google Scholar). This binds Ca2+cooperatively and provides regulation of the exchanger through the I2 mechanism, in which increased levels of cytoplasmic Ca2+ activate the enzyme (19Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 404-420Crossref Scopus (204) Google Scholar). The regulatory Ca2+ binding region involves the β-1 repeat and extends through the variable region between the two β repeats to the beginning of β-2 (18Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar). Two acidic triads, one at the C terminus of β-1 (Asp478(446)-Asp-Asp) and one in the variable region, (Asp530(498)-Asp-Asp), just N-terminal to β-2, are important components of this high affinity Ca2+ binding site (18Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar).The isolated cardiac exchanger shows three bands on silver-stained SDS gels: a glycosylated 120-kDa species corresponding to the native exchanger, a glycosylated 160-kDa polypeptide representing oxidized exchanger, and an unglycosylated polypeptide of 70 kDa, which arises by proteolysis of the 120-kDa protein at the Asp289(257)–Gly or Asp303(270)–Gly bonds in the large cytoplasmic domain (11Philipson K.D. Longoni S. Ward R. Biochim. Biophys. Acta. 1988; 945: 298-306Crossref PubMed Scopus (159) Google Scholar, 21Durkin J.T. Ahrens D.C. Aceto J.F. Condrescu M. Reeves J.P. Ann. N. Y. Acad. Sci. 1991; 639: 189-201Crossref PubMed Scopus (6) Google Scholar, 22Santacruz-Toloza L. Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2000; 275: 182-188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 23Iwata T. Galli C. Carafoli E. Cell Calcium. 1995; 17: 263-269Crossref PubMed Scopus (22) Google Scholar). Digestion of the exchanger with chymotrypsin leads to a loss of regulatory function, probably through proteolysis localized in the cytoplasmic f loop (4Nicoll D.A. Quednau B.D. Qui Z. Xia Y.-R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar).The study of the protein chemistry of the Na+-Ca2+ exchanger has been hampered by its low abundance; even in cardiac muscle, the NCX1 exchanger represents only 0.1–0.2% (w/w) of the sarcolemmal membrane protein (24Cheon J. Reeves J.P. J. Biol. Chem. 1988; 263: 2309-2315Abstract Full Text PDF PubMed Google Scholar). The mitochondrial exchanger is present at 0.4 μg/mg total protein (10Li W. Shariat-Madar Z. Powers M. Sun X. Lane R.D. Garlid K.D. J. Biol. Chem. 1992; 267: 17983-17989Abstract Full Text PDF PubMed Google Scholar). Thus, silver staining has often been necessary to detect the protein on SDS gels (21Durkin J.T. Ahrens D.C. Aceto J.F. Condrescu M. Reeves J.P. Ann. N. Y. Acad. Sci. 1991; 639: 189-201Crossref PubMed Scopus (6) Google Scholar).In the past, a number of studies of the Ca-ATPase from the cross-striated adductor muscle of the deep sea scallop have used a deoxycholate-extracted membrane fraction enriched in fragmented SR (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar, 26Kalabokis V.N. Bozzola J.J. Castellani L. Hardwicke P.M.D. J. Biol. Chem. 1991; 266: 22044-22050Abstract Full Text PDF PubMed Google Scholar, 27Hardwicke P.M.D. Huvos P. J. Musc. Res. Cell Motil. 1989; 10: 229-244Crossref PubMed Scopus (6) Google Scholar, 28Hardwicke P.M.D. Bozzola J.J. J. Musc. Res. Cell Motil. 1989; 10: 245-253Crossref PubMed Scopus (7) Google Scholar). In the course of examining the effect of trypsin on this preparation, it was observed that soluble polypeptides were released by the action of the protease in sufficient amounts for them to be detected by conventional staining of SDS gels with Coomassie Blue. Sequencing of these tryptic fragments showed that they were not derived from the Ca-ATPase but instead possessed N termini identifying them as overlapping stretches of polypeptide originating in the large cytoplasmic domain (f loop) of a Na+-Ca2+exchanger. Because this region of the Na+-Ca2+exchanger is crucial for its regulation through physiological mechanisms and because it may represent a possible target for pharmacological intervention, the polypeptides were further investigated.EXPERIMENTAL PROCEDURESDeep sea scallops (Placopecten magellanicus) were obtained from the Marine Biology Laboratory (Woods Hole, MA).Preparation of Native Membranes Enriched in Fragmented Sarcoplasmic ReticulumThis was carried out essentially as described previously (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar, 26Kalabokis V.N. Bozzola J.J. Castellani L. Hardwicke P.M.D. J. Biol. Chem. 1991; 266: 22044-22050Abstract Full Text PDF PubMed Google Scholar, 27Hardwicke P.M.D. Huvos P. J. Musc. Res. Cell Motil. 1989; 10: 229-244Crossref PubMed Scopus (6) Google Scholar, 28Hardwicke P.M.D. Bozzola J.J. J. Musc. Res. Cell Motil. 1989; 10: 245-253Crossref PubMed Scopus (7) Google Scholar, 29Hardwicke P.M.D. Ryan C. Kalabokis V.N. Biochim. Biophys. Acta. 1999; 1417: 1-8Crossref PubMed Scopus (2) Google Scholar, 30Kalabokis V.N. Hardwicke P.M.D. Biochim. Biophys. Acta. 1993; 1147: 35-41Crossref PubMed Scopus (3) Google Scholar). Total scallop muscle membranes were separated into fractions enriched in SL (B1 fraction) and SR (B2 fraction) by layering the crude total membranes, suspended in 0.32 m sucrose, 0.1 m KCl, 1 mm CaCl2, 20 mm MOPS-Na, pH 7.0, onto a discontinuous gradient comprised of a layer of 0.8 msucrose on 1.3 m sucrose, both in 0.1 m KCl, 1 mm CaCl2, 20 mm MOPS-Na, pH 7.0 (29Hardwicke P.M.D. Ryan C. Kalabokis V.N. Biochim. Biophys. Acta. 1999; 1417: 1-8Crossref PubMed Scopus (2) Google Scholar, 30Kalabokis V.N. Hardwicke P.M.D. Biochim. Biophys. Acta. 1993; 1147: 35-41Crossref PubMed Scopus (3) Google Scholar). The SL-enriched fraction (B1) banded at the 0.32–0.8 m sucrose interface, and the SR-enriched fraction (B2) banded at the 0.8–1.3 m interface (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar,29Hardwicke P.M.D. Ryan C. Kalabokis V.N. Biochim. Biophys. Acta. 1999; 1417: 1-8Crossref PubMed Scopus (2) Google Scholar, 30Kalabokis V.N. Hardwicke P.M.D. Biochim. Biophys. Acta. 1993; 1147: 35-41Crossref PubMed Scopus (3) Google Scholar). The B2 fraction was collected.Preparation of Membranes Enriched in SarcolemmaThe B1 fraction was prepared as described above (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar, 29Hardwicke P.M.D. Ryan C. Kalabokis V.N. Biochim. Biophys. Acta. 1999; 1417: 1-8Crossref PubMed Scopus (2) Google Scholar,30Kalabokis V.N. Hardwicke P.M.D. Biochim. Biophys. Acta. 1993; 1147: 35-41Crossref PubMed Scopus (3) Google Scholar).Preparation of Deoxycholate-extracted Scallop Membrane FractionsThis was carried out when necessary with both the SL-rich (B1) and SR-rich (B2) fractions, as described previously (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar).Preparation of Soluble Fragments from Tryptic DigestsMembranes were typically suspended at 5–10 mg ml−1 in standard media of 20% (v/v) ethylene glycol (Pierce), 0.15 m KCl, 1 mmCaCl2 or 10 mm EGTA-Na, 50 mm MOPS-Na, pH 7.0. Digestions were at room temperature for 10–15 min. with N-tosyl-l-phenylalanyl chloromethyl ketone-treated trypsin (Sigma, dissolved in 1 mm HCl at 12,000 units/mg) added in a 1:30 (w/w) ratio to total membrane protein (giving 400 units of activity/mg total membrane protein). Digestions were terminated by addition of 4-(2-aminoethyl)benzenesulfonyl fluoride (Calbiochem) to a final concentration of 20 mm, followed by transfer of the sample to ice. The samples were centrifuged at 16,000 × g for ½ h at 4 °C in an Eppendorf microcentrifuge. The supernatant was collected and recentrifuged for 1 h at 105 × g in a Beckman TL100 bench top ultracentrifuge at 4 °C to remove traces of contaminating membranes.Concentration of Soluble Peptides for Use in SDS-PAGESodium deoxycholate was added to the high speed supernatant from the tryptic digest to a final concentration of 0.025% (w/v), followed by trichloroacetic acid to a final concentration of 6% (w/v). The trichloroacetic acid also served to completely inactivate the trypsin. After incubation on ice for 15 min, the preparation was centrifuged at 16,000 × g for ½ h, the supernatant was carefully removed, and the pellet was washed with 200 μl of diethyl ether. After drying, the pellet was dissolved in Tricine or Laemmli sample denaturation buffer.Gel ElectrophoresisDiscontinuous SDS-PAGE was carried out with glycine or Tricine as the trailing anion (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205881) Google Scholar, 32Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10429) Google Scholar). Sodium thioglycholate (0.1 mm) was present in the sample denaturation medium and added to the cathode buffer to scavenge the gel ahead of the peptides for free radicals and oxidants.N-terminal SequencingThe proteolytic fragments were separated by SDS-PAGE, and the gels were electroblotted onto Immobilon-PSQ polyvinylidene difluoride membrane (Millipore) in a medium of 10% (v/v) MeOH, 10 mm CAPS-Na, pH 11 at 4 °C. After lightly staining with Coomassie Blue, the bands of interest were cut out and sent to the Protein Chemistry Core of the University of Florida.Phosphorylation of the Membranes with Protein Kinase AThe DOC-extracted B2 membrane fraction (1 mg) was incubated with 50 units of the catalytic subunit of PKA in a medium of 0.27m sucrose, 80 mm KCl, 5 mm EGTA, 0.08 mm CaCl2, 29 mm NaF, 8.3 mm MgCl2, 20 mm MOPS-Na, pH 7.0 containing 0.1 mm [γ-32P]ATP (500 dpm pmol−1) for 2 min at 30 °C. The membranes were sedimented at 16,000 × g and washed free of unbound radioactivity. The phosphorylated membranes were then divided into two parts, one of which was resuspended in 20% (v/v) ethylene glycol, 0.4m KCl, 1 mm CaCl2, 50 mm MOPS-Na, pH 7.0, while the other was resuspended in 20% (v/v) ethylene glycol, 0.4 m KCl, 10 mm EGTA, 50 mm MOPS-Na, pH 7.0. Digestion with trypsin was then carried out as described above. The soluble products were separated from insoluble (membrane-bound) material and concentrated by the DOC-trichloroacetic acid procedure described above. The phosphorylated samples were electrophoresed in the Tricine SDS gel system, and autoradiography of the dried gel was carried out using Kodak X-Omat film.Production of Rabbit Polyclonal Antibody against the ∼37-kDa Soluble Tryptic FragmentThe soluble tryptic fraction formed in the presence of Ca2+ was run on a Tricine SDS gel. The gel was then lightly stained with Coomassie Brilliant Blue, and the ∼37-kDa band was cut out with a razor blade. The gel slice was homogenized in PBS (0.15 m NaCl, 10 mm sodium phosphate, pH 7.2) and Freund's complete adjuvant before injection into New Zealand White rabbits. Booster injections were made every third week using Freund's incomplete adjuvant. Partial purification of antibody from serum was by ammonium sulfate fractionation, followed by DEAE-Sephacel anion exchange chromatography to separate IgG and IgM (33Dunbar B.S. Schwoebel E. Methods Enzymol. 1990; 182: 663-670Crossref PubMed Scopus (88) Google Scholar).Western BlottingSDS gels were first blotted onto polyvinylidene difluoride (Immobilon PSQ), as in the procedure for N-terminal sequencing (see above). Nonspecific binding was blocked with 5% w/v nonfat milk in PBS-T (0.1 m NaCl, O.1% v/v Tween-20, 0.1 m NaPi, pH 7.4) for 1 h at room temperature. The blots were washed twice quickly in PBS-T, followed by one wash for 15 min and two washes for 5 min in PBS-T. The blots were incubated in rabbit anti-37-kDa antibody diluted 1:1000 with 5% (w/v) nonfat dried milk, 0.1% v/v Tween-20 in PBS-for 1 h at room temperature with gentle agitation. The blots were then washed as before with PBS-T. The blots were then incubated for 1 h in goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase (Amersham Pharmacia Biotech) that had been diluted 1:1,500 in 5% (w/v) dried milk in PBS-T. After washing again with PBS-T, the blots were incubated with the ECL luminol system (Amersham Pharmacia Biotech).Protein ConcentrationThis was by the bicinchoninic acid method (34Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18417) Google Scholar).RESULTSThe starting materials for the work reported here were membrane fractions prepared from the cross-striated part of the adductor muscle of the deep sea scallop. One of these (the B2 fraction; see “Experimental Procedures”) is enriched in fragmented SR but still contaminated by membranes derived from the SL (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar, 29Hardwicke P.M.D. Ryan C. Kalabokis V.N. Biochim. Biophys. Acta. 1999; 1417: 1-8Crossref PubMed Scopus (2) Google Scholar). This fraction can be extracted with low (nonsolubilizing) concentrations of DOC to remove peripheral membrane proteins and other contaminants (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar). On SDS gels of the DOC-extracted B2 fraction, ∼90% of the Coomassie Blue-staining material has a mobility corresponding to a molecular mass of 105–115 kDa (Fig. 1and Ref. 25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar), consistent with the size of the Ca-ATPase (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar, 35Castellani L. Hardwicke P.M.D. Vibert P. J. Mol. Biol. 1985; 185: 579-594Crossref PubMed Scopus (63) Google Scholar, 36Shi X. Chen M. Huvos P.E. Hardwicke P.M.D. Comp. Biochem. Physiol. Part B. 1998; 120: 359-374Crossref PubMed Scopus (12) Google Scholar). The second scallop muscle fraction (B1), which was used later in the studies, is enriched in SL (29Hardwicke P.M.D. Ryan C. Kalabokis V.N. Biochim. Biophys. Acta. 1999; 1417: 1-8Crossref PubMed Scopus (2) Google Scholar).Tryptic digests of the DOC-extracted scallop muscle B2membrane fraction were examined for soluble peptide fragments released by the action of the protease. It was found that a polypeptide of ∼37 kDa was the major soluble species formed in the presence of 1 mm Ca2+ (Fig. 2), although smaller amounts of soluble ∼16- and ∼19-kDa peptides were sometimes also present. In the presence of 10 mm EGTA, the pattern of the products was reversed, so that the ∼37-kDa fragment was either absent or present in much smaller amounts, and the two smaller polypeptides were the main soluble products. The relative proportions of ∼16- and ∼19-kDa peptides varied; sometimes the two species were present in approximately the same amounts, whereas in other digests one form predominated. This may mean that the 16- and 19-kDa fragments are susceptible to further proteolysis; in contrast, the ∼37-kDa peptide was relatively resistant to the further action of trypsin. All three soluble peptides could be clearly visualized on the gels with standard Coomassie Blue stain. Occasionally, larger soluble polypeptides of ∼60 and 40 kDa were observed.Figure 2Effect of Ca2+ concentration on soluble tryptic fragments produced from a deoxycholate-treated scallop muscle membrane fraction. Digestion of deoxycholate-extracted scallop muscle membrane B2 was carried out in the presence of EGTA and Ca2+ as described under “Experimental Procedures.” The soluble peptides formed in the presence of CaCl2 and EGTA were concentrated by the DOC-trichloroacetic acid method and electrophoresed in the Tricine SDS-PAGE system. The bands labeled t in lanes 1 and 2 are trypsin (molecular mass, 23.8 kDa) and of two its autolyzis products.Lane 1, soluble peptides formed from 190 μg of total membrane protein in the presence of EGTA. Lane 2, soluble peptides formed from 190 μg of total membrane protein in the presence of Ca2+. Lane 3, markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The precursor polypeptide for the soluble peptides had to be very close in size to the scallop SERCA (molecular mass, 110 kDa), because only material of 105–115 kDa was present in sufficient amounts in the starting preparation to account for the amounts of soluble peptides formed (Fig. 1 and Ref. 25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar). There are usually only small and variable traces of other integral protein membrane components on Coomassie Blue stained SDS gels of the B2 fraction after DOC extraction, the most significant of these being a 28-kDa protein associated with the SL (25Kalabokis V. Hardwicke P.M.D. J. Biol. Chem. 1988; 263: 15184-15188Abstract Full Text PDF PubMed Google Scholar, 29Hardwicke P.M.D. Ryan C. Kalabokis V.N. Biochim. Biophys. Acta. 1999; 1417: 1-8Crossref PubMed Scopus (2) Google Scholar). The latter peptide is too small to be the precursor of the 37-kDa soluble tryptic fragment and in any case is present only at low levels.The results of N-terminal sequencing of the soluble fragments are shown in Table I, where the data have been aligned to show the apparent relationships between the peptides and some sodium-calcium exchangers. The ∼37-kDa peptide produced in the presence of Ca2+ and the ∼16-kDa polypeptide formed by digestion in the absence of Ca2+ had the same N-terminal sequence. Comparison of this N-terminal sequence with known sequences using the EMBL data base (Blitz) indicated that its start corresponded to residue 401(369) of the NCX1 Na+-Ca2+exchanger, at the N-terminal end of the β-1 repeat in the large cytoplasmic domain (loop f) (2Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-564Crossref PubMed Scopus (627) Google Scholar, 4Nicoll D.A. Quednau B.D. Qui Z. Xia Y.-R. Lusis A.J. Philipson K.D. J. Biol. Chem. 1996; 271: 24914-24921Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). A lysyl or arginyl residue must precede this sequence for the 37- and 16-kDa fragments to be produced by the action of trypsin. There was no significant similarity to any part of the scallop Ca-ATPase polypeptide chain (36Shi X. Chen M. Huvos P.E. Hardwicke P.M.D. Comp. Biochem. Physiol. Part B. 1998; 120: 359-374Crossref PubMed Scopus (12) Google Scholar). Assuming an average residue mass of 110 Da, the ∼16-kDa fragment was approximately 145 residues long and was likely to correspond closely to the segment of loop f in NCX1 that starts and ends with the two acidic triads, Asp478(446)-Asp-Asp at the carboxyl end of β-1 and Asp530(498)-Asp-Asp preceding the β-2 repeat. The Ca2+-insensitive cleavage site on the scallop precursor represented by the N terminus of the ∼16- and ∼37-kDa soluble fragments will be designated the T1 site. The C terminus of the ∼16-kDa fragment must be located in a stretch of polypeptide chain inaccessible to trypsin when Ca2+ is bound to the precursor protein but more susceptible to proteolysis when Ca2+is unbound. The Ca2+-sensitive proteolytic cleavage site represented by the C terminus of the ∼16-kDa fragment will be designated the T2 site. Satisfactory N-terminal sequencing of the ∼19-kDa band peptide was not possible, because of microheterogeneity within that band on SDS gels, but the size of the fragment is consistent with it representing that part of the 37-kDa fragment which is C-terminal to the T2 cleavage site. There is other evidence supporting this conclusion from the immunological and phosphorylation studies reported below. The tryptic cleavage site corresponding to the C terminus of the 37-kDa fragment, and possibly the C terminus of the 19-kDa fragment, will be designated the T3 site.Table IComparison of the N-terminal sequences of tryptic fragments from scallop muscle membranes with some sodium-calcium exchangersR/KDQFTTKVFFDPGHYTVMENScallop soluble 37 kDaR/KDQFTTKVFFDPGHYTVMENScallop soluble 16 kDaPDEI-TRVSFDPGHYTVMEN390Squid NCX-SQI (12)ADD-PIRMYFEPGHYTVMEN456Calx(Drosophila) (10)NDPV-SKIFFEQGTYQCLEN418DogNCX1 (6)NDPV-SKVFFEPGTYQCLEN418RatNCX1 (9)DDDGASRIFFEPSLYHCLEN408RatNCX2 (7)PEDFASKVFFDPCSYQCLEN410RatNCX3 (8) Open table in a new tab Polyclonal Antibody against the 37-kDa Soluble Fragment Cross-reacts with the 16- and 19-kDa Soluble Tryptic Fragments and Also a 105–110-kDa Polypeptide in the Undigested MembranesThe soluble fragments formed in the presence of Ca2+ were separated on a Tricine SDS gel, and a Western blot was obtained using polyclonal rabbit IgG raised against the ∼37-kDa peptide. All three peptides bound the antibody, indicating common epitopes between the 37-kDa peptide and the 16-kDa peptide and between the 37-kDa peptide and the 19-kDa peptide (Fig. 3).Figure 3Western blot of the soluble tryptic peptides formed in the presence of Ca2+ using rabbit anti-scallop 37-kDa fragment IgG. The deoxycholate-extracted B2scallop muscle membrane fraction was digested with trypsin, the soluble fraction was concentrated by the DOC-trichloroacetic acid method, and the sample was electrophoresed in the Tricine SDS system. Western blotting using rabbit anti-37-kDa fragment IgG as probe was carried out as described under “Experimental Procedures.” Both the 16- and 19-kDa fragments shared epitopes with the 37-kDa peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Western blots of Tricine SDS gels of the undigested DOC extracted B2 membrane fraction probed with rabbit anti-37 kDa" @default.
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• W1986247804 title "A Ca2+-dependent Tryptic Cleavage Site and a Protein Kinase A Phosphorylation Site Are Present in the Ca2+ Regulatory Domain of Scallop Muscle Na+-Ca2+ Exchanger" @default.
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