Abstract
Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na(+)-Ca(2+) exchanger. In the presence of 1 mm Ca(2+), the major product was a peptide of approximately 37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, approximately 16- and approximately 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 Ca(2+). Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [gamma-(32)P]ATP led to incorporation of (32)P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca(2+) regulatory domain of a scallop muscle Na(+)-Ca(2+) exchanger that mediates direct modulation of secondary Ca(2+) regulation by cAMP.
Highlights
The Naϩ-Ca2ϩ exchangers of the plasma membrane catalyze a secondary active transport process dependent on the Naϩ electrochemical gradient generated by the Naϩ,Kϩ-ATPase and play a major role in cellular Ca2ϩ homeostasis in many tissues [1]
The isolated cardiac exchanger shows three bands on silverstained 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 120kDa protein at the Asp289(257)–Gly or Asp303(270)–Gly bonds in the large cytoplasmic domain [11, 21,22,23]
Tryptic digests of the DOC-extracted scallop muscle B2 membrane fraction were examined for soluble peptide fragments released by the action of the protease
Summary
Deep sea scallops (Placopecten magellanicus) were obtained from the Marine Biology Laboratory (Woods Hole, MA). Phosphorylation of the Membranes with Protein Kinase A—The DOCextracted B2 membrane fraction (1 mg) was incubated with 50 units of the catalytic subunit of PKA in a medium of 0.27 M 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 [␥-32P]ATP (500 dpm pmolϪ1) for 2 min at 30 °C. 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 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. Protein Concentration—This was by the bicinchoninic acid method [34]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
