caa a22 4500 477119557 CHVBK 20180405111632.0 cr unu---uuuuu 170330e19960301xx s 000 0 eng 10.1007/BF00240027 doi (NATIONALLICENCE)springer-10.1007/BF00240027 Cooperative interaction between Ca2+ binding sites in the hydrophilic loop of the Na+-Ca2+ exchanger [Elektronische Daten] [Dmitri Levitsky, Bodvaël Fraysse, Claude Leoty, Debora Nicoll, Kenneth Philipson] A high affinity Ca2+-binding domain which is located in a middle portion of the large intracellular loop of the Na+-Ca2+ exchanger contains two highly acidic sequences, each characterized by three consecutive aspartic acid residues (Levitsky DO, Nicoll DA, and Philipson KD (1994) J Biol Chem 269: 22847-22852). This portion of the protein provides secondary Ca2+ regulation of the exchanger activity. To determine number of Ca2+ binding sites participating in formation of the high affinity domain, we isolated polypeptides of different lengths encompassing the domain and measured 45Ca2+ binding. The fusion proteins containing the high affinity domain were obtained in a Ca2+-bound form and as evidenced by shifts in there mobility in SDS-polyacrylamide gels after EGTA treatment. The Ca2+ binding curves obtained after equilibrium dialysis reached saturation at 1 μM free Ca2+, Kd value being approx. 0.4 μM. The Ca2+ binding occured in a highly cooperative manner. Upon saturation, the amount of Ca2+ ion bound varied from 1.3-2.1 mot per mot protein. Proteins with an aspartate in each acidic sequence mutated lacked the positive cooperativity, had lower Ca2+ affinity and bound two to three times less Ca2+. Na+-Ca2+ exchangers of tissues other than heart though different from the cardiac exchanger by molecular weight most likely possess a similar Ca2+ binding site. It is concluded that, by analogy with Ca2+ binding proteins of EF-type, the high Ca2+-affinity domain of the Na+-Ca2+ exchanger is comprised of at least two binding sites interacting cooperatively. Kluwer Academic Publishers, 1996 Na+-Ca2+ exchanger in different tissues nationallicence high affinity Ca2+ binding nationallicence Levitsky Dmitri Laboratory of General Physiology, URA CNRS 1340, University ofNantes, Nantes, France aut Fraysse Bodvaël Laboratory of General Physiology, URA CNRS 1340, University ofNantes, Nantes, France aut Leoty Claude Laboratory of General Physiology, URA CNRS 1340, University ofNantes, Nantes, France aut Nicoll Debora Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, UCLA School of Medicine, 90095-1760, Los Angeles, CA, USA aut Philipson Kenneth Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, UCLA School of Medicine, 90095-1760, Los Angeles, CA, USA aut Molecular and Cellular Biochemistry Kluwer Academic Publishers; http://www.wkap.nl 160-161/1(1996-03-01), 27-32 0300-8177 160-161:1<27 1996 160-161 11010 https://doi.org/10.1007/BF00240027 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00240027 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Levitsky Dmitri Laboratory of General Physiology, URA CNRS 1340, University ofNantes, Nantes, France aut NATIONALLICENCE 700 1- Fraysse Bodvaël Laboratory of General Physiology, URA CNRS 1340, University ofNantes, Nantes, France aut NATIONALLICENCE 700 1- Leoty Claude Laboratory of General Physiology, URA CNRS 1340, University ofNantes, Nantes, France aut NATIONALLICENCE 700 1- Nicoll Debora Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, UCLA School of Medicine, 90095-1760, Los Angeles, CA, USA aut NATIONALLICENCE 700 1- Philipson Kenneth Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, UCLA School of Medicine, 90095-1760, Los Angeles, CA, USA aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Kluwer Academic Publishers; http://www.wkap.nl 160-161/1(1996-03-01), 27-32 0300-8177 160-161:1<27 1996 160-161 11010 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer