Immunological identification of the synaptic plasma membrane Na+-Ca2+ exchanger.
The protein moiety responsible for Na+-Ca2+ exchange activity was identified in synaptic plasma membranes (SPM). This was done by raising polyclonal antibodies in rabbits against each one of the detectable proteins present in the purified preparation containing the enriched specific transport activity. Two of the antibody preparations bound specifically to native SPM: antibodies which were raised against the 70,000-Da protein (the most prominent species consistently present in the purified preparation) and antibodies raised against a 33,000-Da protein (inconsistently present in variable amounts in the purified preparation). Both antibodies bound exclusively to a protein of 70,000 Da in native SPM. When, however, the purified 33,000- and 70,000-Da proteins were used as antigens, each one of the antibody preparations bound to both proteins. In addition, both antibody preparations immunoprecipitated Na+ gradient-dependent Ca2+ transport activity from detergent-solubilized SPM. This was obtained by incubation of solubilized SPM with a complex containing antibodies bound to Protein A-Sepharose beads, reconstitution of the material excluded from the beads, and determination of the residual transport activity. The decrease in Na+ gradient-dependent Ca2+ transport activity paralleled the amount of antibody bound to Protein A-Sepharose beads and could reach 82% as compared to the activity remaining in control experiments using preimmune sera. In comparison, ATP-dependent Ca2+ transport activity was unimpaired. These results indicate that the 70,000-Da protein in SPM contains the catalytic Na+-Ca2+ antiport activity. The presence of the 33,000-Da protein in some preparations and its properties may be explained by its being either a degradation product or a subunit of the 70,000-Da protein.
Barzilai
A
A
Spanier
R
R
Rahamimoff
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis*; D002846:Chromatography, Affinity; D003429:Cross Reactions; D003902:Detergents; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D012995:Solubility; D013570:Synaptic Membranes / Q000032:analysis*
D000906:Antibodies; D002352:Carrier Proteins; D003902:Detergents; D019831:Sodium-Calcium Exchanger; D000255:Adenosine Triphosphate; D012964:Sodium; D002118:Calcium