Identification of the Na+/CA2+ exchanger of calf heart sarcolemma with the help of specific antibodies.

The Na+/Ca2+ exchanger of calf heart sarcolemma has been identified in solubilized membrane preparations with the help of specific antibodies as a molecule of approximate Mr of 30 KDa. The conclusion supports the previous proposal by Soldati et al. (J. Biol. Chem. 260, 13321-13327, 1985) that the exchanger is a molecule of Mr about 33 KDa. Antibodies (IgG) were raised in rabbits by injecting proteins electroeluted from different regions of preparative SDS gels of solubilized heart sarcolemma. After purification the IgG against the proteins of the 30 KDa region recognized the 33 KDa component but also proteins of Mr about 70 and 140 KDa. Conversely, antibodies against the 140 KDa protein(s) also recognized the 70 and the 33 KDa proteins. However, if the solubilized sarcolemma extract was treated with DTT prior to the transfer to nitrocellulose the 140 KDa protein was not seen. Both the antibodies against the 30 KDa and those against the 140 KDa proteins inhibited the Na+/Ca2+ exchange activity of sarcolemma vesicles. It is proposed that the basic unit of the Na+/Ca2+ exchanger of heart sarcolemma is a monomer of Mr about 33 KDa, the functionally active exchanger being a tetramer in which the four 33 KDa subunits are held together by disulfide bonds. In the monomer-tetramer transition an intermediate dimeric state of Mr 70 KDa is also formed.