THE 70-KD COMPONENT OF THE HEART SARCOLEMMAL NA+ CA2+-EXCHANGER PREPARATION IS THE C-TERMINAL PORTION OF THE PROTEIN/

The cardiac sarcolemmal Na+/Ca2+-exchanger was expressed in COS-7 cell s by the vaccinia virus system as a fusion protein with a poly-His tag at its C-terminus. Extracts of cells expressing the exchanger constru ct without the His-tag reacted with an antiserum against the C-termina l portion of the main intracellular loop of the exchanger: in agreemen t with the finding routinely made on heart sarcolemma and on plasma me mbrane of cells expressing the cardiac exchanger gene, three bands of about 160, 120, and 70 kD were detected in Western blots. All three ba nds shifted to higher molecular masses when the construct with the His -tag was expressed, indicating that the three proteins had the same C- terminus. Thus, the 70 kD protein, whose nature has always been obscur e, appears to be a degradation product of one of the two larger protei ns. N-terminal sequencing of partially purified exchanger preparations has identified the cleavage site(s) producing the 70 kD protein in th e 257-269 residue region of the exchanger molecule.