Regulatory processes on the cytoplasmic surface of the Na(+)/Ca(2+) exchanger from lobster exoskeletal muscle.
A partially purified preparation of the lobster muscle Na(+)/Ca(2+) exchanger was reconstituted with, presumably, random orientation in liposomes. Ca(2+) efflux from (45)Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na(+) (Na(ev))-dependent Ca(2+) efflux depended directly upon the extravesicular Ca(2+) concentration ([Ca(2+)](ev)) with a half-maximal activation at [Ca(2+)](ev) = 0.6 microm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca(2+), as in most other species. In contrast, at low [Na(+)](ev), the Ca(ev)-binding site (i.e., on the cytoplasmic surface) for Ca(2+) transported via Ca(2+)/Ca(2+) exchange was half-maximally activated by about 7.5 microm Ca(2+). Mild proteolysis of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin also upregulated the Na(ev)-dependent Ca(2+) efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca(2+). Proteolytic digestion in the presence of 1.9 microm free ev Ca(2+), however, induced only a 1. 6-fold augmentation of Ca(2+) efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca(2+) also inhibited proteolytic degradation of the Na(+)/Ca(2+) exchanger measured by immunoblotting. These data suggest that Ca(2+), bound to a high affinity binding site, protects against the activation of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin. Additionally, we observed a 6-fold increase in the Na(+)/Ca(2+) exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids.
Eisenrauch
A
A
Max-Planck-Institut für Biophysik, Department of Biophysical Chemistry, Kennedyallee 70, D-60596 Frankfurt am Main, Germany.
Juhaszova
M
M
Blaustein
M P
MP
D000818:Animals; D002132:Calcium Radioisotopes / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D008121:Nephropidae; D011510:Proteolipids; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*
D002132:Calcium Radioisotopes; D011510:Proteolipids; D019831:Sodium-Calcium Exchanger; C020485:proteoliposomes; D002918:Chymotrypsin; C117608:alpha-chymotrypsin