Datensatz

Zusammenfassung

A partially purified preparation of the lobster muscle Na⁺/Ca²⁺ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca²⁺ efflux from 45Ca-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 Ca²⁺ efflux depended directly upon the extravesicular Ca²⁺ concentration ([Ca²⁺]_ev) with a half-maximal activation at [Ca²⁺] ev = 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca²⁺, as in most other species. In contrast, at low [Na⁺]_ev , the Ca ev -binding site (i.e., on the cytoplasmic surface) for Ca²⁺ transported via Ca²⁺/Ca²⁺ exchange was half-maximally activated by about 7.5 μm Ca²⁺. Mild proteolysis of the Na⁺/Ca²⁺ exchanger by α-chymotrypsin also upregulated the Na_ev -dependent Ca²⁺ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca²⁺. Proteolytic digestion in the presence of 1.9 μm free ev Ca²⁺, however, induced only a 1.6-fold augmentation of Ca²⁺ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca²⁺ also inhibited proteolytic degradation of the Na⁺/Ca2⁺ exchanger measured by immunoblotting. These data suggest that Ca²⁺, bound to a high affinity binding site, protects against the activation of the Na⁺/Ca²⁺ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na⁺/Ca²⁺ 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.