Fig. 6.

a, crystallographic models of the Ca²⁺ ATPase in the E2 · TG (left, Protein Data Base code 1iwo) and in the E1 · 2 Ca²⁺ (right, Protein Data Base code 1su4). The sites for initial digestion by proteinase K are in the loops intervening between the A domain and the M2 and M3 transmembrane segments. Note their displacement as the A domain rotates as a consequence of the E1 to E2 transition. Digestion patterns of ATPase with proteinase K in the absence (b) and in the presence of Ca²⁺ (c). The first lane on the left shows a sample with proteinase K denatured before addition of ATPase. The E1 pattern yields complementary 83-kDa (carboxyl terminus) and 28-kDa (amino terminus) bands, whereas the E2 pattern yields additional 95-kDa (carboxyl terminus) and 14-kDa (amino terminus) bands. Note that the E2 pattern is obtained even in the presence of Ca²⁺ when TG (1.0 μM), CPA (10 μM), or DBHQ (20 μM) are added to the reaction mixture, whereas the E1 pattern is obtained in the presence of TITU (100 μM). Digestion (30 min at 25°C) and electrophoresis as explained under Materials and Methods. The free Ca²⁺ concentration, when present, was 50 μM. Total protein loaded per well was 30 μg. SERCA protein and fragments thereof were evidenced by Western blotting with specific antibodies. digest., digestion.