FIGURE 7.

Equilibrium levels of phosphoenzyme obtained through utilization of [³²P]P_i by recombinant SERCA and E309Q mutant at acid or alkaline pH. Effects of K⁺ and Ca²⁺ are shown. Microsomes derived from COS1 cells expressing SERCA or mutant (E309Q) protein were diluted (50 μg/ml) in medium containing 50 mm MES (pH 6.0) or HEPES (pH 7.5), 20% Me₂SO₄, and 10 mm MgCl₂. 2 mm EGTA, 100 mm KCl, and 1 mm CaCl₂ (in the absence of EGTA) were added as indicated in the figure. The reaction was started by the addition of 50 μm [³²P]P, at 30 °C temperature and acid-quenched after 2 min. The quenched samples were processed for determination of radioactive protein by electrophoretic analysis and detection of radioactivity.