Biochemical characterization of the plasma membrane Ca²⁺ pumping ATPase activity present in the gill cells of Mytilus galloprovincialis LAM

1. 1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca²⁺-activated ATPase activity is present. The ATPase has high Ca²⁺ affinity (K_ma = 0.3 μM). 2. 2. The optimum assay conditions to evaluate the enzymatic activity of the Ca²⁺-stimulated ATPase at 19°C are: 120-300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca²⁺ ATPase activity is stimulated by DTT (0.5-1 mM) and it is inhibited by low concentrations of vanadate (10-50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive. 3. 3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca²⁺ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La³⁺; (c) the mol. wt of Ca²⁺ ATPase is about 140 kDa. 4. 4. Low Ca²⁺ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca²⁺ uptake by plasma membrane inside-out vesicles. 5. 5. The results indicate that the Ca²⁺ pump present in the gill plasma membranes could be responsible for Ca²⁺ extrusion and therefore involved in maintaining the cytosolic Ca²⁺ concentration within physiological levels.