Lanthanum as a calcium-substituting ion for binding to sarcoplasmic reticulum ATPase

Ca2+ binding and internalization in sarcoplasmic reticulum ATPase can be investigated by the use of La3+ as a Ca2+ analog. Displacement kinetics of Ca2+ bound by La3+ in native vesicles is a slow biphasic process (k1 = 0.55 s-1 and k2 = 0.05 s-1) that is consistent with the existence of two Ca2+ binding populations whereas in leaky vesicles there appears to be a single population (k = 0.57 s-1). Rapid quench experiments demonstrate that Ca2+ internalization occurs with an initial burst (approximately 8 nmol/mg protein) associated with the presence of a phosphate-donor substrate in the reaction medium. While acid quenching for measurements of phosphoenzyme is instantaneous, La3+ quenching allows completion of one catalytic and transport cycle due to the slow La3+ exchange with Ca2+. This explains the apparent inconsistencies in the kinetics and stoichiometry of phosphoenzyme formation and Ca2+ internalization that are observed under certain experimental conditions.