Difference in Kinetic Properties of Phosphorylated Intermediates Formed in the Forward and Backward Reactions of Solubilized Ca²⁺-ATPase of Sarcoplasmic Reticulum

Abstract

Solubilized Ca²⁺-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C₁₂E₈) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with P₁.
The time course of decay of E³²P formed with 4 μM AT³²P in the presence of 10mM CaCl₂ and 10mM MgCl₂ (forward reaction) was measured by adding 0.4mM unlabeled ATP and 10mM P₁ at pH 6.0 and 30°C. The rate of E³²P decay was accelerated by 0.4mM ADP. On the other hand, when the time course of decay of E³²P formed with 10mM ³²P₁ in the presence of 5mM EGTA and 10mM MgCl₂ (backward reaction) was measured by adding 0.4mM unlabeled ATP and 15mM CaCl₂, the rate of E³²P decay was unaffected by 0.4mM ADP.
AT³²P was produced on adding ADP to E³²P formed with AT³²P in the presence of 10mM CaCl₂ and 10mM MgCl₂, while no AT³²P was produced on adding ADP to E³²P formed with ³²P₁ in the presence of 5mM EGTA and 10mM MgCl₂, even when 15mM CaCl₂ was added simultaneously with ADP.