Affinity Purification of μ-Calpain from Erythrocytes on an Immobilized Peptide from the Plasma Membrane Calcium Pump

Abstract

The plasma membrane Ca²⁺-ATPase (Ca²⁺ pump) (1) is a ubiquitously distributed P-type ATPase (2), consisting of a single polypeptide chain of about 134 kDa, which spans the cellular membrane 10 times. A large cytosolic loop (between transmembrane segments 4/5) contains the catalytic region of the enzyme. A peculiarity of the Ca²⁺ pump among P-type ATPases is an extended C-terminal tail of about 160 residues, which is the target of many regulators of the pump’s activity, such as calmodulin, some kinases, and calpain. Under resting conditions, the C-terminal tail acts as an autoinhibitory domain covering the active site. Energy from ATP hydrolysis allows the extrusion of cytosolic Ca²⁺ by the pump against a 10,000-fold concentration gradient. Since the maintenance of a submicromolar Ca²⁺ concentration in the cytosol is vital to its second messenger function, the role played by the Ca²⁺-ATPase in cellular regulation is obvious.