Purification from Synaptosomal Plasma Membranes of Calpain I, a Thiol Protease Activated by Micromolar Calcium Concentrations

Abstract

Synaptosomal plasma membranes (SPMs) were prepared from whole rat brain and assayed for calcium-stimulated proteolytic activity. Addition of calcium to SPMs caused a dose-dependent increase in trichloroacetic acid-soluble protein. Two peaks of protease activity directed against a casein substrate were detectable when SPMs were incubated with low-ionic-strength buffer and the extract was fractionated on DEAE-cellulose. The enzyme in peak 1 required less than 1/10 the calcium concentration for activation as the peak 2 protease (K_act1= 35 μM; A_act2= 500 μM). The specific thiol-protease inhibitors leupeptin and antipain and the alkylator iodoacetate blocked enzyme activity. The low-sensitivity protease was converted to a high-sensitivity enzyme (K_act= 20 μM) by substrate affinity chromatography in the presence of calcium. This protease was purified 550-fold from SPMs. The high- and low-sensitivity membrane-associated calcium-dependent proteases are part of a family of enzymes, the calpains, previously reported in cytosolic fractions of several tissues.