title: 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 (Kact1 = 35 microM; Kact2 = 500 microM). 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 (Kact = 20 microM) 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.
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002154:Calpain; D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D008297:Male; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013570:Synaptic Membranes / Q000201:enzymology*
chemical_list: D011480:Protease Inhibitors; D010450:Endopeptidases; D002154:Calpain; D002118:Calcium