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Purification from Synaptosomal Plasma Membranes of Calpain I, a Thiol Protease Activated by Micromolar Calcium Concentrations
Author(s) -
Siman Robert,
Baudry Michel,
Lynch Gary
Publication year - 1983
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1983.tb09039.x
Subject(s) - leupeptin , antipain , protease , chemistry , calpain , calcium , biochemistry , enzyme , ionic strength , iodoacetamide , proteases , membrane , chromatography , pmsf , cytosol , proteolysis , substrate (aquarium) , dithiothreitol , enzyme assay , casein , cysteine , biology , organic chemistry , aqueous solution , ecology
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 μ M; A act2 = 500 μ M ). 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 μ M ) 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.