Micromolar Ca2+ requiring protease from human platelets: purification, partial characterization and effect on the cytoskeletal proteins.

A calcium-activated neutral protease (CANP) has been purified 2,800 fold, to near homogeneity, from human platelets. The purification procedure involved ammonium sulfate fractionation of the platelet cytosol followed by chromatography on Sephacryl S-200, DEAE-Sephacel, Agarose-Hexylamine, Agarose-Octylamine and alpha-casein-Sepharose 4B affinity gel. The protease consisted of two polypeptides of Mr = 74,000 and 28,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzed [methyl-14C] alpha-casein at a significant rate of 37 degrees C which was, therefore, used as an exogenous substrate. Microtubules and intermediate filament proteins were also susceptible to hydrolysis by the purified protease. It attained maximum activity at 0.06 uM CaCl2 and displayed two pH maxima: one at 5.5 and the other at 6.5. The protease was fully active in the presence of MnCl2 and was about 75% active with BaCl2 and SrCl2. Among the actinomycete protease inhibitors, leupeptin, antipain and pepstatin, the order of inhibition was: leupeptin greater than antipain greater than pepstatin. The protease was also inhibited by sulfhydryl modifying agents.