Purification and characterization of high Ca2+-requiring neutral proteases from porcine and bovine brains.

Porcine and bovine brain high Ca2+-requiring neutral proteases were purified to homogeneity by the same isolation procedures, and their properties were compared. A high degree of similarity existed between the two proteases. The purification procedures included ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose CL-4B, second DEAE-cellulose chromatography, second phenyl-Sepharose CL-4B chromatography, and gel filtration on Ultrogel AcA 34. Both purified enzymes were composed of Mr 75,000 and 29,000 subunits, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes required 250 microM Ca2+ for half-maximal activity and 700 microM Ca2+ for maximal activity. Sr2+ and Ba2+, but not Mg2+ or Mn2+, also activated both enzymes but not as effectively as Ca2+. Both enzymes displayed maximum activity at pH 7.5-8.0. Leupeptin, antipain, and trans-epoxysuccinyl-L-leucylagmatine inhibited both enzymes. Neurofilament triplet proteins and microtubule-associated proteins were extensively hydrolyzed by both proteases, but tubulin and actin were not hydrolyzed. The amino acid compositions of the two proteases were very similar. Antisera against bovine brain protease cross-reacted with porcine brain protease when examined by immunoelectrotransfer blot techniques.