Purification and characterization of a calcium-activated neutral protease from monkey brain and its action on...

A calcium-activated neutral protease was purified from Japanese monkey brain by ammonium sulfate fractionation and sequential column chromatographies monitored by assay of caseinolytic activity. The purified enzyme gave a single protein band on non-denaturing polyacrylamide gel electrophoresis, and consisted of two subunits with molecular weights of 74,000 and 20,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required millimolar order calcium ions for activation, and was optimally active at pH 7.5-8.0. Upon incubation with various neuropeptides as substrates, the enzyme preferentially cleaved the peptide bonds with Arg, Lys, or Tyr at the P1 position and an amino acid residue with a bulky aliphatic side chain, such as Leu, Val, or Ile, at the P2 position. The hydrolytic activity toward neuropeptides as well as casein was strongly inhibited by various thiol protease inhibitors. These results suggested that the brain calcium-activated neutral protease may participate in the degradation of neuropeptides in vivo.