Purification of a novel type of calcium-activated neutral protease from rat brain. Possible involvement in production of the neuropeptide kyotorphin from calpastatin fragments.

We have found a novel type of Ca2(+)-activated neutral protease in rat brain cytosol which cleaves -Tyr-Arg-containing calpastatin fragments to release the neuropeptide kyotorphin. This enzyme was purified about 26,000-fold by column chromatography as follows: DE52 cellulose, Ultrogel AcA 44, thiopropyl-Sepharose 6B, second DE52 cellulose, Ultrogel AcA 34, and blue Sepharose CL-6B. The molecular mass of the enzyme was estimated to be 65-75 kDa by gel filtration. The purified enzyme gave a single band of 74 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some properties of this enzyme were similar to those of the calpains, i.e. an absolute requirement for Ca2+, maximal activity at neutral pH, and inhibition by sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and N-ethylmaleimide. However, it differs from the calpains in that it possesses no caseinolytic activity, separates from the calpains on the first DE52 column, and is insensitive to leupeptin and E-64 (N-[N-(L-3-trans-carboxyoxrian-2-carbonyl)-L-leucyl]agmatine). Thus, the molecular mass, the substrate specificity, the chromatographic behavior, and the inhibitor spectrum all suggest that this enzyme is a novel type of Ca2(+)-activated neutral protease.