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A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.

Two forms (CANP1 and CANP2) of a calcium-activated neutral protease (CANP) have been purified, 1,950- and 1,250-fold, respectively, to near homogeneity from calf brain. The purification procedure involves ammonium sulfate fractionation of the brain cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, and alpha-casein-CH-Sepharose 4B affinity gel. A protein with apparent Mr = 17,000 copurifies with each of the proteases. This protein was separated by chromatography on a reactive red-120 agarose. Preliminary experiments indicate that, in the absence of this protein, the activity of each of the proteases was reduced. These observations raise the possibility that the 17,000-Da protein may regulate the activity of these proteases. Each of the proteases have similar apparent Mr = 78,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Except for casein, hemocyanin, and hemoglobin, no other exogenous proteins tested were significantly hydrolyzed by either of the proteases. [ methyl-14C ]alpha-Casein or methemoglobin was routinely used as a substrate for both of the enzymes. The endogenous proteins, neurotubules (microtubule-associated proteins and tubulin), neurofilament triplet proteins and desmin from smooth muscle were extensively hydrolyzed by both of the proteases. A marked difference was found in their requirement for CaCl2. CANP1 was maximally active at 700 microM while CANP2 exhibited highest activity at 2 microM CaCl2. Both displayed maximum activity at pH 7.5, although the overall pH profiles were slightly different. Among the actinomycete protease inhibitors, antipain, leupeptin, and pepstatin, leupeptin was highly effective in inhibiting the activities of both enzymes. Both of the proteases were also inhibited by sulfhydryl modifying agents. Metal ions other than CaCl2 were poor activators of the activity of either protease.