Identification and Characterization of Calcium‐Dependent Metalloproteases in Rat Brain

Abstract

We have begun to identify and characterize brain protease activities separated by and assayed in substrate-containing polyacrylamide gels. In the present report, we focus on four proteolytic activities identified from rat brain that are dependent on micromolar and millimolar Ca2+ concentrations for activity. In contrast to the previously described Ca2+-dependent neutral cysteine proteases (calpains), all four activities appear to be metalloproteases based on their inhibition by EDTA, EGTA, and 1,10-o-phenanthroline, but not by blockers of serine, cysteine, or aspartic proteases. In the presence of excess Ca2+ and the Zn2+-chelating inhibitor, 1,10-o-phenanthroline, activity of the enzymes was reconstituted by addition of lower concentrations of Zn2+, and inhibited by higher Zn2+ concentrations. The four metalloproteases were disignated MP-112, MP-92, MP-70, and MP-65 on the basis of their apparent molecular masses in kilodaltons. The MP-70, the major activity detected, had an apparent kcat for Ca2+ > 100 .mu.M versus 10-25 .mu.M for MP-65 and 50-100 .mu.M for MP-92. MP-112 was a minor activity for which Ca2+ activation levels were not determined. MP-112, MP-70, and MP-65 were similar in being most active in the soluble fraction of 7-day neonate forebrain. In contrast, MP92 activity was highest in the particulate fraction of adult forebrain. About half of the MO-92 activity and lower levels of the other three activities were still detectable in particulate fractions after detergent extraction of membrane, suggesting an association with cytoskeletal or other structural proteins. These results indicate the presence in CNS of a class of Ca2+-dependent proteases that are distinct from the previously described calpains, and that may be important in Ca2+-mediated neural events.