Identification and characterization of calcium-dependent metalloproteases in rat brain.
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 designated MP-112, MP-92, MP-70, and MP-65 on the basis of their apparent molecular masses in kilodaltons. MP-70, the major activity detected, had an apparent kact for Ca2+ greater than 100 microM versus 10-25 microM for MP-65 and 50-100 microM 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, MP-92 activity was highest in the particulate fraction of adult forebrain. About half of the MP-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Nelson
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Neuroscience Group, DuPont Company, Wilmington, DE 19898.
Siman
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D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002413:Cations, Divalent / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D008666:Metalloendopeptidases; D015032:Zinc; D002118:Calcium