title: Third form of calcium-activated neutral proteinase from calf brain: purification, partial characterization and comparison of properties with other forms.
abstract: A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.
authors.affiliation: Department of Pathological Biochemistry, New York State Office of Mental Retardation and Developmental Disabilities, Staten Island.
authors.forename: M N
authors.initials: MN
authors.lastname: Malik
authors.forename: M D
authors.initials: MD
authors.lastname: Fenko
authors.forename: A M
authors.initials: AM
authors.lastname: Sheikh
authors.forename: R J
authors.initials: RJ
authors.lastname: Kascsak
authors.forename: M S
authors.initials: MS
authors.lastname: Tonna-DeMasi
authors.forename: H M
authors.initials: HM
authors.lastname: Wisniewski
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002154:Calpain / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D007527:Isoenzymes; D002154:Calpain