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.
Yoshihara
Y
Y
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Ueda
H
H
Fujii
N
N
Shide
A
A
Yajima
H
H
Satoh
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004723:Endorphins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D004723:Endorphins; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; C019448:kyotorphin; C033668:calpastatin; D002154:Calpain; D002118:Calcium