calpain and kyotorphin

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.

Topics: Amino Acid Sequence; Animals; Brain; Calcium; Calcium-Binding Proteins; Calpain; Chromatography; Cytosol; Electrophoresis, Polyacrylamide Gel; Endorphins; Humans; Hydrogen-Ion Concentration; Male; Molecular Sequence Data; Molecular Weight; Peptide Fragments; Rabbits; Rats; Rats, Inbred Strains; Substrate Specificity; Sulfhydryl Reagents

Topics: Analgesics; Animals; Brain; Calpain; Endorphins; Neurotransmitter Agents

Kyotorphin (Tyr-Arg) accumulation in the dialysed synaptosol from the rat brain in the presence of an inhibitor of kyotorphin-degrading enzyme, was maximal at neutral pH. This accumulation was activated by calcium ions, but was inhibited by leupeptin and SH-blocking agents, a finding which suggests the involvement of calcium-activated neutral protease (CANP or calpain). In addition, the kyotorphin-precursor protein, being processed by purified mu- or m-CANP, was detected at about 160 kDa on Sephacryl S-300 chromatography of the synaptosol. The present findings seem to be the first evidence for the role of CANP as a processing enzyme of neuropeptide-precursor in nerve terminals.

Topics: Animals; Brain; Calcium; Calpain; Endorphins; Hydrogen-Ion Concentration; Leucine; Male; Rats; Rats, Inbred Strains; Sulfhydryl Reagents; Synaptosomes; Time Factors