Identification of Ca2+-activated neutral protease in the peripheral nerve and its effects on neurofilament...

Rat sciatic nerve segments were incubated in five different media. Disappearance of neurofilament (NF) triplet proteins (200K, 160K, and 68K MW) occurred in medium containing Ca2+ and was inhibited by the addition of E-64-c or leupeptin. Therefore, the presence in the peripheral nerve of an enzyme whose properties are similar to those of Ca2+-activated neutral protease (CANP) is suggested. The extraction of crude CANP from rat sciatic nerve was performed. CANP activity was completely recovered (0.129 +/- 0.008 U/g) in the precipitate salted out by the addition of 0 to 50% saturated ammonium sulfate to the soluble fraction of the peripheral nerve (crude CANP). Properties of the crude CANP were examined using NF as a substrate and were found to be similar to those of the CANP extracted from skeletal muscle. Identification of the crude CANP with the CANP extracted from rat skeletal muscle was performed using the immunoreplica method. Bands corresponding to 73K were detected in both CANPs.