Ca2+-activated protease activity in frog sciatic nerve: Characterization and effect on rapidly transported axonal proteins

Protease activity was studied in the frog sciatic nerve. The activity was measured as the release of TCA-soluble radioactivity from either ³H-labelled proteins transported by rapid axonal transport (AXT) or ³H-labelled ganglionic proteins. In nerve homogenates containing transported substrates, protease activity exhibited two peaks, one around pH 5 and one around pH 8. Ca²⁺ at 100 μM or higher concentrations only stimulated the latter, which was inhibited by 1 mM parachloromercuric benzoate, a sulphydryl reagent, but unaffected by ATP (1 mM). The proteolytic activity was recovered in the 10⁵ g supernatant of the homogenate. In desheathed nerves containing ³H-labelled transported proteins, the protease activity could be activated by exposing the nerve to a Ca²⁺-ionophore, X-537 A, or to an elevated Ca²⁺-concentration (50 mM). These conditions were also shown to increase the influx and efflux of ⁴⁵Ca²⁺ in the nerves. The results indicate the presence within axons of a Ca²⁺-activated soluble protease, which degrades rapidly transported proteins. The finding that the protease degraded ganglionic soluble proteins to about the same extent suggests a broad substrate specificity. The present system should be useful for further characterization of protease activity during various physiological conditions.