Neurofilament proteolysis in rat peripheral nerve. Homologies with calcium-activated proteolysis of other tissues

Abstract

Alterations of nerve proteins were studied in desheathed segments of rat sciatic nerves which were freeze-thawed and incubated in solutions containing calcium or EGTA. Calcium caused a selective loss of 69,000, 150,000, and 200,000 MW neurofilament triplet proteins as well as some loss of 55,000–57,000 MW proteins when nerve proteins were examined by SDS gel electrophoresis. The loss of nerve proteins was accompanied by a variable appearance of 25,000, 36,000, and 72,000 MW proteins. The calcium-induced changes did not occur in nerve segments which had been heated to 60°C for 30 min and were prevented by preincubation of tissues with 1 mM p-chloromercuribenzoate (PCMB), N-ethyl-malemide (NEM), or iodoacetamide. The calcium-induced alterations of nerve proteins were attributed to a calcium-activated thiol protease which preferentially degrades neurofilament proteins. Neurofilament protease of rat peripheral nerve was characterized by studying the alterations of nerve proteins under different incubational conditions. Calcium activated proteolysis was demonstrated between pH 6.0 and 8.8 and at 4, 20, and 37°C. Protein breakdown occurred with 50 μM calcium, and could be simulated by strontium and, to a lesser extent, by barium and lanthanum. Other metals had strong (Hg, Zn, Cd, Cu, Co), weak (Pb and Ag), or no (Al, Mg, and Mn) inhibitory effects on enzymatic activity. Proteolysis was also strongly inhibited by TLCK (1 mM) and by TPCK (1 mM) but not by PMSF (1 mM). The properties of neurofilament protease closely resemble those of calcium-activated proteases described in several tissues.