IDENTIFICATION, CHARACTERIZATION AND PARTIAL-PURIFICATION OF A THIOL-PROTEASE WHICH CLEAVES SPECIFICALLY THE SKELETAL-MUSCLE RYANODINE RECEPTOR CA2+ RELEASE CHANNEL

A 94 kDa large subunit thiol-protease, as identified by anti-calpain a ntibodies, has been isolated from skeletal muscle junctional sarcoplas mic reticulum (SR). This protease cleaves specifically the skeletal mu scle ryanodine receptor (RyR)/Ca2+ release channel at one site resulti ng in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease deg rades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about, was absolutely dep endent on the presence of thiol reducing reagents, and was completely inhibited by HgCl2, leupeptin and the specific calpain I inhibitor. Ho wever, while the SR membrane-associated protease requires Ca2+ at a su bmicromolar concentration, the isolated thiol-protease has lost the Ca 2+ requirement. The 94 kDa thiol-protease had no effect on ryanodine b inding but modified the channel activity of RyR reconstituted into pla nar lipid bilayer: in a time-dependent manner, the channel activity de creases and within several minutes the channel is converted into a sub conducting state. The protease-modified channel activity is still Ca2-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross r eact with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94.