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 antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca2+ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades 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 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl2, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca2+ at a submicromolar concentration, the isolated thiol-protease has lost the Ca2+ requirement. The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca2+-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94.
Shevchenko
S
S
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Feng
W
W
Varsanyi
M
M
Shoshan-Barmatz
V
V
D000818:Animals; D001921:Brain / Q000378:metabolism; D002154:Calpain / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004533:Egtazic Acid; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D011817:Rabbits; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity
D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D004533:Egtazic Acid; D002154:Calpain