Calcium-activated neutral protease effects upon skeletal muscle sarcoplasmic reticulum protein structure and calcium release.
In this study, the effects of Ca(2+)-activated neutral protease (CANP) upon skeletal muscle heavy sarcoplasmic reticulum (HSR) structure and function were investigated. CANP was immunolocalized to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent-insoluble fraction of purified HSR membranes. Ca2+ activation of the endogenous membrane-bound CANP produced a characteristic partial fragmentation of the HSR 565-kDa Ca2+ release channel. Similarly, the major substrate for both micromolar and millimolar Ca(2+)-sensitive isoforms of exogenous CANP was the Ca2+ release channel with proteolysis of a 88-kDa HSR protein also observed. Ca2+ release channel proteolysis was initiated at a single cleavage site with coincidental production of 410- and 150-kDa peptide fragments. Appearance of 160- and 137-kDa limiting peptides accompanied secondary proteolysis of the primary 410- and 150-kDa fragments, respectively. Despite extensive proteolysis of the Ca2+ release channel, CANP did not dramatically alter the Ca2+ handling and ryanodine binding properties of HSR membranes. The association of CANP with isolated HSR membranes suggests that, in vivo, this protease may modify an additional property of the Ca2+ release channel. This may be related to the CANP-susceptible structural association of the Ca2+ release channel with dihydropyridine receptors at T-tubule/sarcoplasmic reticulum junctions.
Gilchrist
J S
JS
Faculty of Graduate Studies, University of British Columbia, Vancouver, Canada.
Wang
K K
KK
Katz
S
S
Belcastro
A N
AN
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000276:immunology / Q000378:metabolism*; D002458:Cell Fractionation; D002793:Cholic Acids; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013053:Spectrophotometry
D002793:Cholic Acids; D008565:Membrane Proteins; D012433:Ryanodine; D000255:Adenosine Triphosphate; D002154:Calpain; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium