Removal of Z-lines and alpha-actinin from isolated myofibrils by a calcium-activated neutral protease.
A calcium-activated factor (CaAF) has been isolated and partially purified from the post-myofibrillar supernatant fraction of rabbit skeletal muscle. The 200-fold purified CaAF hydrolyzed denatured casein, [3-H]acetyl hemoglobin, and N-ethyl[3-H]maleimide-labeled alpha-actinin. The proteolytic activity has a pH optimum at 6.9 and is dependent on the presence of Ca2+ (optimum concentration, 10 mM). Digestion of isolated myofibrils with CaAF results in removal of Z-lines and in a parallel loss of a 90, 000-dalton protein that has a mobility identical with that of alpha-actinin as determined by polyacrylamide gel electrophoresis. A protein with the properties of alpha-actinin (identical electrophoretic mobility, and ability to accelerate the Mg2+-activated ATPase of reconstituted actomyosin) was isolated from the supernatant of CaAF-treated myofibrils. The release of alpha-actinin from myofibrils by the calcium-activated neutral protease occurs in the absence of detectable change in the electrophoretic profiles of the other myofibrillar proteins, or in the ethylene glycol bis(beta-aminoethyl ether)-N, N' tetraacetic acid (EGTA) sensitivity of Mg2+-activated ATPase. In contrast to the specific removal of Z-lines and of alpha-actinin by CaAF, trypsin treatment of myofibrils results in extensive degradation of myosin heavy chains and of the inhibitory component of troponin (TN-I), and in loss of EGTA sensitivity of myofibrillar ATPase. The degradation of TN-I and loss of EGTA sensitivity occur before the Z-line disappearance.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D009124:Muscle Proteins / Q000032:analysis*; D009132:Muscles / Q000032:analysis / Q000201:enzymology*; D009210:Myofibrils / Q000032:analysis / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D014357:Trypsin
D009124:Muscle Proteins; D004533:Egtazic Acid; D010447:Peptide Hydrolases; D014357:Trypsin; D005033:Ethylmaleimide; D002118:Calcium