Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase.
Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.
D000185:Actinin / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain; D002413:Cations, Divalent / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D008566:Membranes / Q000187:drug effects; D008673:Metals, Alkaline Earth / Q000494:pharmacology; D008857:Microscopy, Interference; D008858:Microscopy, Phase-Contrast; D009124:Muscle Proteins / Q000378:metabolism*; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D013324:Strontium / Q000494:pharmacology
D002413:Cations, Divalent; D008673:Metals, Alkaline Earth; D009124:Muscle Proteins; D000185:Actinin; D004533:Egtazic Acid; D010450:Endopeptidases; D002154:Calpain; D002118:Calcium; D013324:Strontium