Studies of the alpha-actinin/actin interaction in the Z-disk by using calpain.
Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.
Goll
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DE
Muscle Biology Group, University of Arizona, Tucson 85721.
Dayton
W R
WR
Singh
I
I
Robson
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RM
D000185:Actinin / Q000302:isolation & purification / Q000378:metabolism*; D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002154:Calpain / Q000494:pharmacology*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009210:Myofibrils / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits; D013552:Swine
D000199:Actins; D000596:Amino Acids; D000185:Actinin; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D043424:Carboxypeptidase B; D002154:Calpain