In previous work, we presented evidence that Ca2+ activation of skeletal myofilaments depends on a specific actin domain. We showed that rabbit skeletal thin filaments reconstituted with actin modified at Lys-237 activate heavy meromyosin·Mg2+-ATPase activity independently of the Ca2+ ion concentration. The modification, which apparently blocks the inhibitory effects of troponin-tropomyosin (Tn·Tm), on acto-heavy meromyosin·Mg2+-ATPase activity, consisted of conversion of Lys-237 to an enamine by reaction of purified actin with 2,4-pentanedione (PD). In experiments reported here, we have treated myofibrils with PD with the idea of altering actin in its native state within the myofilament lattice. Preparations of native and Tn·Tm free ('desensitized') myofibrils were incubated with PD (100 mol/mol of actin lysine) under rigorous conditions (10 mM 4-morpholinepropanesulfonic acid, pH 7.0, 2.0 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.4 mM dithiothreitol, and 0.15 mM NaN3). Actin isolated from PD·myofibrils contained 0.5 mol of enamine/mol. In the presence of Ca2+, the Mg2+-ATPase activity of PD-treated myofibrils was 110-120% of the maximum Ca2+-stimulated Mg2+-ATPase activity of untreated control myofibrils. In low free Ca2+ (pCa > 8), the Mg2+-ATPase activity of the PD-treated myofibrils was not suppressed and remained at 100-106% of the maximum activity of the control myofibrils. Ca2+ sensitivity of the PD-treated myofibrils was restored following treatment with hydroxylamine, which hydrolyzes enamine's products. Preparations of desensitized myofibrils reconstituted with PD-modified or unmodified Tn·Tm demonstrated the same Ca2+-sensitive ATPase activities. On the other hand, preparations reconstituted with unmodified or PD-modified Tn·Tm and PD-modified desensitized myofibrils were insensitive to Ca2+ ion concentration. The Mg2+-ATPase activity of preparations of myosin treated with PD was not activated by modified or unmodified actin. Our results indicate that it is possible to produce an active state(s) of the myofibrils in the absence and presence of Ca2+ by specific alteration of the actin·Tm interaction following modification of myofibrillar actin most likely at Lys-237.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1986|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology