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Maleimidobenzoyl actin: its biochemical properties and in vitro motility
T Hozumi1, M Miki, S Higashi-Fujime
1Department of Physiology, Nagoya City University Medical School, Mizuho-ku, Aichi.
Abstract:
Muscle G-actin treated with the hetero-bifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), denoted as MBS-G-actin, is not induced to polymerize into F-actin by salt and myosin subfragment 1 [Bettache, N., Bertrand, R., and Kassab, R. (1989) Proc. Natl. Acad. Sci. USA 86, 6028-6032]. However, the addition of salt and phalloidin together allowed MBS-G-actin to polymerize and the resulting polymer (P-MBS-G-actin) could activate the Mg2+ -ATPase of S-1 [Miki, M. and Hozumi, T. (1991) Biochemistry 30, 5625-5630]. When F-actin was treated with MBS (MBS-F-actin), unlike MBS-G-actin, intercross-links between monomers in F-actin occurred. The MBS-F-actin could activate the Mg2+ -ATPase of heavy meromyosin (HMM): its maximum turnover rate, Vmax, was almost the same as that of native F-actin, but the affinity of HMM for MBS-F-actin in the presence of ATP, Km, was about 3 times higher. Electron microscopy showed that both P-MBS-G-actin and MBS-F-actin had the double stranded structures of F-actin and formed the arrowhead structures when combined with HMM. By in vitro motility assay, the sliding velocities of P-MBS-G-actin and MBS-F-actin were found to be slightly slower than that of native F-actin. But the critical concentration of KCl, over which the sliding movement was not observed, for MBS-modified actins was considerably higher than for native F-actin. When MBS-modified actins were regulated by tropomyosin-troponin complex, they were less sensitive to the Ca2+ concentration for HMM ATPase activation and sliding movement. These results showed that the modification of some of the lysine residues in the actin molecule leads to change in the biochemical properties of F-actin.
Insights
Modification of muscle actin with MBS alters its polymerization and biochemical properties, affecting muscle contraction regulation. MBS-G-actin polymerization requires phalloidin, and MBS-F-actin shows altered myosin binding affinity and Ca2+ sensitivity.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Muscle actin polymerization is crucial for muscle contraction.
- Chemical modification of actin can reveal insights into its structure-function relationships.
- Previous studies showed MBS-G-actin polymerization is salt- and myosin subfragment 1-independent.
Purpose of the Study:
- To investigate the effects of modifying actin's lysine residues with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) on its polymerization and biochemical properties.
- To understand how MBS modification impacts actin's interaction with myosin and its regulation by tropomyosin-troponin.
Main Methods:
- Treatment of G-actin and F-actin with MBS.
- Polymerization assays with salt, phalloidin, and myosin subfragment 1 (S-1).
- Enzyme kinetic assays (Mg2+-ATPase activity of S-1 and heavy meromyosin (HMM)).
- Electron microscopy for structural analysis.
- In vitro motility assays to measure sliding velocity.
- Ca2+-dependent regulation studies with tropomyosin-troponin.
Main Results:
- MBS-G-actin polymerization into F-actin required phalloidin.
- MBS-F-actin exhibited similar Mg2+-ATPase Vmax with HMM as native F-actin, but a 3-fold higher Km.
- Both polymerized MBS-actins formed F-actin-like structures and arrowhead complexes with HMM.
- Sliding velocities of MBS-actins were slightly slower than native F-actin, with a higher critical concentration for movement.
- MBS-modified actins showed reduced Ca2+ sensitivity in tropomyosin-troponin regulated assays.
Conclusions:
- Modification of lysine residues in actin with MBS alters its polymerization behavior and biochemical characteristics.
- MBS-induced changes in actin affect its interaction with myosin and its calcium-dependent regulation.
- These findings highlight the importance of specific lysine residues in maintaining actin's native functional properties.