Maleimidobenzoyl actin: its biochemical properties and in vitro motility.
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.
Department of Physiology, Nagoya City University Medical School, Mizuho-ku, Aichi.
T
T
Hozumi
M
M
Miki
S
S
Higashi-Fujime
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D008239:Lysine; D008854:Microscopy, Electron; D015879:Myosin Subfragments / Q000378:metabolism; D013388:Succinimides; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism
D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D013388:Succinimides; D014335:Tropomyosin; D014336:Troponin; C066969:maleimidobenzoyl-G-actin; C072252:m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; D000251:Adenosine Triphosphatases; D008239:Lysine; D002118:Calcium