Specific cross-linking of the SH1 thiol of skeletal myosin subfragment 1 to F-actin and G-actin.
Recently, we reported that (maleimidobenzoyl)-G-actin (MBS-G-actin), which was resistant to the salt and myosin subfragment 1 (S-1) induced polymerizations, reacts reversibly and covalently in solution with the S-1 heavy chain at or near the strong F-actin binding region [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. Here, we have readily converted the MBS-G-actin into MBS-F-actin in the presence of phalloidin and salts. The binding of S-1 to the two actin derivatives carrying on their surface free reactive maleimidobenzoyl groups was investigated comparatively in cross-linking experiments performed under various conditions to probe further the molecular structure of the actin-heavy chain complex before and after the polymerization process. Like MBS-G-actin, the isolated MBS-F-actin, which did not undergo any intersubunit cross-linking, bound stoichiometrically to S-1, generating two kinds of actin-heavy chain covalent complexes migrating on electrophoretic gels at 180 and 140 kDa. The relative extent of their production was essentially dependent on pH for both G-and F-actins. At pH 8.0, the 180-kDa species was predominant, and at pH 7.0, the amount of the 140-kDa adduct increased at the expense of the 180-kDa entity. The cross-linking of MBS-F-actin to S-1 led to the superactivation of the MgATPase substantiating the ability of this derivative to stimulate the S-1 ATPase as the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Bettache
N
N
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, France.
Bertrand
R
R
Kassab
R
R
D000199:Actins / Q000737:chemistry*; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D009132:Muscles / Q000737:chemistry*; D015879:Myosin Subfragments / Q000737:chemistry*; D011817:Rabbits; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000199:Actins; D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D015879:Myosin Subfragments; D013388:Succinimides; D013438:Sulfhydryl Compounds; C013097:3-maleimidobenzoyl N-hydroxysuccinimide