[Cleavage of DNA-binding loops of actin by subtilisin prevent formation of a strong type of myosin binding with actin].
In order to elucidate the role of DNA-binding loop of actin (amino acid residues 38-52) in mechanisms of muscle contraction, polarizational fluorimetry and ghost muscle fibers, containing thin filaments reconstructed by intact and subtilisin-cleaved G-actin were used. The thin filaments were modified by fluorescent probes rhodamin-phalloidin and 1,5-IAEDANS. Changes in orientation and mobility of the probes were considered as an indication of changes in actin conformation. The stage AM of ATP hydrolysis cycle was simulated. For this purpose, thin filaments were decorated by myosin subfragment-1 (S1) in the absence of nucleotide. It has been shown that S1 binding to actin is accompanied by changes in orientation and mobility of the fluorescent probes. For intact filaments, the changes of these parameters indicate the formation of a strong binding between S1 and actin. Cleavage of DNA-binding loop by subtilisin markedly inhibits this effect. The cleavage of actin by subtilisin has also been shown to diminish the changes in fiber birefringence, which takes place at the formation of F-actin-S1 complex in the muscle fiber. The spatial organization of the actin DNA-binding loop is suggested to play an important role in determining the character of myosin interaction with actin in the ATP hydrolysis cycle.
Khoroshev
M I
MI
Institute of Cytology RAS, St. Petersburg, Russia.
Morachevska
I
I
Strzhelska-Golashevska
Kh
Kh
Borovikov
Iu S
IuS
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008854:Microscopy, Electron; D009218:Myosins / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011485:Protein Binding; D013381:Subtilisins / Q000378:metabolism*
D000199:Actins; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D004247:DNA; D013381:Subtilisins; D009218:Myosins
Rasshcheplenie subtilizinom DNK-sviazyvaiushcheĭ petli aktina predotvrashchaet formirovanie sil'noĭ formy sviazyvaniia miozina s aktinom.