title: Actin cross-linking and inhibition of the actomyosin motor.
abstract: Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.
authors.affiliation: Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
authors.forename: Eldar
authors.initials: E
authors.lastname: Kim
authors.forename: Elena
authors.initials: E
authors.lastname: Bobkova
authors.forename: György
authors.initials: G
authors.lastname: Hegyi
authors.forename: Andras
authors.initials: A
authors.lastname: Muhlrad
authors.forename: Emil
authors.initials: E
authors.lastname: Reisler
mesh_terms: D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000502:physiology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D066298:In Vitro Techniques; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000737:chemistry; D009218:Myosins / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D005973:Glutamine; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D009218:Myosins; D008239:Lysine; D003545:Cysteine