title: Actomyosin interaction at low ATP concentrations.
abstract: In vitro motility assay (IVMA) experiments were performed to analyze the movement of actin filaments sliding on a pavement of myosin molecules at different [ATP] and [ADP]. In standard experimental conditions at [ATP] = 2 mM, about 80% of the actin filaments move in unloaded conditions with a constant velocity. However, a fraction of at least 20% static actin filaments is always present. The accepted explanation is the occurrence of damaged "rigor"-like myosin heads that do not undergo the normal ATP-dependent cycling motion. However, in a series of IVMA experiments performed at different [ATP] we observed that the mobility of actin filaments increased with lowering [ATP]. We investigated the influence of [ATP] on the number of mobile actin filaments. IVMA experiments were performed at controlled nucleotide concentrations and the percentage of mobile filaments accurately determined by specific operator-guided software. The value of ΔGATPinvolved was determined. Results showed that the number of mobile actin filaments sliding on type 2B heavy meromyosin isoform (2B HMM) increased at very low [ATP] accompanied by less negative ΔGATPvalues. Similar results were obtained by increasing [ADP]. Performing experiments at the same [ATP] with different myosin types, we found a higher number of mobile actin filaments on slow type 1 HMM with respect to type 2B HMM while the highest number of mobile actin filaments was found on single-head myosin (S1 fraction). We also found that [ATP] did not influence the percentage of mobile actin filaments sliding on S1. Our results reveal novel aspects of actomyosin interaction.
authors.affiliation: Department of Molecular Medicine, University of Pavia, Via Forlanini 6, 27100, Pavia, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Maffei
authors.affiliation: Fondazione Salvatore Maugeri (IRCCS), Scientific Institute of Pavia, Pavia, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Longa
authors.affiliation: Department of Chemistry, University of Firenze, Sesto Fiorentino, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Sabatini
authors.affiliation: Department of Chemistry, University of Firenze, Sesto Fiorentino, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Vacca
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, Via San Donatoi 15, 40127, Bologna, Italy. stefano.iotti@unibo.it.|National Institute of Biostructures and Biosystems, Rome, Italy. stefano.iotti@unibo.it.
authors.forename: Stefano
authors.identifier: http://orcid.org/0000-0003-1426-6400
authors.initials: S
authors.lastname: Iotti
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D006868:Hydrolysis; D009038:Motion; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D013816:Thermodynamics
chemical_list: D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D000205:Actomyosin
keywords: ATP concentration; ATP hydrolysis; Actomyosin interaction; Gibbs free energy; In vitro motility assay