title: Sucrose increases the activation energy barrier for actin-myosin strong binding.
abstract: To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin-myosin (A-M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A-M dissociation, kT (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A-M strong binding by 37±3%, indicating that sucrose inhibits the rate of A-M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A-M rigor binding (71%) are comparable in magnitude to sucrose's effects on both V (79±33% decrease) and maximal actin-activated ATPase, kcat, (81±16% decrease), indicating that the rate of A-M strong bond formation significantly influences both kcat and V.
authors.affiliation: Department of Biomedical Engineering, University of Nevada, Reno, NV, United States.
authors.forename: Del R
authors.initials: del R
authors.lastname: Jackson
authors.affiliation: Department of Biomedical Engineering, University of Nevada, Reno, NV, United States.
authors.forename: Milad
authors.initials: M
authors.lastname: Webb
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV, United States.
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Stewart
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV, United States.
authors.forename: Travis
authors.initials: T
authors.lastname: Phillips
authors.affiliation: Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Carter
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV, United States.
authors.forename: Christine R
authors.initials: CR
authors.lastname: Cremo
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV, United States. Electronic address: jebaker@unr.edu.
authors.forename: Josh E
authors.initials: JE
authors.lastname: Baker
mesh_terms: D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D013395:Sucrose / Q000494:pharmacology*; D013549:Sweetening Agents / Q000494:pharmacology*
chemical_list: D000199:Actins; D013549:Sweetening Agents; D013395:Sucrose; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins
keywords: ATPase; Actin; Binding; Motility; Myosin; Sucrose