title: Measurement of nucleotide release kinetics in single skeletal muscle myofibrils during isometric and isovelocity contractions using fluorescence microscopy.
abstract: Rabbit psoas muscle myofibrils, in the presence of the fluorescent nucleotide analog 2'(3')-O-[N-[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP), showed selective fluorescence staining of the A-band with a reduced fluorescence at the M-line. Addition of Cy3-EDA-ATP to a myofibril in the presence of Ca2+ caused auxotonic shortening against a compliant glass microneedle. These results indicate that Cy3-EDA-ATP is a substrate for myosin in the myofibril system. The kinetics of nucleotide release from a single myofibril, held isometrically between two needles, were measured by the displacement of prebound Cy3-EDA-ATP on flash photolysis of caged ATP. The A-band fluorescence of the myofibril decayed exponentially with a rate constant of 0.3 s(-1) at 8 degrees C, an order of magnitude faster than that for isolated thick filaments in the absence of actin. When a myofibril was imposed to shorten with a constant velocity by a piezoelectric actuator, the nucleotide displacement rate constant initially increased to 0.7 s(-1) with increasing shortening velocity and then declined with a further increase in shortening velocity. These results demonstrate that the displacement rates of Cy3-EDA-nucleotides bound to the cross-bridges in the contracting myofibril reflect a process that shows strain dependence.
authors.affiliation: Department of Physiology, School of Medicine, Teikyo University, Tokyo, Japan. chaen@med.teikyo-u.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Chaen
authors.forename: I
authors.initials: I
authors.lastname: Shirakawa
authors.forename: C R
authors.initials: CR
authors.lastname: Bagshaw
authors.forename: H
authors.initials: H
authors.lastname: Sugi
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D001703:Biophysics; D005456:Fluorescent Dyes / Q000378:metabolism*; D066298:In Vitro Techniques; D007211:Indoles / Q000378:metabolism*; D007537:Isometric Contraction / Q000502:physiology; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D009119:Muscle Contraction / Q000502:physiology*; D009210:Myofibrils / Q000378:metabolism / Q000502:physiology; D016658:Psoas Muscles / Q000378:metabolism* / Q000502:physiology*; D011817:Rabbits
chemical_list: C106705:3'(2')-O-(N-(2-((2-((1-ethyl-3,3-dimethyl-5-sulfoindol-2-yl)prop-2-enylidene)-3,3-dimethyl-5-sulfoindol-1-yl)amino)ethyl)carbamoyl)-adenosine triphosphate; D005456:Fluorescent Dyes; D007211:Indoles; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases