caa a22 4500 475745329 CHVBK 20180406123511.0 cr unu---uuuuu 170329e20000701xx s 000 0 eng 10.1023/A:1005690106951 doi (NATIONALLICENCE)springer-10.1023/A:1005690106951 A flash photolysis fluorescence/light scattering apparatus for use with sub microgram quantities of muscle proteins [Elektronische Daten] [Stefan Weiss, Igor Chizhov, Michael Geeves] Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However, these methods traditionally require relatively large amounts of protein (10−3 g) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g., Dictyostelium). We have investigated the use of flash photolysis of an inert precursor of ATP (cATP) to initiate the dissociation of acto.S1 and acto.myosin and the subsequent ATP turnover reaction. Using a sample volume of 10 μl we show that a significant amount of information on the transient and steady-state kinetics of the system can be obtained from a sample containing just 50 nM of acto.myosin or acto.S1 complex in solution. Therefore in presence of excess of one protein component the measurements require only 250 ng myosin, 62 ng S1 or 25 ng actin. This is therefore the method of choice for kinetic analysis of acto.myosins which are only available in microgram quantities. We report for the first time the determination of the second order rate constant of ATP-induced dissociation of actin from the myosin extracted from a single fibre from a rabbit psoas muscle. Kluwer Academic Publishers, 2000 Weiss Stefan Research School of Biosciences, University of Kent, Canterbury, CT2 7NJ, Kent, UK aut Chizhov Igor Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany aut Geeves Michael Research School of Biosciences, University of Kent, Canterbury, CT2 7NJ, Kent, UK aut Journal of Muscle Research & Cell Motility Kluwer Academic Publishers 21/5(2000-07-01), 423-432 0142-4319 21:5<423 2000 21 10974 https://doi.org/10.1023/A:1005690106951 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1005690106951 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Weiss Stefan Research School of Biosciences, University of Kent, Canterbury, CT2 7NJ, Kent, UK aut NATIONALLICENCE 700 1- Chizhov Igor Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany aut NATIONALLICENCE 700 1- Geeves Michael Research School of Biosciences, University of Kent, Canterbury, CT2 7NJ, Kent, UK aut NATIONALLICENCE 773 0- Journal of Muscle Research & Cell Motility Kluwer Academic Publishers 21/5(2000-07-01), 423-432 0142-4319 21:5<423 2000 21 10974 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer