tailieunhanh - Báo cáo khoa học: Equilibrium distribution of skeletal actin–tropomyosin– troponin states, determined by pyrene–tropomyosin fluorescence

Actin–tropomyosin–troponin has three structural states, but the functional properties of regulation can be explained with models having two func-tional states. As a step towards assigning functional properties to all the structural states, we examined fluorescent probes that monitor changes in troponin and tropomyosin. | ỊFEBS Journal Equilibrium distribution of skeletal actin-tropomyosin-troponin states determined by pyrene-tropomyosin fluorescence Boris Gafurov1 and Joseph M. Chalovich2 1 Uniformed Services University of the Health Sciences Department of Pharmacology Bethesda MD USA 2 Department of Biochemistry and Molecular Biology Brody Schoolof Medicine at East Carolina University Greenville NC USA Keywords parallel pathway model pyrene iodoacetamide regulation of contraction tropomyosin troponin Correspondence Joseph M. Chalovich Department of Biochemistry and Molecular Biology Brody Schoolof Medicine at East Carolina University 5E-122 Brody Bldg Greenville NC 27834 USA Fax 1 252 7443383 Tel 1 252 7442973 E-mail chalovichj@ Website http biochemistry Received 11 December 2006 revised 10 February 2007 accepted 1 March 2007 doi Actin-tropomyosin-troponin has three structural states but the functional properties of regulation can be explained with models having two functional states. As a step towards assigning functional properties to all the structural states we examined fluorescent probes that monitor changes in troponin and tropomyosin. Tropomyosin labeled with pyrene-iodoacetamide is thought to reflect the transition to the most active state whereas N- 2-iodoacetoxy ethyl -N-methyl -amino-7-nitrobenz-2-oxa-1 3-diazole-labeled troponin I is thought to monitor the transition to any state other than the inactive state. The fraction of actin in an active state determined from pyrene excimer fluoresecence agreed with that calculated from lightscattering measurements of myosin subfragment 1 S1 -ADP to regulated actin in both the presence and absence of Ca2 over a range of ionic strength conditions. The only exceptions were conditions where the binding of S1-ADP to actin was too strong to measure accurately. Pyrene-tropo-myosin excimer fluorescence was Ca2 dependent and so reflected the change in population caused by .