Development of tools for the kinetic study of myosin

• Main Author: Rezavandi, Heresh
• Published: University of Kent 2011
• Subjects: 572.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594229

Rapid reaction techniques such as the stopped-flow apparatus have been fundamental in elucidating the molecular events in the myosin cross bridge cycle. The use compounds that regulate the ATPase activity of myosin (myosin regulators) are valuable tools in myosin research and have the potential to be lead compounds in the development of therapeutic agents. The advances in our knowledge of myosin are owed to the development of these scientific tools. There are many important myosin isofonns that remain to be kinetically characterised. This is largely due to the amounts of these myosins available, the concentration of myosin and the volumes of solution required to perform transient kinetic characterisations with the stopped-flow apparatus. Therefore in collaboration with TgK Scientific we have investigated methods to reduce the amount of solution required to perform an experiment (sample volume) with the stopped-flow apparatus from 500 )ll to 50-100 )ll. We have developed external components (MVCs) that attach to the existing SF- 61DX2 stopped-flow apparatus that demonstrate it is feasible to reduce the sample volume without having to redesign and build a stopped-flow apparatus from new. The stopped-flow apparatus was used for the kinetic characterisation of the mouse cardiac S I a isoform (mcSia). The actin affinity for mcSIa., ADP affinity for acto.mcSla., ATP binding to acto.mcS I a and the ATP hydrolysis step for mcS 10. were found to be more similar to a fast MHC-I I- 2X isofonn rather than a slow MHC-II-I isoform, despite mcS la shari ng greater sequence homology to a MHC-II-1 isoform. It has been reported that the compound Resveratrol has a potentiating effect on both the basal activated ATPase activity and the actin activated ATPase activity. It was found that Resveratrol did not affect the stoichiometry between actin and S I, neither with the ATP induced dissociation of acto.SI nor with the actin activated ATPase and the steady state activated ATPase activity. Instead Resveratrol may weakly inhibit the basal activity. Thus the results suggest that there is no potentiating effect by Resveratrol and an inhibitory effect remains to be determ ined.