Probing nucleotide dissociation from myosin in vitro using microgram quantities of myosin

Clark, R.J.B. and Nyitrai, M. and Webb, M.R. and Geeves, M.A. (2003) Probing nucleotide dissociation from myosin in vitro using microgram quantities of myosin. J Muscle Res Cell Motility, 24 (4-6). pp. 315-321. ISSN 0142-4319. (The full text of this publication is not available from this repository)

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Abstract

The detailed kinetic analysis of novel myosin motors is often limited by the quantity of stable protein available for study. We show here that the use of coumarin based fluorescent ADP analogues allows the assay of ADP affinities and dissociation rate constants in a flash photolysis apparatus using microg quantities of the rabbit muscle myosin S1. We go on to use the analogues to characterise two other rat muscle myosin S1 and the motor domain of Dictyostelium cytoplasmic myosin II. The results show that the fluorescence change for the binding of a coumarin based ADP analogue to a myosin motor domain is variable in sign as well as amplitude for the different proteins. The analysis also provided estimates of the affinities of caged-ATP for S1 which were < or = 10 microM for muscle S1s and > 200 microM for the non-muscle myosin.

Item Type: Article
Additional information: 0142-4319 (Print) Journal Article
Uncontrolled keywords: Adenosine Diphosphate/analogs & derivatives/metabolism/pharmacology Animals Coumarins/metabolism/pharmacology Myosins/administration & dosage/*metabolism Nucleotides/*metabolism Protein Binding/drug effects/physiology Rabbits Rats Research Support, Non-U.S. Gov't
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: Michael Geeves
Date Deposited: 14 Mar 2009 16:09
Last Modified: 14 Mar 2009 16:27
Resource URI: http://kar.kent.ac.uk/id/eprint/13192 (The current URI for this page, for reference purposes)
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