Isolation and kinetic characterisation of myosin and myosin S1 from the Drosophila indirect flight muscles

Silva, R. and Sparrow, J.C. and Geeves, M.A. (2003) Isolation and kinetic characterisation of myosin and myosin S1 from the Drosophila indirect flight muscles. J Muscle Res Cell Motil, 24 (8). pp. 489-498. ISSN 0142-4319. (The full text of this publication is not available from this repository)

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Abstract

The potential to explore myosin function through the alternative exons and mutations of the single muscle myosin heavy chain gene, Mhc of Drosophila requires detailed kinetic analysis of the myosins. We have obtained microgram quantities of enzymatically active Drosophila myosin and subfragment 1 (S1) from dissected indirect flight muscles. Using recent developments in stopped-flow and flash-photolysis methods combined with fluorescent/light scattering technologies we have determined some of the key kinetic parameters of actin-myosin and myosin-nucleotide interactions. The rate of ATP-induced dissociation of actin from Drosophila myosin (0.23 microM(-1) s(-1)) and subfragment 1 (S1, 0.82 microM(-1) s(-1)) are both fast and similar to values measured for mammalian skeletal muscle myosins and S1 fragments respectively. The ATP-induced cross bridge dissociation of Drosophila acto.S1 is expected to be fast since, for a rapidly contracting muscle like the Drosophila flight muscle, the post power stroke cross bridge must detach rapidly from actin or become a drag on the contracting filament. ATP-induced detachment is preceded by ADP release and this is proposed as the rate-limiting step that defines muscle shortening velocity. We show that the affinity of ADP for acto.S1 at 400 microM is 2-3 fold weaker than fast vertebrate myosins. This leads to an estimate of the ADP release rate constant of 4000 s(-1). We show that this predicts a maximum shortening velocity very similar to that obtained from in vivo estimates of indirect flight muscle shortening. The data is therefore compatible with ADP dissociation limiting the in vivo shortening velocity.

Item Type: Article
Additional information: 0142-4319 (Print) Journal Article
Uncontrolled keywords: Actins/metabolism Actomyosin/*metabolism Adenosine Diphosphate/metabolism Adenosine Triphosphate/metabolism Animals Drosophila/*enzymology Muscles/*enzymology Myosin Subfragments/*metabolism Myosins/*metabolism Protein Binding Rabbits 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 16:25
Last Modified: 14 Mar 2009 16:25
Resource URI: http://kar.kent.ac.uk/id/eprint/13196 (The current URI for this page, for reference purposes)
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