The principle aim of the work proposed here is to determine which step or step(s) of the nucleotide triphosphate hydrolysis mechanism limits shortening velocity in muscle. Recent work in my laboratory has shown that ADP dissociation from actomyosin-S1 is sufficiently slow to be the molecular step that limits unloaded shortening in mammalian muscle. To further test this hypothesis the rate constants of the dissociation of a series of nucleoside disphosphates (NDPs) from actomyosin-S1, will be measured using the stopped flow light scattering method. We have shown in preliminary studies that the complementary series of nucleoside triphosphates (NTPs) differ widely in their ability to support shortening and motility as measured in skinned muscle fibers and using the in vitro motility assay of Kron and Spudich. NTPs that poorly support contraction and motility, GTP and lTP, show novel steady state kinetics in which the rate of hydrolysis is strongly inhibited at moderately high actin concentrations. This indicates that one or more steps of the mechanism between attached crossbridge state is much slower for NTPs that support contraction poorly than for NTPs that support contraction well. It therefore appears that a sufficiently rapid rate of a critical attached crossbridge step is essential support normal contraction. We propose to use the methods of stopped-flow fluorescence, rapid chemical quenching, and intermediate exchange to determine which step or steps of the actomyosin hydrolysis mechanisms are changed for a series of NTPs. Altering the structure of the nucleotide substrates provides a critical test of which step(s) of the actomyosin NTP hydrolysis mechanism are essential for contraction and/or limit shortening velocity. An increased understanding of mechanism by which the chemical energy of nucleotide triphosphate hydrolysis is coupled to the production of mechanical work in muscle will improve our understanding of the molecular mechanisms that underly some of the alterations in contractility observed in heart disease and other muscle disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL041776-01
Application #
3359516
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1988-12-01
Project End
1993-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Eastern Virginia Medical School
Department
Type
Schools of Medicine
DUNS #
City
Norfolk
State
VA
Country
United States
Zip Code
23501
Heeley, David H; Belknap, Betty; White, Howard D (2006) Maximal activation of skeletal muscle thin filaments requires both rigor myosin S1 and calcium. J Biol Chem 281:668-76
Heeley, David H; Belknap, Betty; White, Howard D (2002) Mechanism of regulation of phosphate dissociation from actomyosin-ADP-Pi by thin filament proteins. Proc Natl Acad Sci U S A 99:16731-6
Ikebe, R; Reardon, S; Mitsui, T et al. (1999) Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase. J Biol Chem 274:30122-6
Kambara, T; Rhodes, T E; Ikebe, R et al. (1999) Functional significance of the conserved residues in the flexible hinge region of the myosin motor domain. J Biol Chem 274:16400-6
White, H D; Belknap, B; Webb, M R (1997) Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate. Biochemistry 36:11828-36
Persechini, A; White, H D; Gansz, K J (1996) Different mechanisms for Ca2+ dissociation from complexes of calmodulin with nitric oxide synthase or myosin light chain kinase. J Biol Chem 271:62-7
Walker, M; Trinick, J; White, H (1995) Millisecond time resolution electron cryo-microscopy of the M-ATP transient kinetic state of the acto-myosin ATPase. Biophys J 68:87S-91S
Walker, M; White, H; Belknap, B et al. (1994) Electron cryomicroscopy of acto-myosin-S1 during steady-state ATP hydrolysis. Biophys J 66:1563-72
Cooke, R; White, H; Pate, E (1994) A model of the release of myosin heads from actin in rapidly contracting muscle fibers. Biophys J 66:778-88
Pate, E; Franks-Skiba, K; White, H et al. (1993) The use of differing nucleotides to investigate cross-bridge kinetics. J Biol Chem 268:10046-53

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