Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue.

Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. Research Abstract Details 

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Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. Abstract Text:

Eva Forgacs,Suzanne Cartwright, ,Takeshi Sakamoto,James R Sellers,John E T Corrie,Martin R Webb,Howard D White,

We have used a new fluorescent ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine-5'-triphosphate (deac-aminoATP), to study the ATP hydrolysis mechanism of the single headed myosinV-S1. Our study demonstrates that deac-aminoATP is an excellent substrate for these studies. Although the deac-amino nucleotides have a low quantum yield in free solution, there is a very large increase in fluorescence emission ( approximately 20-fold) upon binding to the myosinV active site. The fluorescence emission intensity is independent of the hydrolysis state of the nucleotide bound to myosinV-S1. The very good signal-to-noise ratio that is obtained with deac-amino nucleotides makes them excellent substrates for studying expressed proteins that can only be isolated in small quantities. The combination of the fast rate of binding and the favorable signal-to-noise ratio also allows deac-nucleotides to be used in chase experiments to determine the kinetics of ADP and Pi dissociation from actomyosin-ADP-Pi. Although phosphate dissociation from actomyosinV-ADP-Pi does not itself produce a fluorescence signal, it produces a lag in the signal for deac-aminoADP dissociation. The lag provides direct evidence that the principal pathway of product dissociation from actomyosinV-ADP-Pi is an ordered mechanism in which phosphate precedes ADP. Although the mechanism of hydrolysis of deac-aminoATP by (acto)myosinV-S1 is qualitatively similar to the ATP hydrolysis mechanism, there are significant differences in some of the rate constants. Deac-aminoATP binds 3-fold faster to myosinV-S1, and the rate of deac-aminoADP dissociation from actomyosinV-S1 is 20-fold slower. Deac-aminoATP supports motility by myosinV-HMM on actin at a rate consistent with the slower rate of deac-aminoADP dissociation.

Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. Publishing Authors By Initials

E Forgacs,S Cartwright,M ,T Sakamoto,JR Sellers,JE Corrie,MR Webb,HD White,

For similar investigative techniques: chemistry, analytical: photometry: luminescent measurements: fluorometry: spectrometry, fluorescence research abstracts see: investigative techniques: chemistry, analytical: photometry: luminescent measurements: fluorometry: spectrometry, fluorescence research

PUBMED ID PMID:

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Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemistry

VOLUME: 45

Page Numbers: 13035-45

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 31

MONTH: Oct

YEAR: 2006

Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 370623

Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. Keywords Mesh Terms:

KEYWORDS: Spectrometry, Fluorescence

MESH TERMS: methods

Chemical & Substance for Abstract: Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue. Information

Substance Name: Myosins

Registry Number: EC 3.6.1.4

Grant and Affiliation Information for Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue.

AFFILIATION: Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.

Country: United States

AGENCY: United States NIBIB

GRANT: EB00209

ACRONYM: EB

MEDLINETA: Biochemistry

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