What question did this study set out to answer?

To evaluate whether ribose-modified fluorescent nucleotide analogs, in combination with vanadate, can serve as affinity labels for probing the myosin ATPase active site.

January 1, 1984The Journal of Biochemistry

Affinity Labeling of the Myosin ATPase with Ribose-Modffied fluorescent Nucleotides and Vanadate

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Population

Myosin ATPase and myosin subfragment-1 (S-1)

Comparison

Ribose-modified fluorescent nucleotide analogs… vs Natural nucleoside triphosphates (NTPs)

Design

Preclinical

Authors

THToshiaki HiratsukaAsahikawa Medical College Hospital

Discussion

Member takes

Implication

May aid myosin labeling in basic research; leaves open translation to cardiac muscle studies or therapy.

Key Points

To evaluate whether ribose-modified fluorescent nucleotide analogs, in combination with vanadate, can serve as affinity labels for probing the myosin ATPase active site.
Synthesized 3′-O-anthraniloyl and 3′-O-(N-methylanthraniloyl) derivatives of multiple nucleoside triphosphates (NTPs) and diphosphates (NDPs).
Measured actomyosin superprecipitation and myosin subfragment-1 (S-1) ATPase activity in the presence of 1 mM vanadate ion.
Isolated labeled S-1 complexes using gel filtration and evaluated binding stability via native and dissociating polyacrylamide gel electrophoresis.
Exposure of S-1 ATPase to fluorescent NTPs and 1 mM vanadate resulted in progressive enzymatic inhibition exceeding 60% over 1 hour, with apparent second-order rate constants of 0.14–0.44 M⁻¹·s⁻¹.
Gel filtration yielded stable fluorescent NDP-labeled S-1 complexes with stoichiometries of 0.6–1.1 analog per active site that dissociated under denaturing electrophoresis, demonstrating non-covalent association.
Affinity labeling was abolished by excess ATP and ADP but not by AMP, establishing that the pyrophosphate moiety is essential for complex formation while ribose hydroxyl groups are not.

Structured PICO

Population

Myosin ATPase and myosin subfragment-1 (S-1)

Intervention

Ribose-modified fluorescent nucleotide analogs (3′-O-anthraniloyl and 3′-O-(N-methylanthraniloyl) derivatives of AT(D)P, CIAT(D)P, CT(D)P, UT(D)P, IT(D)P, and GT(D)P) and vanadate ion (V1)

Comparator

Natural nucleoside triphosphates (NTPs)

Outcome

Formation of stable myosin-NDP-V1 complex and inhibition of S-1 ATPasesurrogate

Ribose-modified fluorescent nucleotide analogs and vanadate function together as stable, non-covalent fluorescent affinity labels for the myosin active site.

Cite This Study

Toshiaki Hiratsuka (1984) studied this question.

synapsesocial.com/papers/6a7142cf5d37378ac1de6a63 https://doi.org/10.1093/oxfordjournals.jbchem.a134806

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Affinity Labeling of the Myosin ATPase with Ribose-Modffied fluorescent Nucleotides and Vanadate

Discussion

Member takes

Implication

Key Points

Structured PICO

Cite This Study

Affinity Labeling of the Myosin ATPase with Ribose-Modffied fluorescent Nucleotides and Vanadate

Discussion

Member takes

Implication

Key Points

Structured PICO

Cite This Study

Also Consider

Also Consider