HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fujita, S. Articles by Yamada, K. Search for Related Content PubMed PubMed Citation Articles by Fujita, S. Articles by Yamada, K.

Fluorescence changes of a label attached near the myosin active site on nucleotide binding in rat skeletal muscle fibres

Suguru Fujita, Tomoko Nawata and Kazuhiro Yamada

Trinitrophenyl AMP (TNP-AMP) in the concentration range 10-300 µM induced an increase in fluorescence intensity at around 530 nm in skinned skeletal muscle fibres freshly obtained from rat psoas muscle.

The fluorescence intensity of the fibres depended on TNP-AMP concentration up to ~200 µM. The K_d of TNP-AMP binding to the muscle fibres was 38·0 ± 8·4 µM (mean ± s.d., n = 4 measurements) in three fibres. TNP-AMP fluorescence was readily washed out.

Various nucleotides affected the fluorescence of the fibres incubated in 20 µM TNP-AMP. MgATP (1 mM) and caged ATP (5 mM) reduced the fluorescence in 20 µM TNP-AMP by more than 40 % of the value measured in the absence of nucleotide.

When the fibres were stretched to almost no filament overlap, the extent of the quenching of the TNP-AMP (20 µM) fluorescence due to ATP binding was reduced by 14 %. This might be explained by assuming that the association of the thin filament affected the TNP-AMP fluorescence in muscle fibres.

The distance between the active site and the specific site for TNP was measured by the fluorescence resonance energy transfer between N-methylanthraniloyl-ATP (Mant-ATP) bound to the active site and the TNP-AMP bound to the TNP-specific site in muscle fibres. The results showed that the distance between the two may be less than 2 nm.

It may be concluded that the fluorescence intensity at 530 nm in skinned muscle fibres in low concentrations of TNP-AMP changes directly reflecting the conformational state of the nucleotide-binding region that is determined by the binding of nucleotides.