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

Abstract

1. Trinitrophenyl AMP (TNP_AMP) in the concentration range 10-300 ìÒ induced an increase in fluorescence intensity at around 530 nm in skinned skeletal muscle fibres freshly obtained from rat psoas muscle. 2. The fluorescence intensity of the fibres depended on TNP_AMP concentration up to•200 ìÒ. The Kd of TNP_AMP binding to the muscle fibres was 38·0 ± 8·4 ìÒ (mean ± s.d., n = 4 measurements) in three fibres. TNP_AMP fluorescence was readily washed out. 3. Various nucleotides affected the fluorescence of the fibres incubated in 20 ìÒ TNP_AMP.MgATP (1 mÒ) and caged ATP (5 mÒ) reduced the fluorescence in 20 ìÒ TNP_AMP by more than 40 % of the value measured in the absence of nucleotide. 4. When the fibres were stretched to almost no filament overlap, the extent of the quenching of the TNP_AMP (20 ìÒ) 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. 5. 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. 6. 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.8726