Key Points
- Investigate nucleotide-induced, site-specific fluorescent modification of myosin subfragment-1 using the serine-reactive reagent 9-anthroylnitrile.
- Incubated myosin subfragment-1 (S-1) with the serine-reactive probe 9-anthroylnitrile (ANN) in the presence and absence of ATP and ADP.
- Characterized fluorescence and absorption spectra and quantified alterations in K+-, Ca2+-, and Mg2+-ATPase activity.
- Mapped the modified residue location through tryptic cleavage of labeled S-1 into 23-kDa, 50-kDa, and 20-kDa peptide fragments.
- ATP exposure enabled maximal incorporation of 0.98 mol of 9-anthroyl group per mole of S-1, producing an emission maximum at 462 nm characteristic of serine adducts.
- Incorporation of 0.98 mol of the label reduced K+- and Ca2+-ATPase activities to 30% of original values while increasing Mg2+-ATPase activity to 220%.
- Tryptic digestion isolated the 9-anthroyl label exclusively on the NH2-terminal 23-kDa peptide of the heavy chain, leaving 20-kDa and 50-kDa fragments unlabeled.
Structured PICO
PPopulationMyosin subfragment-1 (S-1) and various amino acids
IInterventionIncubation with the fluorescent reagent 9-anthroylnitrile (ANN) in the presence of ATP and ADP
OOutcomeCovalent incorporation of the 9-anthroyl (AN) group into the S-1 heavy chain and resulting ATPase activitiessurrogate
The fluorescent reagent 9-anthroylnitrile specifically labels a serine residue in the 23-kDa tryptic peptide of the myosin S-1 heavy chain, a reaction enhanced by nucleotides.