title: Nucleotide-induced specific fluorescent labeling of the 23-kDa NH2-terminal tryptic peptide of myosin ATPase by the serine-reactive reagent 9-anthroylnitrile.
abstract: The fluorescent reagent 9-anthroylnitrile (ANN) reacted preferentially with serine among various amino acids tested. When the myosin subfragment-1 (S-1) was incubated with ANN, the 9-anthroyl (AN) group was covalently incorporated into the S-1 heavy chain. The incorporation of the AN group was enhanced by the presence of ATP and ADP. In the presence of ATP, 0.98 mol of the AN group was maximally incorporated into S-1. The resulting S-1 derivative exhibited four absorption maxima in the range of 300-400 nm and fluoresced strongly with an emission maximum at 462 nm upon excitation at 390 nm. The spectral properties were similar to those of the AN-derivatives of serine and polyserine. When 0.98 mol of the AN group was incorporated into S-1, the K+- and Ca2+-ATPase activities decreased to 30%, while the Mg2+-ATPase activity increased to 220% of the original value. Tryptic digestion of the labeled S-1 revealed that the AN group was attached only to the NH2-terminal 23-kDa tryptic peptide of the S-1 heavy chain. Neither the 20-nor the 50-kDa peptide was labeled with ANN. The results suggest that a serine residue, which becomes more reactive in the presence of the nucleotide, is located in the 23-kDa tryptic peptide of S-1.
authors.affiliation: Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Hiratsuka
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012694:Serine; D013050:Spectrometry, Fluorescence; D014357:Trypsin
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D010446:Peptide Fragments; D012694:Serine; D014357:Trypsin; D009218:Myosins