8-Anilino-1-naphthalenesulphonate, a fluorescent probe for the regulatory light chain binding site of scallop myosin.
Regulatory light chain (RLC) dissociation from scallop myofibrils, myosin or its subfragments was accompanied by an increase in binding of the hydrophobic fluorophore, 8-anilino-1-naphthalene-sulphonate (ANS) to the denuded proteins. The binding was monitored by the large increase in fluorescence emission at 460 nm when excited directly at 380 nm or via energy transfer from nearby tryptophan residues at 295 nm. ANS thus provides a convenient probe for following the kinetics of RLC dissociation in the presence of EDTA and its association in the presence of divalent metal ions. The observed RLC dissociation rate constant for myosin at 20 degrees C was 7.5 X 10(-3)S-1. The association rate constant, which was independent of the RLC concentration, was 5 X 10(-3) S-1. Subfragment 1, prepared by digestion of myosin in the presence of divalent metal ions to protect the light chains [S1(+LC)], showed reversible ANS binding qualitatively similar to the parent molecule. However when prepared in the presence of EDTA, subfragment 1 lacked RLC [S1(-LC)], its heavy chain molecular weight was reduced by about 4000 and it lacked the ANS binding region attributed to the RLC site. The tryptic digestion pattern of of S1(+LC) and S1(-LC) suggested that the 4000 difference peptide is at the C-terminus. Tryptic digestion of S1(+LC) has been shown to lead to the production of a regulatory peptide, comprising the two light chains and a heavy chain fragment, which displayed reversible ANS binding on addition of EDTA. Evidence is presented which suggests that this domain is at the C-terminus of subfragment 1.
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008974:Mollusca / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; C027132:1-anilino-8-naphthalenesulfonate; C006421:1,5-I-AEDANS; D009218:Myosins; D008274:Magnesium