Association of melittin with the isolated myosin light chains.
Melittin is a 26-residue peptide which undergoes high-affinity calcium-dependent binding by calmodulin [Barnette, M.S., Daly, R., & Weiss, B. (1983) Biochem. Pharmacol. 32, 2929; Comte, M., Maulet, Y., & Cox, J.A. (1983) Biochem. J. 209, 269; Anderson, S.R., & Malencik, D.A. (1986) Calcium Cell Funct. 6, 1]. The results in this paper show that three different types of myosin light chain--the smooth muscle regulatory light chain, the smooth muscle essential light chain, and the skeletal muscle regulatory 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chain--also associate with melittin. The resulting complexes have dissociation constants ranging from 1.1 to 2.5 microM in the presence of 0.10 M NaCl and from approximately 50 to approximately 130 nM in solutions of 20 mM 3-(N-morpholino)propanesulfonic acid alone. The regulatory smooth muscle myosin light chain exhibits two equivalent melittin binding sites while each of the others displays only one. The myosin light chains evidently contain elements of structure related to the macromolecular interaction sites present in calmodulin and troponin C but not in parvalbumin. The association of melittin and other peptides with the light chains requires consideration whenever assays of the calmodulin-dependent activity of myosin light chain kinase are used to determine peptide binding by calmodulin. The binding measurements performed on the DTNB light chain and melittin necessitated derivation of the equation relating complex formation to the observed fluorescence anisotropy of a solution containing three fluorescent components. This analysis is generally applicable to equilibria involving the association of two fluorescent molecules emitting in the same wavelength range.
Malencik
D A
DA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Anderson
S R
SR
D000818:Animals; D001514:Bee Venoms / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D005454:Fluorescence Polarization; D005895:Gizzard, Avian / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008555:Melitten / Q000378:metabolism*; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D014422:Turkeys
D001514:Bee Venoms; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D008555:Melitten; D009218:Myosins; D008274:Magnesium; D002118:Calcium