Different actin affinities of human cardiac essential myosin light chain isoforms.
The N terminus of myosin light chain 1 (MLC-1) of skeletal muscle bind to the C terminus of actin. We investigated whether the N termini of human cardiac MLC-1 isoforms likewise bind to actin. Furthermore, we investigated whether the N-terminal sequence 5-15 (P5-14) of MLC-1 of human atrium (ALC-1) and ventricle (VLC-1) bind with different affinities to actin. Affinity beads were produced by covalently coupling a synthetic peptide corresponding to the N-terminal sequence 4-14 of human VLC-1 to aminohexylagarose in order to bind G-actin. We found, that G-actin specifically binds to the affinity beads. Furthermore, preincubation of G-actin with P5-14 of both ALC-1 and VLC-1 decreased the amount of G-actin recovered from the affinity beads in a concentration-dependent manner. The half-maximal effective concentrations, however were significantly (p < 0.01) different being 0.32 +/- 0.02 microM and 0.71 +/- 0.02 microM for the VLC-1 and ALC-1 peptide, respectively. The appropriate scrambled peptides were without effect up to 3 microM. These results demonstrate the specific interaction between the N-terminal domains of human cardiac MLC-1 isoforms and actin and reveal different actin affinities of MLC-1 isoforms. Weak binding of ALC-1 to actin could explain the higher cycling kinetics of cross-bridges with ALC-1 compared to those with VLC-1.
Morano
I
I
Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Haase
H
H
D000199:Actins / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D006325:Heart Atria / Q000737:chemistry; D006352:Heart Ventricles / Q000737:chemistry; D006801:Humans; D009206:Myocardium / Q000737:chemistry*; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism
D000199:Actins; D018994:Myosin Light Chains; D010446:Peptide Fragments; C076659:myosin light chain I; C024903:aminohexyl-sepharose; D012685:Sepharose