Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils.
Myosin subfragment 1 (S1) can be specifically modified at Lys-553 with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) (Bertrand, R., J. Derancourt, and R. Kassab. 1995. Biochemistry. 34:9500-9507), and solvent quenching of FHS-S1 with iodide has been shown to be sensitive to actin binding at low ionic strength (MacLean, Chrin, and Berger, 2000. Biophys. J. 000-000). In order to extend these results and examine the fraction of actin-bound myosin heads within the myofilament lattice during calcium activation, we have modified skeletal muscle myofibrils, mildly cross-linked with EDC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) to prevent shortening, with FHS. The myosin heavy chain appears to be the predominant site of labeling, and the iodide quenching patterns are consistent with those obtained for myosin S1 in solution, suggesting that Lys-553 is indeed the primary site of FHS incorporation in skeletal muscle myofibrils. The iodide quenching results from calcium-activated FHS-myofibrils indicate that during isometric contraction 29% of the myosin heads are strongly bound to actin within the myofilament lattice at low ionic strength. These results suggest that myosin can be specifically modified with FHS in more complex and physiologically relevant preparations, allowing the real time examination of cross-bridge interactions with actin in in vitro motility assays and during isometric and isotonic contractions within single muscle fibers.
Cooper
W C
WC
Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vermont 05405-0068, USA.
Chrin
L R
LR
Berger
C L
CL
D000199:Actins / Q000737:chemistry* / Q000502:physiology*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D003497:Cyclic N-Oxides / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D008239:Lysine; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D009210:Myofibrils / Q000187:drug effects / Q000502:physiology*; D015879:Myosin Subfragments / Q000737:chemistry* / Q000502:physiology*; D011193:Potassium Iodide / Q000494:pharmacology; D011817:Rabbits; D013050:Spectrometry, Fluorescence
C115825:6-(fluorescein-5-carboxamido)hexanoic acid succimidyl ester; D000199:Actins; D003497:Cyclic N-Oxides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D011193:Potassium Iodide; D000251:Adenosine Triphosphatases; D008239:Lysine; C003959:TEMPO