Regulation of binding of subfragment 1 in isolated rigor myofibrils.
A steric-hindrance model has been used to explain the regulation of muscle contraction by tropomyosin-troponin complex. The regulation of binding was studied by microscopic observation of mixtures of fluorescent subfragment 1 (S1) with rigor myofibrils at different actin-to-S1 ratios and in the presence and absence of calcium. Procedures were adapted to protect the critical thiols of S1 before conjugation to thiol-specific fluorochromes, this giving fluorescent S1 with unaltered enzyme activity. S1 binding was greatest in the I band (except at the Z-lines) in the presence of calcium regardless of the [S1]. The patterns in the absence of calcium depended on the actin-to-S1 ratios: low [S1], binding in the myosin-actin overlap region; intermediate [S1], highest binding at the A-I junction; high [S1], greatest binding in the I-band. The two distinct binding patterns observed at low [S1] were demonstrated by dual-channel fluorescence microscopy when myofibrils were sequentially incubated with fluorescent S1 without calcium followed by a different fluorescent S1 with calcium. These observations support the concept of rigor activation of actin sites. The change in the pattern upon increasing [S1] without calcium demonstrate cooperative interactions along the thin filament. However, these interactions (under the conditions used without calcium) do not appear to extend over greater than 2-3 tropomyosin-troponin-7 actin functional units.
Swartz
D R
DR
University of Wisconsin, Muscle Biology Laboratory, Madison 53706.
Greaser
M L
ML
Marsh
B B
BB
D000009:Abdominal Muscles; D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology; D002417:Cattle; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000502:physiology*; D009210:Myofibrils / Q000378:metabolism* / Q000648:ultrastructure; D015879:Myosin Subfragments / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D002118:Calcium