Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol.
We have exploited solvent perturbation to probe the coupling of Ca2+ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4 degrees C, the glycol had little effect on the myofibrillar ATPase at low [Ca2+], but at high [Ca2+] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca2+) was reduced only 3-4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca2+. Further experiments revealed that in 40% ethylene glycol, Ca2+ does initiate shortening but only with the aid of strong interactions and at temperatures above 15 degrees C. This confirms that in the organized and intact myofibril, Ca2+ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1.
Stehle
R
R
INSERM U 128, CNRS, Montpellier, France.
Lionne
C
C
Travers
F
F
Barman
T
T
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D019855:Ethylene Glycol / Q000494:pharmacology*; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism*; D016658:Psoas Muscles / Q000378:metabolism*; D011817:Rabbits
D003432:Cross-Linking Reagents; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D019855:Ethylene Glycol; D002118:Calcium