NUCLEOTIDE TURNOVER RATE MEASURED IN FULLY RELAXED RABBIT SKELETAL-MUSCLE MYOFIBRILS

Steady state measurements of the ATP turnover rate of myosin crossbrid ges in relaxed living mammalian muscle or in in vitro systems are comp licated by other more rapid ATPase activities. To surmount these probl ems we have developed a technique to measure the nucleotide turnover r ate of fully relaxed myosin heads in myofibrils using a fluorescent an alogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, we re rapidly mixed with an equal volume of solution containing 80 mu M m ant-ATP and injected into a fluorimeter. As bound ADP is released, a f raction of the myosin active sites bind mant-ATP and fluorescence emis sion rises exponentially, defining a rate of nucleotide turnover of 0. 03 +/- 0.001 s(-1) at 25 degrees C (n = 17). This rate was approximate ly equal to one half that of purified myosin. The turnover rates for m yosin and myofibrils increased between 5 degrees and 42 degrees C, rea ching 0.16 +/- 0.04 s(-1) and 0.06 +/- 0.005 s(-1), respectively, at 3 9 degrees C, the body temperature of the rabbit. If the rate observed for purified myosin occurred in vivo, it would generate more heat than is observed for resting living muscle. When myosin is incorporated in to the myofilament lattice, its ATPase activity is inhibited, providin g at least a partial explanation for the low rate of heat production b y living resting muscle.