Nucleotide turnover rate measured in fully relaxed rabbit skeletal muscle myofibrils

Abstract

Steady state measurements of the ATP turnover rate of myosin cross-bridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in myofibrils using a fluorescent analogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, were rapidly mixed with an equal volume of solution containing 80 μM mant-ATP and injected into a fluorimeter. As bound ADP is released, a fraction of the myosin active sites bind mant-ATP and fluorescence emission rises exponentially, defining a rate of nucleotide turnover of 0.03 ± 0.001 s-1 at 25°C (n = 17). This rate was approximately equal to one half that of purified myosin. The turnover rates for myosin and myofibrils increased between 5° and 42°C, reaching 0.16 ± 0.04 s-1 and 0.06 ± 0.005 s-1, respectively, at 39°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 into the myofilament lattice, its ATPase activity is inhibited, providing at least a partial explanation for the low rate of heat production by living resting muscle.