Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[C y3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of cag ed ATP, Use of myofibrils from this slow twitch muscle allowed better resol ution of the kinetics of nucleotide exchange than previous studies with pso as muscle myofibrils (Chaen et al., 1997, Biophys. J 73:2033-2042), Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-A DP) showed selective fluorescence staining of the A-band. The K-m for Cy3-E DA-ATP and the K-d for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 mu M and 3.8 mu M, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 mu M and 50 m u M, respectively). After flash photolysis of caged ATP, the A-band fluores cence of the myofibril in the Cy3-EDA-ATP solution under isometric conditio ns decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoa s myofibrils. When a myofibril was allowed to shorten with a constant veloc ity, the nucleotide displacement rate constant increased from 0.066 s(-1) ( isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocit y up to 0.1 myofibril length/s (V-max, the shortening velocity under no loa d was similar to 0.2 myofibril lengths/s), The rate constant was not signif icantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s , These results suggest that the cross-bridge kinetics are not significantl y affected at higher strain during lengthening but depend on the lower stra in during shortening. These data also indicate that the interaction distanc e between a cross-bridge and the actin filament is at least 16 nm for a sin gle cycle of the ATPase.