Biophys J, February 2000, p. 918-926, Vol. 78, No. 2

Measurement of Nucleotide Exchange Rate Constants in Single Rabbit Soleus Myofibrils during Shortening and Lengthening Using a Fluorescent ATP Analog

Ibuki Shirakawa,^* Shigeru Chaen,^* Clive R. Bagshaw, and Haruo Sugi^*

 ^*Department of Physiology, School of Medicine, Teikyo University, Tokyo 173, Japan, and  Department of Biochemistry, University of Leicester, Leicester LE1 7RH, England

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas 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-ADP) showed selective fluorescence staining of the A-band. The K_m for Cy3-EDA-ATP and the K_d for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 µM and 3.8 µM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 µM and 50 µM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 ± 0.007 s¹ (n = 32) at 10°C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s¹ (isometric) to 0.14 s¹ at 20°C with increasing shortening velocity up to 0.1 myofibril length/s (V_max, the shortening velocity under no load was ~0.2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.

Biophys J, February 2000, p. 918-926, Vol. 78, No. 2
© 2000 by the Biophysical Society   0006-3495/00/02/918/09  $2.00

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