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pubmed:abstractText |
Shortening and ATPase rates were measured in Ca2+-activated myofibrils from frog fast muscles in unloaded conditions at 4 degrees C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load (V0) were estimated by quenching reaction mixtures 50 ms to 10 s old at pH 3.5 and measuring sarcomere lengths under the optical microscope. As with the rabbit psoas myofibrils (C. Lionne, F. Travers, and T. Barman, 1996, Biophys. J. 70:887-895), the ATPase progress curves had three phases: a transient Pi burst, a fast linear phase (kF), and a deceleration to a slow phase (kS). Evidence is given that kF is the ATPase rate of shortening myofibrils. V0 is in good agreement with mechanical measurements in myofibrils and fibers. Under the same conditions and at saturation in ATP, V0 and kF are 2.4 microm half-sarcomere(-1) s(-1) and 4.6 s(-1), and their Km values are 33 and 200 microM, respectively. These parameters are higher than found with rabbit psoas myofibrils. The myofibrillar kF is higher than the fiber ATPase rates obtained previously in frog fast muscles but considerably lower than obtained in skinned fibers by the phosphate-binding protein method (Z. H. He, R. K. Chillingworth, M. Brune, J. E. T. Corrie, D. R. Trentham, M. R. Webb, and M. R. Ferenczi, 1997, J. Physiol. 50:125-148). We show that, with frog as with rabbit myofibrillar ATPase, phosphate release is the rate-limiting step.
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