ATPase and Shortening Rates in Frog Fast Skeletal Myofibrils by Time-Resolved Measurements of Protein-Bound and Free P i

Abstract

Shortening and ATPase rates were measured in Ca 2+-activated myofibrils from frog fast muscles in unloaded conditions at 4°C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load ( V o) 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 P i burst, a fast linear phase ( k F), and a deceleration to a slow phase ( k S). Evidence is given that k F is the ATPase rate of shortening myofibrils. V o is in good agreement with mechanical measurements in myofibrils and fibers. Under the same conditions and at saturation in ATP, V o and k F are 2.4 μm half-sarcomere −1 s −1 and 4.6 s −1, and their K m values are 33 and 200 μM, respectively. These parameters are higher than found with rabbit psoas myofibrils. The myofibrillar k F 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.