Time-resolved measurements of phosphate release by cycling cross-bridges in portal vein smooth muscle

Biophys J. 1998 Dec;75(6):3031-40. doi: 10.1016/S0006-3495(98)77744-6.


The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism
  • Animals
  • Biophysical Phenomena
  • Biophysics
  • In Vitro Techniques
  • Kinetics
  • Muscle Contraction / physiology
  • Muscle, Smooth, Vascular / metabolism*
  • Muscle, Smooth, Vascular / physiology
  • Myosins / metabolism
  • Permeability
  • Phosphates / metabolism*
  • Phosphorylation
  • Photolysis
  • Portal Vein / metabolism
  • Portal Vein / physiology
  • Rabbits


  • Phosphates
  • P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • Myosins