*Muscle Research Unit, Department of Anatomy and Histology, +Department of Biochemistry, The University of Sydney, N.S.W. 2006.
Actin is often present in the cytoplasm of eucaryotic cells at concentrations up to several hundred umolar, higher than that of any other protein. In a physiological environment, such concentrations of purified actin polymerise very rapidly, leaving less than 1% in the monomeric globular form (G-actin) (Pollard, 1984). However, in cells, only part of the actin is polymerised. This is due to the abundance of intracellular actin-binding proteins (ABPs) which allow the concentration of G-actin to be maintained substantially above the threshold at which polymerisation occurs.
Using a neutral, hydrophilic coated, 75 um i.d. capillary we have analysed the binding affinities of native proteins in solution using capillary zone electrophoresis. The data were fit to a series of equations to give a Kd that agrees with the previously determined constant. Further determination of equilibrium constants for the binding of other ABPs to actin (i.e. actin depolymerising factor, thymosin b4, myosin subfragment-1, and profilin) may provide information on the interrelationships between these ABPs and give some indication as to how their interaction with actin is regulated.
Capillary electrophoresis is an invaluable experimental technique because it is quick, uses only very small amounts of sample, the protein is not chemically altered.
1. Pollard, T.D. (1984) Nature 312, 403.