Practice question

F actin formation prerequisites can be recapitulated in vitro using purified muscle actin from rabbit skeletal muscle. G actin in Ca ATP state low ionic strength is monomeric. Initiation requires exchange of Ca2+ for Mg2+ at high affinity site by adding 2 millimolar MgCl2 and 50 to 100 millimolar KCl to screen electrostatic repulsion, plus 1 millimolar ATP to maintain ATP actin. Crucially thermodynamic requirement total G actin concentration must exceed critical concentration about 0.1 micromolar at plus end. Below this no polymer forms, above this equilibrium free monomer remains at Cc and excess converts to filament, detected by increase in pyrene fluorescence, light scattering or sedimentation. High cofilin raises effective Cc by increasing off rate and severing, low salt prevents nucleation by failing to neutralize surface charges, absence ATP yields ADP actin with higher Cc and unstable filaments. Experiments routinely use 5 micromolar actin in F buffer containing MgCl2 KCl ATP, often adding spectrin actin seeds or formin FH2 to bypass slow nucleation phase, ensuring rapid polymerization for motility assays and structural studies.