Effect of villin on the kinetics of actin polymerization.
The effect of villin on the critical concentration of actin and on the kinetics of its polymerization has been measured. In the presence of villin and 10 microM calcium, the critical concentration of actin increased from 0.2 to 0.9 microM. This effect of villin on the critical concentration was shown to be the result of its well-documented ability to block the "barbed" end of actin filaments, i.e., the "high-affinity end" of a polymer with a different monomer binding constant at each end. Thus, below 0.8 microM actin polymerization was prevented when the ratio of villin to actin was about 1 in 1000. Furthermore, the effect of villin was saturable; i.e., the critical concentration remained constant with increasing villin concentration once the maximal change had been obtained. In addition, fragmentation of actin filaments previously capped with villin, producing uncapped filaments, caused a rapid, transient fall of the monomer concentration. With the disappearance of the uncapped filaments the actin monomer concentration returned to that measured before fragmentation. The binding constant of villin to the barbed end of the actin filament was calculated to be greater than 10(11) M-1. The rate constants of elongation and of depolymerization at each end of an actin filament were measured. The depolymerization rate constant from the barbed end was about 10 times greater under conditions leading to complete depolymerization than under steady-state conditions. We discuss a possible explanation for the finding and its implication for possible regulatory mechanisms.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D011721:Pyrenes / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D000199:Actins; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D011721:Pyrenes; C026615:villin; D004533:Egtazic Acid; C030984:pyrene; D004492:Edetic Acid; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide; D002118:Calcium