Dissociation of F-actin induced by hydrostatic pressure.
F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits.
Garcia
C R
CR
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
Amaral Júnior
J A
JA
Abrahamsohn
P
P
Verjovski-Almeida
S
S
D000199:Actins / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006874:Hydrostatic Pressure; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008854:Microscopy, Electron; D009132:Muscles / Q000737:chemistry; D011721:Pyrenes; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D008024:Ligands; D011721:Pyrenes; C030984:pyrene; D008274:Magnesium; D002118:Calcium