Decavanadate interactions with actin: cysteine oxidation and vanadyl formation.
Incubation of actin with decavanadate induces cysteine oxidation and oxidovanadium(IV) formation. The studies were performed combining kinetic with spectroscopic (NMR and EPR) methodologies. Although decavanadate is converted to labile oxovanadates, the rate of deoligomerization can be very slow (half-life time of 5.4 h, at 25 degrees C, with a first order kinetics), which effectively allows decavanadate to exist for some time under experimental conditions. It was observed that decavanadate inhibits F-actin-stimulated myosin ATPase activity with an IC(50) of 0.8 microM V(10) species, whereas 50 microM of vanadate or oxidovanadium(IV) only inhibits enzyme activity up to 25%. Moreover, from these three vanadium forms, only decavanadate induces the oxidation of the so called "fast" cysteines (or exposed cysteine, Cys-374) when the enzyme is in the polymerized and active form, F-actin, with an IC(50) of 1 microM V(10) species. Decavanadate exposition to F- and G-actin (monomeric form) promotes vanadate reduction since a typical EPR oxidovanadium(IV) spectrum was observed. Upon observation that V(10) reduces to oxidovanadium(IV), it is proposed that this cation interacts with G-actin (K(d) of 7.48 +/- 1.11 microM), and with F-actin (K(d) = 43.05 +/- 5.34 microM) with 1:1 and 4:1 stoichiometries, respectively, as observed by EPR upon protein titration with oxidovanadium(IV). The interaction of oxidovanadium(IV) with the protein may occur close to the ATP binding site of actin, eventually with lysine-336 and 3 water molecules.
Ramos
Susana
S
FCT, University of Algarve, Faro, Portugal.
Duarte
Rui O
RO
Moura
José J G
JJ
Aureliano
Manuel
M
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009218:Myosins / Q000378:metabolism; D010084:Oxidation-Reduction; D014638:Vanadates / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D014638:Vanadates; D000255:Adenosine Triphosphate; D009218:Myosins; D003545:Cysteine