Kinetics of the interaction of myosin subfragment-1 with G-actin. Effect of nucleotides and DNaseI.
The kinetics of interaction of monomeric pyrenyl-labeled G-actin with myosin subfragment-1 (S1 (A1) and S1(A2) isomers) has been examined in the stopped-flow at low ionic strength. The data confirm the previously reported existence of binary GS and ternary G2S complexes. The increase in pyrenyl-actin fluorescence which monitors the G-actin-S1 interactions is linked to the isomerization of these complexes following rapid equilibrium binding steps. The rates of isomerization are approximately 200 s-1 for GS and approximately 50 s-1 for G2S at 4 degrees C and in the absence of ATP. DNaseI and S1 bind G-actin essentially in a mutually exclusive fashion. Both GS and G2S are dissociated by MgATP and MgADP. The kinetics and mechanism of ATP-induced dissociation of G2S are quantitatively close to the ATP-induced dissociation of F-actin-S1, which indicates the G2S is a good model for the F-actin-S1 interface. GS and G2S display different kinetic behaviors in response to nucleotides, GS being less efficiently dissociated than G2S by MgATP. This result suggests that different mechanical properties of the cross-bridge might correlate with different orientations of the myosin head and different actin/myosin binding ratios.
Blanchoin
L
L
Laboratoire d'Enzymologie du CNRS, Gif-sur-Yvette, France.
Fievez
S
S
Travers
F
F
Carlier
M F
MF
Pantaloni
D
D
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D003850:Deoxyribonuclease I / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D011817:Rabbits
D000199:Actins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I