The kinetics of the interaction between the actin-binding domain of alpha-actinin and F-actin.
Measurement of the binding equilibrium for the interaction of alpha-actinin with F-actin is complicated by secondary reactions involving cross-linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin-binding domain (ABD) of chick smooth muscle alpha-actinin with F-actin. Stopped-flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F-actin yielding second order rate constants for association of 2 x 10(5), 1.8 x 10(6) and 4 x 10(6) M-1.s-1 at 5 degrees C, 15 degrees C and 25 degrees C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6s-1, giving equilibrium constants of 0.83 and 2.4 microM, respectively. Optical changes on mixing intact alpha-actinin with F-actin were dominated by secondary bundling events.
Kuhlman
P A
PA
Department of Biochemistry, University of Leicester, UK.
Ellis
J
J
Critchley
D R
DR
Bagshaw
C R
CR
D000185:Actinin / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004926:Escherichia coli; D007700:Kinetics; D008027:Light; D009130:Muscle, Smooth / Q000737:chemistry; D009132:Muscles / Q000737:chemistry; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D000199:Actins; D011994:Recombinant Proteins; D000185:Actinin; D014364:Tryptophan