A. Ooi et K. Mihashi, EFFECTS OF SUBTILISIN CLEAVAGE OF MONOMERIC ACTIN ON ITS NUCLEOTIDE-BINDING, Journal of Biochemistry, 120(6), 1996, pp. 1104-1110
The kinetics of ATP exchange on subtilisin-cleaved G-actin was investi
gated by measuring the fluorescence of 1,N-6-ethenoadenosine 5'-tripho
sphate. The apparent dissociation rate of ATP (k(-ATP)) was 2.8-fold l
arger than that of intact G-actin in the presence of 300 mu M free Ca2
+. Analysis of the dependence of k(-ATP) on free Ca2+ showed that the
dissociation rate constant of tightly bound Ca2+ was not significantly
changed by subtilisin cleavage, On the other hand, an equilibrium bin
ding study using 2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline N,
N,N',N'-tetraacetic acid (Quin 2) showed that the affinity of tightly
bound Ca2+ for G-actin was reduced by about 13-fold after subtilisin t
reatment. Consequently, the stabilization by Ca2+ of ATP was weak in c
leaved G-actin. Furthermore, the kinetic analysis of ATP exchange reve
aled that the binding equilibrium between ATP and divalent cat ion-fre
e cleaved G-actin was much slower than that in the case of intact G-ac
tin.