Effects of subtilisin cleavage of monomeric actin on its nucleotide binding.
The kinetics of ATP exchange on subtilisin-cleaved G-actin was investigated by measuring the fluorescence of 1,N6-ethenoadenosine 5'-triphosphate. The apparent dissociation rate of ATP (k-ATP) was 2.8-fold larger than that of intact G-actin in the presence of 300 microM 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 binding study using 8-amino-2-[(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 treatment. Consequently, the stabilization by Ca2+ of ATP was weak in cleaved G-actin. Furthermore, the kinetic analysis of ATP exchange revealed that the binding equilibrium between ATP and divalent cation-free cleaved G-actin was much slower than that in the case of intact G-actin.
Ooi
A
A
Faculty of Bioresources, Mie University, Tsu. ooi@bio.mie-u.ac.jp
Mihashi
K
K
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D011817:Rabbits; D013381:Subtilisins / Q000378:metabolism*
D000199:Actins; D004985:Ethenoadenosine Triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D013381:Subtilisins; D002118:Calcium