Tubulin dimer dissociation and proteolytic accessibility.
The alpha and beta subunits of the tubulin dimer each possess a distal C-terminal subtilisin cleavage site which, when cleaved, releases an acidic, small peptide. In addition, each possesses an internal site, cleaved by trypsin in alpha and chymotrypsin in beta, which connects the amino and carboxyl structural domains. A model of the dimer is presented which suggests that the beta C-terminal subtilisin site may be more accessible in the monomer than in the dimer. Kinetics of cleavage at this site on the dimer yield straight-line plots of log (undigested fraction) versus time, from which pseudo-first-order rate constants are obtained. Temperature effects on the rate constant are due to changes in the activity of subtilisin, not to temperature-induced unfolding around this site. The rate constant is proportional to the subtilisin/tubulin ratio, whether this is varied by changing the concentration of subtilisin or of tubulin. However, if the rate constant increases due to decreasing tubulin concentration, the extrapolated zero time intercept decreases. The decrease in zero time intercept is interpreted as being due to the appearance of a rapidly digested fraction upon dilution of tubulin. The increase observed in this fast fraction with dilution of tubulin is fully reversible upon reconcentration. It is suggested that this fast fraction represents monomeric beta-tubulin and the concentration dependence of this fast fraction indicates a dissociation constant of about 1.5 X 10(-7) M.
Sackett
D L
DL
National Institute of Diabetes, Digestive, and Kidney Diseases, Bethesda, Maryland 20894.
Zimmerman
D A
DA
Wolff
J
J
D000818:Animals; D001921:Brain / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D011487:Protein Conformation; D051381:Rats; D013381:Subtilisins / Q000378:metabolism; D013816:Thermodynamics; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D014404:Tubulin; D013381:Subtilisins; D002918:Chymotrypsin