Subtilisin cleavage of tubulin heterodimers and polymers.
Native pig brain tubulin in heterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last 50 amino acids of the highly variable carboxyl-termini of the alpha and beta subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 M glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, alpha' and beta', under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native alpha,beta tubulin to 7-9 for subtilisin-cleaved alpha',beta' tubulin. When tubulin heterodimers were cleaved, a single major beta' isotype was evident; however, when tubulin polymerized in 4 M glycerol was cleaved, two major beta' isotypes were found. Monoclonal antibodies that recognize a beta carboxyl-terminal peptide, residues 410-430, reacted with both major beta' isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major beta' isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the beta subunit, near residues 430-435, with different accessibility to the enzyme in the heterodimer and polymer form.
Lobert
S
S
School of Nursing, University of Mississippi Medical Center, Jackson 39216.
Correia
J J
JJ
D000818:Animals; D001921:Brain / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D013381:Subtilisins / Q000378:metabolism*; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D014404:Tubulin; D013381:Subtilisins