MULTIPLE SITES FOR SUBTILISIN CLEAVAGE OF TUBULIN - EFFECTS OF DIVALENT-CATIONS

Limited digestion of pig brain GDP-tubulin by subtilisin was carried o ut in the presence of Mg2+, Mn2+, Ca2+ , Zn2+ , or Be2+. Isoelectric f ocusing, followed by SDS-PAGE, revealed characteristic divalent cation -dependent changes in the alpha- and beta-tubulin cleavage patterns. P revious studies revealed that the beta-cleavage pattern is different f or heterodimers and microtubules [Lobert and Correia, 1992: Arch. Bioc hem. Biophys. 296:152-160]. Divalent cation effects on subtilisin dige stion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and elect ron microscopy reveal that GDP-tubulin cleaved by subtilisin in the pr esence of Mg2+, Ca2+ , or Mn2+ forms sheets of rings. Mn2+ induces rin g formation in uncleaved GDP-tubulin. Isotype-depleted tubulin was gen erated by the removal of class III beta-tubulin using immunoaffinity c hromatography. Subtilisin digestion of the depleted fraction and the p urified class III beta-tubulin demonstrates that cleavage occurs at th ree to four distinct sites. Thus, subtilisin-digested tubulin is more heterogeneous than was previously reported and the cleavage sites depe nd on solution conditions, divalent cations, and the state of assembly . This has important implications for experiments that utilize subtili sin-digested tubulin for studying microtubule-associated protein bindi ng.