FIGURE 9.

Characterization of various molecular forms of Bcl_xL. A, immunoblot analysis of the Bcl_xL n (native-unmodified) and i (isomerized) forms. The latter was generated through alkali treatment (see “Experimental Procedures”). B, evaluation of PCMT1 substrate capability of Bcl_xL i form. IsoAsp residues in the Bcl_xL i form were identified by human recombinant PCMT1 assay in the presence of methyl labeled S-adenosylmethionine (see “Experimental Procedures”). The result is expressed as percentage of the fraction of methylesterified Bcl_xL molecules over total. Inset, MALDI/TOF spectrum of the Bcl_xL i form. Arrows mark the peptides containing deamidated Asn⁵² and Asn⁶⁶ residues. C, electrophoretic behavior of the Bcl_xL m form compared with the n and i forms. Bcl_xL m form was obtained through enzymatic methylation of the i form (see “Experimental Procedures”) by human recombinant PCMT1 in the presence of unlabeled S-adenosylmethionine.