Protein Protocols Protein Protocols

Carboxyl Methyltransferase 623
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Identification of Proteins Modified by Protein
(D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferase
Darin J. Weber and Philip N. McFadden
1. Introduction
The several classes of S-adenosylmethionine-dependent protein methyltransferases
are distinguishable by the type of amino acid they modify in a substrate protein. The
protein carboxyl methyltransferases constitute the subclass of enzymes that incorporate
a methyl group into a methyl ester linkage with the carboxyl groups of proteins. Of
these, protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase, EC 2.1.1.77 (PCM)
specifically methyl esterifies aspartyl residues that through age-dependent alterations
are in either the D-aspartyl or the L-isoaspartyl configuration (1,2). There are two major
reasons for wishing to know the identity of protein substrates for PCM. First, the proteins
that are methylated by PCM in the living cell, most of which have not yet been
identified, are facets in the age-dependent metabolism of cells. Second, the fact that
PCM can methylate many proteins in vitro, including products of overexpression systems,
can be taken as evidence of spontaneous damage that has occurred in these proteins
since the time of their translation.
The biggest hurdle in identification of substrates for PCM arises from the extreme
base-lability of the incorporated methyl esters, which typically hydrolyze in a few hours
or less at neutral pH. Thus, many standard biochemical techniques for separating and
characterizing proteins are not usefully applied to the identification of these methylated
proteins. In particular, the electrophoresis of proteins by the most commonly
employed techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) results in a complete loss of methyl esters incorporated by PCM, owing
to the alkaline pH of the buffers employed. Consequently, a series of systems employing
polyacrylamide gel electrophoresis at acidic pH have been utilized in efforts to
identify the substrates of PCM. A pH 2.4 SDS system (3) using a continuous sodium
phosphate buffering system has received the most attention (1,3–14). The main drawback
of this system is that it produces broad electrophoretic bands. Acidic discontinuous
gel systems using cationic detergents, (15), have proven useful in certain situations
(16–21) and can be recommended if the cationic detergent is compatible with other
procedures that might be utilized by the investigator (e.g., immunoblotting, protein
sequencing). Recently, we have developed an electrophoresis system that employs SDS
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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