J. Biol. Chem., Vol. 258, Issue 20, 12135-12138, Oct, 1983
Selective isolation of polypeptide chains bearing multiple types of postsynthetic modifications. Recovery of simultaneously acetylated and phosphorylated forms of histone H2A and high mobility group proteins 14 and 17
R Sterner and VG Allfrey
In order to study coordinate or simultaneous modifications of chromosomal
proteins by phosphorylation and acetylation, duck erythrocytes were
incubated with [32P]orthophosphate and the thiol- containing acetate
analogue, 2-mercaptoacetate. Enzymatic transfer of the analogue to the
epsilon-amino groups of lysine residues permits the selective recovery of
the newly thio-derivatized polypeptide chains by Hg-affinity
chromatography, and this acetylated subpopulation can then be analyzed for
[32P]phosphate uptake. The histones and high mobility group proteins were
extracted from cell nuclei, purified, and finally analyzed for
incorporation of [32P]phosphate and 2-mercaptoacetate. Several of the
nuclear proteins, in particular histone H2A and the high mobility group
proteins HMG-14 and HMG-17, were subjected to organomercurial-agarose
chromatography. Significant amounts of these cysteine-free proteins were
retained on the affinity column, and by this criterion were shown to have
incorporated mercaptoacetate. The mercaptoacetylated proteins were further
analyzed and found to contain the 32P label as well. These observations
provide incontrovertible evidence that individual molecules of chromosomal
proteins can carry postsynthetic modifications in the form of
phosphorylation and acetylation at the same time, and also establish that
both types of modification must have occurred during the short period in
which the cells were exposed to the two precursors.
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