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.
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.
D000107:Acetylation; D000818:Animals; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000302:isolation & purification; D004372:Ducks; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis; D006609:High Mobility Group Proteins; D006657:Histones / Q000235:genetics* / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
D002868:Chromosomal Proteins, Non-Histone; D006609:High Mobility Group Proteins; D006657:Histones; D010455:Peptides