In vitro mercaptoacetylation of chromosomal proteins. Selective recovery of newly modified protein molecules.
Duck erythrocytes were incubated with the thiol-containing acetate analog 2-mercaptoacetate. The high mobility group proteins and histones were then extracted from the cell nuclei and the resultant protein mixtures subsequently were fractionated to their individual components by established procedures of selective trichloracetic acid precipitation and ion exchange and gel exclusion chromatography. The protein mixtures and the proteins derived from them were then subjected to mercury-affinity chromatography on organomercurial agarose columns, and it was found that significant amounts of all of the high mobility group proteins and core histones were bound by the columns. Furthermore, chymotryptic digestion of histone H4 from mercaptoacetate-treated cells and subsequent mercury-affinity chromatography revealed that the only peptides which bound to the column were the NH2-terminal peptides, the ones containing the lysines known to be the normal sites of in vivo acetylation. Carboxymethylation of mercaptoacetate-treated H4 with iodo[3H) acetate acid yielded 3H-labeled H4 and subsequent chymotryptic digestion showed that the only radioactive peptides were again those containing the lysines known to be the normal sites of enzymatic acetylation. These results and other biochemical evidence strongly indicate that the proteins of the histone and high mobility group classes which are known to be subject to postsynthetic acetyl transfer reactions can undergo postsynthetic mercaptoacetylation in vivo and that the sites of such modification are the same as those for normal acetylation. The phenomenon of mercaptoacetylation of chromosomal proteins affords a means for the selective recovery of newly modified protein molecules and their associated chromatin.
D000215:Acylation; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism; D004372:Ducks; D004912:Erythrocytes / Q000378:metabolism*; D006609:High Mobility Group Proteins; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D013864:Thioglycolates / Q000378:metabolism*
D002868:Chromosomal Proteins, Non-Histone; D006609:High Mobility Group Proteins; D006657:Histones; D013864:Thioglycolates; C017487:2-mercaptoacetate