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1. 1. Three enzymes which transferred acetate from acetyl coenzyme A specifically to histones were extracted from rat thymus nuclei or chromatin by sonication in the presence of 1 M (NH 4 ) 2 SO 4 . 2. 2. Two fractions having histone acetyltransferase activity (A and B) were separated by DEAE-cellulose chromatography. Enzyme Fraction B was separated into two enzymes (B 1 and B 2 ) by gel filtration on Sephadex G-200. All three acetyltransferases were further purified by chromatography on hydroxylapatite. 3. 3. The approximate molecular weights according to Sephadex G-200 chromatography and sucrose gradient centrifugation were 99 000, 110 000 and 92 000 for the enzymes A, B 1 and B 2 , respectively. 4. 4. The p I of the enzyme A as estimated by isoelectric focusing was 5.9, the enzymes B 1 and B 2 were isoelectric at pH 4.75. 5. 5. p -Chloromercuribenzoate inhibited the activity of the B enzymes and the formation of an acyl-enzyme complex. 6. 6. Acetyltransferase A preferentially acetylated histone Fraction fr and in addition poly- l -lysine, resulting in the formation of e - N -acetyllysine. The enzymes B 1 and B 2 preferred histone f2a 1 as their substrate, histone f3 and poly- l -lysine were not acetylated. Two as yet unidentified acetylated amino acid derivatives were obtained from a digest of histone f2a 1 acetylated by the B enzymes.

Previous work has attempted to localize nuclear histone acetyltransferase activity in the cromatin. Evidence was presented indicating that the transfer of 14 C-acetate from 14 C-acetyl CoA to histones in chromatin was an enzymatic process. We now report on the extraction of part of the histone acetyltransferase activity from rat liver chromatin, employing a procedure originally described for extraction of DNA-dependent RNA polymerase. The K m of the extracted transferase activity for the substrate acetyl CoA was 5 × 10 −7 , the Q 10 : 1.8 and the optimal pH: 7.1. Serum albumine, protamine and polylysine were poor substrates as compared to histones. Activity of extracted or heated chromatin was not restored upon incubation in the presence of extract. Also the selectivity exhibited by the transferase activity in unextracted chromatin towards arginine-rich histones, was much less pronounced in the extracts prepared from it. It is possible that the influence of steric factors contributing to this specificity in native chromatin is lost upon isolation of the enzyme from it. Alternatively, a less specific isoenzyme may have been extracted.