Abstract
The major histone acetyltransferase activity from porcine liver nuclei has been isolated and partially purified by a simple, rapid, and reproducible method. Extraction of nuclei in buffered 30% saturated ammonium sulfate and subsequent ammonium sulfate fractionation, chromatography on DEAE-Sephacel and hydroxylapatite, and ultracentrifugation on linear 15-30% glycerol gradients provides an 8650-fold purification (over nuclei) in 42% yield. The molecular weight of the enzyme is approximately 94,000 as determined by glycerol gradient ultracentrifugation and gel filtration on Sephacryl S-200. The optimum pH for the reaction is 7.5 and the activity is inhibited by monovalent and divalent salts and by sulfhydryl blocking reagents. The enzyme activity is substantially protected from thermal denaturation at 37 degrees C by the addition of glycerol to the incubation medium. In the presence of the core histones, the enzyme catalyzes the acetylation reaction in the order H3 greater than H4 greater than H2B greater than H2A; the order for histones bound in nucleosome core particles is H4 greater than H2B greater than H3 greater than H2A. The high mobility group proteins 14 and 17 serve as substrates for the enzyme in vitro, suggesting a possible role for enzymatic high mobility group acetylation in chromatin function.
Highlights
Themajorhistone acetyltransferase activity from 8) and animal systems [9] demonstrated that cells which are porcine liver nucleihas been isolatedandpartially actively engaged in the synthesis of RNA showan increase in purified by asimple, rapid, and reproducible method. the levels of histone acetylation relative to template inactive
More recent work has shown that DNase I and micromonium sulfate andsubsequentammonium sulfate coccal nuclease-sensitive regions of chromatin, which are fractionation, chromatographyon DEAE-Sephacel and known to be actively engaged in transcription [10,11,12,13,14], selechydroxylapatite,and ultracentrifugationon linear 1530%glycerol gradients provides an 8660-fold purification in 42%yield
The optimum pH for the reaction is 7.6 and the activity is inhibited by monovalent anddivalent salts tively release acetylated histones [15,16,17,18,19]. direct proof of the biochemical role of histone acetylation has remained elusive, the exquisite specificity of the acetylation reaction, the relatively rapid turnover of acetate groups, and the physical-chemical consequences of positive charge neutralization resulting from the acetylation of lysine residues in the DNA-binding regions of the nucleosomal histones seem and by sulfhydryl blocking reagents
Summary
The histone proteins that comprise the nucleosome core, acetyltransferase activity from porcine liver, a tissue that is highlyconserved in their primary sequences, are readily available locally upon slaughter and from which large subject to a variety of postsynthetic modifications that pro- scale preparation of highly purified enzyme seemed feasible. It has been suggested that enzymatically regulated processes method for purification of the porcine liver nuclear histone that alter DNA-protein interactions at the level of the nu- acetyltransferase which provides sufficient quantities of encleosome core particle may play an important role in the zyme for detailed in uitro characterization. Assay of Histone AcetyltransferaseActtuity-Enzyme samples were Sulfhydryl Sensitivity-When the enzymewas incubated incubated in capped Microfuge tubes at 37"C in a total volume of 100 pl containing 50 mM potassium phosphate (pH 7.4), 15 mM 2mercaptoethanol, 10% glycerol,0.1 mM PMSF, 20 nCi of [l-''C] acetylcoenzyme A, and 0.5 mg/ml of calf thymus histone substrate. Salt concentration was monitored by conductivity measurements using a Radiometer Copenhagen conductivity meter (type CDM 20).pH measurements were performed a t "C using a Radiometer Copenhagen pH meter, model
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