Porcine liver nuclear histone acetyltransferase. Partial purification and basic properties.

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Histone acetyltransferases (HATs) play crucial roles in regulating gene expression through chromatin modification.
• Understanding the specific HATs involved and their substrate preferences is essential for elucidating epigenetic mechanisms.

Purpose of the Study:

• To isolate and partially purify the major histone acetyltransferase activity from porcine liver nuclei.
• To characterize the basic properties and substrate specificity of the purified enzyme.

Main Methods:

• A rapid and reproducible purification protocol involving ammonium sulfate fractionation, ion-exchange chromatography (DEAE-Sephacel), hydroxylapatite chromatography, and glycerol gradient ultracentrifugation.
• Determination of molecular weight using glycerol gradient ultracentrifugation and gel filtration (Sephacryl S-200).
• Assay of enzyme activity across different pH values and in the presence of various salts and inhibitors.

Main Results:

• An 8650-fold purification of histone acetyltransferase activity with a 42% yield was achieved.
• The enzyme has an estimated molecular weight of approximately 94,000.
• Optimal activity at pH 7.5, with inhibition by salts and sulfhydryl reagents. Glycerol protected against thermal denaturation.
• Preferential acetylation of histones H3, H4, H2B, and H2A in free form, and H4, H2B, H3, and H2A within nucleosome core particles.
• High mobility group proteins 14 and 17 were identified as in vitro substrates.

Conclusions:

• A robust method for partial purification of a major porcine liver nuclear histone acetyltransferase was developed.
• The enzyme exhibits distinct substrate specificities for free histones versus nucleosome-bound histones.
• Enzymatic acetylation of high mobility group proteins suggests a potential role in chromatin regulation.