Abstract
We have assayed several methods to quantitatively recover yeast histone acetyltransferases in an attempt to study the multiplicity of enzymatic activities. Two methods, namely (NH4)2SO4 precipitation and salt dissociation of chromatin in 0.5 M NaCl, yielded convenient preparations of total histone acetyltransferases. DEAE-Sepharose chromatography of the crude extracts resulted in the separation of three peaks of activity when total yeast histones were used as substrate. However, the scanning of the enzymatic activity toward individual histones along the chromatography, achieved by determining the specific activity of the individual histones after incubating whole histones and [14C]acetyl-CoA with the chromatographic fractions, yielded four peaks. The first two peaks showed specificity toward H2B and H3, respectively. Although they partially overlapped, rechromatography on cation exchangers allowed us to resolve the two activities, and several criteria were used to prove that they correspond to different enzyme molecules. The last two peaks were H4-specific, but the present data suggest that one of the activities is chromatin-bound, whereas the other surely corresponds to the cytoplasmic B-form of the enzyme. The enzyme specific for yeast H2B acetylates chicken erythrocyte H2A, rather than H2B. The detected multiplicity of yeast histone acetyltransferases may correspond to the multiplicity of roles proposed for histone acetylation.
Highlights
We have assayed several methods to quantitatively recover yeast histone acetyltransferases in anattempt to study the multiplicity of enzymatic activities
It has been concluded that acetylation of a single lysyl residue is related to histone deposition, whereas the turnover of acetyl groups at other lysines may be connected with other chromatin functions
To quantitatively recover enzymatic activities, we have tested the possibility of applying, to yeast cells, three of the methods currentlyused to prepare histone acetyltransferases. This objective was essential to our purpose of determining the number of molecular species of yeast histone acetyltransferases
Summary
We have assayed several methods to quantitatively recover yeast histone acetyltransferases in anattempt to study the multiplicity of enzymatic activities. Acetylation of the t-amino groups of lysyl residues is the most widely studied reversible modification of histones, but only in the last few years are its functional roles being disclosed These include the deposition and assembly of histones into nucleosomes during DNA replication [1,2,3], the substitution of histones during differentiation (for instance, the replacement of histones by protamines during spermatogenesis) [4,5,6,7], together with the already classical, proposed role in transcription [8,9,10]. Acetyltransferases B arecytoplasmic enzymes, and they seem to be responsible for the acetylation of H4 before chromatin assembly [18,20, 24, 25] It is not yet clear whether histone acetyltransferases are single enzyme molecules; but in view of the multiplicity of roles proposed for histone acetylation (ll),it may be possible that activities A and B are composed of several isoenzymatic forms
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