Abstract
Duck erythrocytes were incubated with [3H]acetate both in the presence and absence of sodium butyrate. Subsequent perchloric acid extraction of the nuclei, followed by selective acetone precipitation, CM-Sephadex ion exchange chromatography, and gel filtration yielded radioactively labeled high mobility group (HMG) proteins HMG-14 and HMG-17 in pure form. Extensive enzymatic degradation of the proteins followed by amino acid analysis of the digests yielded a significant amount of material eluting in the position of epsilon-N-acetyllysine. Furthermore, automated Edman degradation of intact 3H-labeled HMG-14 and HMG-17 identified the specific sites of acetylation of these proteins. In both erythrocyte HMGs isolated from cells not exposed to butyrate, the lysine residue at position 2 was the only one found to be labeled. However, one additional site in HMG-14 and two additional sites in HMG-17 were found in the proteins from cells incubated in butyrate. Finally, studies of the enzymatic deacetylation of HMG-14 and HMG-17 confirmed that both nuclear proteins serve as deacetylase substrates and that butyrate inhibits their deacetylation, just as in the case of other HMG proteins and nucleosomal core histones.
Highlights
From the Department of Cell Biology, The Rockefeller [9] chromatin
Recent data have indicated that HMG-14 and Subsequentperchloricacidextraction of thenuclei, HMG-17 can partially inhibit the activity of protein deacetyfollowed by selective acetone precipitation, CM-Sephadex ion exchange chromatography, andgel filtration yieldedradioactivelylabeled high mobilitygroup (HMG) proteins HMG-14 and HMG-17 in pure form
Radioactive Labeling and Isolation of HMG Proteins-The HMG proteins were preparedfromduckerythrocytes which had been incubatedwithsodium["Hlacetate in the presence and absenceof sodium butyrate.A final purification step employing Sephadex G-50 gel filtration proved to be an effective method for separating either HMG-14 or HMG-17 from contaminatinpgroteins which remained in small amounts following CM-Sephadex chromatography.The result FIG. 1
Summary
Enzymatic Deacetylation of Radioactive HMG Proteins-Identification of the Sites of Acetylation in HMG-14 and The ability of [:'H]acetate-labeled HMG proteins to serve as HMG-17-Automated Edman degradation through the first substrates for calf thymus deacetylasewas tested by incuba- Obtained for HMG-1 and histone H4 were virtually identical Such high levels of deacetylase activity would be expected to to those reported earlier [28],with 40% and 92% of the total complicate studies of [3H]acetyllysineformation in the HMG counts released, respectively, after a 1-h incubation with the proteins as well as in erythrocyte histones.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.