A series of ion-exchange resins were tested for their ability to enrich (ADPR)n-proteins (from isolated nuclei) with respect to other proteins. It was discovered that factors in addition to ion-exchange were involved in the process of enrichment. Methodology was developed such that DEAE Sephadex could be used for the enrichment of (ADPR)n-proteins in good yield. In order to test the effectiveness of the method for proteins ADP-ribosylated in vivo, a procedure was devised whereby cells labelled with [3H]-adenosine could be analysed quantitatively for (ADPR)n. Using this procedure proteins ADP-ribosylated in vivo were found to be substantially enriched from other cellular proteins by DEAE Sephadex chromatography. However, contamination with radioactive nucleic acid made analysis of the enriched proteins difficult. Electrophoresis proved unsuitable in this case but by using gel permeation chromatography, (ADPR)n-proteins of between 8,000 and 40,000 daltons were indicated. When cells were treated with 500M dimethyl sulphate, an enhancement of the ADP-ribosylation of an 8,000 dalton protein was discovered. When DEAE Sephadex chromatography was applied to permea-bilised cells incubated with [3H] -NAD, electrophoresis of the enriched fraction identified a range of acceptor proteins from 15,000 to 160,000 daltons in size. Prior treatment of cells with dimethyl sulphate increased the level of ADP-ribosylation in all bands, but particularly those of low (approximately 8,000 daltons) molecular weight.
|Date of Award||1983|