Isolation of adenosine diphosphoribosyltransferase by precipitation with reactive red 120 combined with affinity chromatography

Apparent Role of Adenosine Diphosphoribosyl Transferase in the Development of Mytilus edulis and the Inhibition of Differentiation by Ligands of the Enzyme Protein

“…separation of mono-ADP-ribosylated ADPRT of Mytilus is shown when 100 n M NAD was the substrate concentration (5,17,18) and the activation of mono-ADP-ribosylation by octamer C is illustrated in Figure 2, Lane 2. In the presence of 200 pMNAD and octamer C, the enzyme was poly(ADP-ribosylated) and its migration retarded due to oligomeric (ADPR)n, as apparent from Figure 2, Lane 3.…”

“…After further extraction for 20 min with mild stirring, the cell debris were removed by centrifugation at 5000g at 4"C, and the supernatant was loaded onto a hydroxylapatite column of 20-ml bed volume and developed as described (1 7). The eluted protein solution was desalted and fractionated on a benzamide-Sepharose afinity column as described for the thymus enzyme (17). The resulting Table I.…”

“…As a marker protein, ADPRT purified from calf thymus by the method of Buki et a/. (17) (0.2 pg) was placed into Lane 2 (Fig. 2B).…”

“…2B). Following electrophoresis, the proteins were transblotted onto nitrocellulose sheets and the ADPRT immunostained as described earlier (17). Lane 1, M. edulis ADPRT; Lane 2, calf thymus ADPRT.…”

“…trophoretic enzyme assays were the same as described earlier (17,18). Coenzymic DNA (2-to 4-kb doublestranded DNA) was the byproduct of enzyme isolation (1 7) and was further purified by phenol extraction.…”