| 4-(Oxyacetyl)phenoxyacetic acid (OAPA) was synthesized and its reactions with methylguanidine, arginine, N;OAPA was coupled to a solid support via its carboxyl group, producing a resin capable of immobilizing proteins specifically via arginine residues. As expected, the capacity of this resin to bind proteins varies according to the arginine content of the proteins, with some proteins able to bind at levels greater than 10 mg protein/packed ml resin. Proteins thus immobilized are stably linked.;A ubiquitin-affinity column, prepared using these techniques, reversibly bound the previously described enzyme ubiquitin carboxyl-terminal hydrolase. The enzyme could be eluted from the column with either a salt gradient or with free ubiquitin.;This affinity resin was then applied to the purification of a newly identified enzyme possessing ubiquitin ethyl ester hydrolytic activity. Combined with the previously described steps of anion exchange and gel filtration chromatography, this affinity chromatographic step, along with one final gel permeation chromatographic separation, yielded active enzyme which displays one band when analyzed by SDS-PAGE.;As expected for an arginine specific reagent, OAPA inactivates equine liver alcohol dehydrogenase in a time-dependent manner. The rate of this inactivation is decreased in the presence of the substrate NADH. OAPA also reacts with arginine residues of the protein ubiquitin. Four distinct OAPA-modified species of ubiquitin were isolated, and the nature of the modifications determined by studying peptide maps and the incorporation of radiolabeled OAPA. The modifications affect the ability of ubiquitin to participate in the various steps of ubiquitin-dependent proteolysis, and define a face of ubiquitin involved in interactions with one or more proteins of the proteolytic complex. |
