ADP-ribosyltransferase is highly conserved: purification and characterization of ADP-ribosyltransferase from a fish and its comparison with the human enzyme

Summary

Covalent modification of proteins by ADP-ribosylation is a major mode of protein regulation in eukaryotic cells. ADP-ribosyltransferases have been characterized from mammals but little is known about these enzymes in lower vertebrates. We purified an ADP-ribosyltransferase (E.C. 2.4.2.30) from trout (Salmo trutta faris) by affinity chromatography and characterized it. The 11700-fold purified activity shows a major protein band at a molecular mass of 75000 kDa in a SDS-polyacrylamide gel.In situ reactivation of SDS gels showed the 75000 kDa protein to be enzymatically active, and additional enzymatically active bands at molecular masses of 115000, 90000 and 87000 kDa, respectively. The enzyme is capable of poly-ADP-ribosylation. It crossreacts with affinity purified antibodies raised against human poly(ADP-ribose)synthetase and, except for the temperature optimum, its properties strongly resemble the mammalian enzymes, indicating the conserved character of nuclear ADP-ribosyltransferases. The trout enzyme is DNA- and histone-dependent, has an optimal pH between 8 and 9 and an apparentK _m for NAD⁺ of 24 μM. The temperature optimum is 10°C compared with 25°C for the human enzyme. Known ADP-ribosyltransferase inhibitors also inhibit the enzyme from trout. ribosyl-)residues from NAD⁺. It plays a central role in processes affecting DNA function such as DNA repair, recombination, differentiation, tumorigenic cell transformation and cell proliferation (Pekala and Moss 1983; Ueda and Hayaishi 1985; Creissen and Shall 1982; Borek et al. 1984; Lunec 1984; Cleaver et al. 1985; Tseng et al. 1987). It seems to be a central controller of cell physiology (Loetscher et al. 1987; Schweiger et al. 1987). ADP-ribosylation is high in proliferating cells whereas it is low in differentiated cells (Surowy and Berger 1983). Due to the important functions of ADP-ribosyltransferase this enzyme has been studied extensively but almost exclusively in mammals (Ueda and Hayaishi 1985). Only little is known about ADP-ribosyltransferases of lower vertebrates and of species of lower phylogenetic families. Hence it appeared of interest to us to study this enzyme in a fish. Here we report the purification to homogeneity and the characterization of ADP-ribosyltransferase from trout liver. We compared the fish enzyme with the human one and found very similar enzymatic properties and immunological crossreactivity. This indicates that ADP-ribosyltransferases are highly conserved.