ADP-ribose acceptors

West RE Jr, Moss J, Vaughan M et al. (1985) Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site. In J. Biol. Chem. 260 14428-14430. 

This assay, which does not require the addition of lipids or detergent, measures the ability of CT or LT to ADP-ribosylate itself in the absence of another ADP-ribose acceptor (except water). ARF added to this assay will also be ADP-ribosylated, but the ADP-ribosylation of it or CT under these conditions does not appear to decrease CT activity or the ability of ARF to stimulate CT (Tsai ef a/., 1991). Reaction products are separated by SDS-PAGE and analyzed by autoradiography as described for Assay 1 (Section 2.3). This assay is the most sensitive of all those listed here and can be used to verify toxin activity observed in other assays. 

Histones have been reported to be required for stimulation of synthetase activity in the presence of DNA (43, 60, 93,100,105,117,125, 148,158,159,220,221,233). As noted, however, near maximal rates of poly(ADP-ribose) formation could be obtained utilizing poly(dA) poly(dT), or "active DNA in the absence of added histones (83). Stimulation of poly ADP-ribosylation was observed when histones were added to intact (100%) or partially denatured calf thymus DNA (400%). Yoshihara suggested (83) that added histone binds to denatured DNA and masks its inhibitory action. Histones did not activate by serving as ADP-ribose acceptor in the enzyme reaction catalyzed by the purified bovine thymus poly(ADP-ribose) synthetase. It was known, however. 

Holtlund J, Lemtland R, Kristensen T (1983) Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase. Eur J Biochem 130 309-314... 

ADP-ribosyltransferase catalyzes the transfer of the ADP-ribose moiety of NAD to acceptors, as arginine and other guanidino compounds and proteins, and forms mono-(ADP-ribose)-acceptor adducts. In eukaryotes, this enzyme was first detected in turkey erythrocyte by Moss and associates who went on to purify and characterize the enzyme [1]. 

I detected some minor ADP-ribose acceptors with apparent mol. wts. of 100,000, 140,000, and 150,000 [5]. The identity of these polypeptides have not been clarified. 

It may be asked whether the changes in the pattern of poly(ADP-ribose) modified proteins are specific for myoblast differentiation. To answer this question we tested a myoblast clone that had lost its capacity to fuse. This clone (Nf-1) was isolated from the E63 myoblast cell line [1 ]. Cultures of Nf-1 were maintained for 8 days and were analyzed for poly(ADP-ribose) modified proteins. Figure 3 shows that the pattern of modified proteins isolated from the nonfusing variant is similar to that of prefusion E63 myoblasts (5 days). The 116 kD modified protein is present in both cultures, whereas the changes in poly(ADP-ribose) acceptor(s) accompanying differentiation are not observed. We have recently observed that the 116 kD modified protein is also formed if differentiation of E63 myoblasts is inhibited by DMSO or UV light. 

Kinetic analysis revealed that the Michaelis constant for NAD increased with increasing concentrations of the ADP-ribose acceptor agmatine (Fig. 1). This result is inconsistent... 

There is a class of mono-ADP-ribosyltransferases that are distinguished by their ability to utilize as ADP-ribose acceptors the free amino acid arginine, other simple guanidino compoimds, and proteins. Several transferases of this type were identified in and purified from turkey erythrocytes (1-3). The enzymes displayed different physical, kinetic and regulatory properties and were localized to the soluble, membrane and nuclear compartments (1-3). Similar NAD arginine ADP-ribosyltransferases have been observed in other tissues and organ systems from a variety of species (1-5). The enzymes are found in vimses, bacteria and animal cells (1-7). 

Poly(ADP-ribosyl)ation at 200 p,M NAD+ as substrate with an excess of whole thymus histones as additional poly(ADP-ribose) acceptors, probably simulating conditions prevailing in chromatin, yielded a less complicated kinetics (Fig. 2). In this system duplex C WAS the best synthetic coenzyme, whereas the activation curve of a sDNA remained sigmoidal. In the cell nucleus, the concentration of DNA is far greater than that of the poly(ADP-ribose) transferase, therefore the observed inhibition in the in vitro model... 

Since the major poly(ADP-ribose) acceptor protein in chromatin and in intact cells is poly(ADP-ribose) polymerase (5, 11, 12), we have studied the interaction of the highly modified polymerase with native and Hl-depleted chromatin in order to define its role as a potential modulator of chromatin structure. When poly(ADP-ribosyl)ated native chromatin was resolved on sucrose gradients, it was found that most of the modified enzyme was... 

We report here on the mechanism of DNA strand breakage and poly(ADP-ribosyl)ation by AO in comparison to the methylating agent N-methyl-N -nitro-N-nitrosoguanidine (MNNG) in promotable JB6 clone 41 cells. We also summarize first results on the identity of the nuclear proteins which serve as poly(ADP-ribose) acceptors in response to AO. We chose xanthine/xanthine oxidase (X/XO) as a source of extracellular AO because it produces a mixture of superoxide and H2O2 which resembles the oxidants released by macrophages and mouse epidermal cells JB6 because promotable (clone 41) and non-promotable (clone 30) variants are available (9). 

Histone- and non-histone poly(ADP-ribose)-acceptors following treatment with active oxygen or MNNG. For the... 

We had shown previously that the phorbolester promoter TPA increased poly(ADP-ribosyl)ation of e same histones in mouse embryo fibroblasts C3H10T1/2 but in this case H2B was the major acceptor (38). H2B was also the major poly(ADP-ribose) acceptor in Yoshida hepatoma cells following exposure to the methylating agent dimethylsulfate (32). However, it should be recognized that no immunoblots were used for the identification of the individual histones in the previous woik. 

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