Mono ADP-ribosyl Transferase Activity Research Articles

Biochemical characterization of mono ADP ribosyl transferase activity of human sirtuin SIRT7 and its regulation

SIRT7, an epigenetic modulator is related to several important cellular processes like aging, genome stability, and metabolism. The mechanistic and regulatory aspect of this enzyme needs to be explored. SIRT7 contains a conserved catalytic core with long flanking N- and C-terminal extensions. We find that the N terminus is involved in substrate binding, thus also in its dual enzyme activity i.e. deacetylation and ADP ribosylation. The C-terminus is not essential for its catalysis. Mutation of certain residues at the active site suggests that mono ADP-ribosylation and deacetylation are two distinct activities of SIRT7. In this study, we also find that the SIRT7 enzyme can specifically transfer a single moiety of ADP ribose on other nuclear proteins, with a preference for NAD+. For this, the ADPr transfer follows the enzymatic reaction mechanism. Nicotinamide and certain metal ions have a significant negative effect on this mono ADP ribosylation process. A comparison of these dual activities suggests SIRT7's preference for the mono ADPr transfer over its deacetylation of H3K18Ac. Mono ADP ribosylation in cells is often linked to different metabolic disease conditions. This kind of modification of transcription factors, p53 and ELK4 by SIRT7 may play a key role in maintaining the tumor phenotype. Thus, SIRT7 becomes an important therapeutic hotspot for drug designing against several diseases. Finally, we can also relate SIRT7 to the DNA repair process through ADP ribosylation of one of its key players, PARP1. Here, SIRT7 positively regulates the PARP1 activity.

An ELISA method to estimate the mono ADP-ribosyltransferase activities: e.g in pertussis toxin and vaccines

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.

An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites.

Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and [32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and [32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since [14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process.

NAD: Guanidino group specific mono ADP-ribosyltransferase activity in skeletal muscle

The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcolemma and sarcoplasmic reticulum from pig skeletal muscle contains NAD:dependent mono ADP-ribosyltransferase activity toward the guanidine analog, P- nitrobenzylidine aminoguanidine. No or little activity could be found in the sarcolemma or sarcoplasmic reticulum derived from canine cardiac muscle. Seventy percent of activity extracted from rabbit skeletal muscle is localized in the sarcoplasmic reticulum. The enzyme has a pH optimum of 7.4, and KM of 0.5 mM and 0.35 mM for NAD and p-nitro benzylidine aminoguanidine, respectively. Inorganic phosphate, KCl, and guanidine derivatives inhibit the reaction. Incubation of the sarcoplasmic reticulum or glycogen pellet with (adenylate-32P) NAD or [adenosine-14C(U)]-labeled NAD results in the incorporation of radioactivity into proteins. A large number of proteins are labeled in the sarcoplasmic reticulum fraction. The major labeled band in the glycogen pellet corresponds to a protein of molecular weight of 83 K.

Assay of mono ADP-ribosyltransferase activity by using guanylhydrazones

Guanylhydrazones of p-nitrobenzaldehyde and methylglyoxal serve as acceptors of ADP-ribosyl groups for the reactions catalyzed by cholera toxin. The absorption spectrum of the ADP-ribosylated p-nitrobenzylidine aminoguanidine is similar to that of a 1:1 mixture of ADP-ribose and p-nitrobenzylidine aminoguanidine. Results from fast atom bombardment mass spectrometry prove that the product is mono-ADP-ribosylated. ADP-ribosylation lowers the p K a of the p-nitrobenzylidine aminoguanidine by 0.7–0.8 pH unit. Assay methods are developed for measuring the ADP-ribosyltransferase reaction by following the rate of disappearance of p-nitrobenzylidine aminoguanidine by high-performance liquid chromatography or spectrophotometrically by monitoring the absorbance increase at 370 nm accompanying ADP-ribosylation of p-nitrobenzylidine aminoguanidine. The high-performance liquid chromatographic system can be utilized to measure ADP-ribosyltransferase activity in animal tissues. By using this procedure, the presence and quantitation of an ADP-ribosyltransferase in a homogenate of rabbit skeletal muscle is reported.