Rafael Alvarez-Gonzalez

[50] Determination of in Vivo levels of polymeric and monomeric ADP-ribose by fluorescence methods

Publisher Summary This chapter describes the determination of in vivo levels of polymeric and monomeric ADP-ribose by fluorescence methods. Two key features that are common to both methods and are crucial in providing the necessary selectivity and sensitivity include the utilization of immobilized boronate resins to selectively and quantitatively adsorb polymeric or monomeric ADP-ribose from cell or tissue extracts and the conversion of adenine-containing compounds to highly fluorescent 1, N 6 -etheno derivatives which can be quantified at the picomole level. For polymeric ADP-ribose, the adenine-containing compounds are formed by the enzymatic hydrolysis of the polymer to generate unique adenosine derivatives from all internal residues. For monomeric ADP-ribose, the method involves chemical release of intact ADP-ribose residues from protein and quantification following conversion to the 1, N 6 -etheno(ADP-ribose). The assay for measurement of polymeric ADP-ribose is designed for up to 10 8 tissue culture cells or for up to 1.3 g (wet weight) of tissue. A real difficulty with regard to the quantification of monomeric ADP-ribose residues covalently bound to proteins is the limited knowledge of the chemical nature of the linkages that exists in vivo . Enzymes from eukaryotic sources have been purified that can catalyze the covalent attachment of single ADP-ribosyl residues to acceptor proteins via N-glycosylic linkages to the guanidino group of arginine residues.

Nuclear matrix associated poly(ADP-ribose) metabolism in regenerating rat liver

We have examined a possible role for protein poly(ADP‐ribosylation) during in vivo DNA replication by studying the metabolism of poly(ADP‐ribose) in the nuclear matrix fraction from normal and regenerating rat liver. This fraction contains the newly replicated DNA and thus allows for the examination of the events closely associated with the replication process. It was found that 55% of the total nuclear protein‐bound poly(ADP‐ribose) and 15–35% of the total nuclear poly(ADP‐ribose)‐polymerase activity were tightly associated with this subnuclear compartment in normal liver. Surgical removal of two‐thirds of the liver initiated a time‐dependent decrease in nuclear matrix associated polymers of ADP‐ribose and poly(ADP‐ribose) polymerase activity which reached a minimum of 40% of control livers after 24 h, before returning to normal levels at 41 h post‐partial hepatectomy. In contrast, the total levels of poly(ADP‐ribose) in intact liver and the total polymerase activity of isolated nuclei exhibited a 2‐fold increase over basal levels. These results are consistent with the conclusion that the nuclear matrix is a major poly(ADP‐ribosylation) site within the nucleus and that this metabolic reaction may be closely connected with the events modulating DNA replication in this fraction.

Poly (3’-deoxyADP-ribosyl)ation of Proteins in Liver Chromatin Isolated from Rats Fed with Hepatocarcinogens

We have recently found that 3’-deoxyNAD is a good substrate for poly(ADP-ribose)polymerase (PADPRP) (1,2). In fact, we observed that PADPRP makes small linear oligomers of 3’-deoxyADP-ribose with an average size of 4 ADP-ribose residues. The main advantage of this approach is that the highly branched and complex polymers of ADP-ribose synthesized with NAD (3) are not observed. Therefore, the electrophoretic identification of poly(ADPribosyl)ated-polypeptides following incubation of biological samples possessing PADPRP activity with [32P] 3’-deoxyNAD is facilitated.

Up-Regulation of Two Distinct p53-DNA Binding Functions by Covalent Poly(ADP-ribosyl)ation: Transactivating and Single Strand Break Sensing

We used a [32P] p53 sequence-specific oligodeoxynucleotide and Electrophoretic-Mobility-Shift-Assays to monitor p53 DNA sequence-specific binding with p53-R267W, a nonbinding point mutant; and p53-Δ30, a deletion-mutant which lacks the carboxy-terminus that recognizes DNA-strand-breaks. Recombinant p53 and poly(ADP-ribose)polymerase-1 (PARP-1) were incubated with labeled βNAD+ with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and βNAD+ and p53 concentration(s). Since p53-Δ30 was efficiently labeled, poly(ADP-ribosyl)ation target site(s) of wt-p53 must reside outside its carboxy-terminal-domain. The poly(ADP-ribosyl)ation of p53-Δ30 did not diminish its DNA binding; Instead, it enhanced DNA-sequence-specific-binding. Therefore, we conclude that DNA-sequence-specific-binding and DNA-nick-sensing of mutant-p53 are differentially regulated by poly(ADP-ribosyl)ation.

Proteolytic Degradation of Poly(ADP-ribose)polymerase by Contaminating Proteases in Commercial Preparations of DNAse I

The stimulation of poly(ADP-ribose)polymerase (PADPRP) activity by the addition of agents that induce DNA damage and formation of DNA strand-breaks in vitro and in vivo is well established (1). Not surprisingly, DNAse I is commonly added enzyme to stimulate PADPRP activity in biological samples such as cell ghosts (2) and nuclei preparations (3). A high level of DNA-strand breaks helps to accurately determine the amount of PADPRP activity in these samples.

Amino Acid Specific Modification of Poly(ADP-ribose)polymerase with Monomers and Polymers of ADP-ribose

The primary protein target for poly(ADP-ribose) modification in mammalian chromatin is poly(ADP-ribose)polymerase (PADPRP) itself (1,2). However, our current understanding of this reaction at the biochemical level is very limited. Here, we have utilized 2’ and 3’-deoxyNAD analogs as substrates for the amino acid-specific covalent modification of PADPRP with monomers and polymers of ADP-ribose. Specific mono(ADP-ribosyl)ation of PADPRP at arginine residues was achieved by incubating pure polymerase with mono(ADP-ribosyl)transferase A (3) from turkey erythrocytes and 2’-deoxyNAD as an ADP-ribosylation substrate (4,5). In contrast, the auto[poly(ADP-ribosyl)ation] of PADPRP was performed with 3’-deoxyNAD. Utilization of this NAD analog is advantageous because it does not alter the physicochemical properties of the polymerase upon modification (6,7).

In Vitro ADP-Ribosylation Utilizing 2′Deoxy-NAD+ as a Substrate

The majority of the mono(ADP-ribosyl) transferases identified to date in animal tissues (1–5) are characterized by their specific modification of the guanidinium group of arginine residues. In contrast, poly(ADP-ribose) polymerase is known only to modify carboxylate groups on protein acceptors, i.e., glutamate (6–9), carboxy-terminal lysine (8) and aspartate (10). Both classes of enzymes have identical substrate stereospecificity in which the β-configuration of the anomeric carbon of NAD+ is converted to the α-configuration in the product (11, 12). No major differences between these two classes of enzymes in substrate structural requirements have been documented. The present study identifies a difference in behavior of an NAD+:arginine mono(ADP-ribosyl) transferase from turkey erythrocytes (2) and poly(ADP-ribose) polymerase from calf thymus (13) toward 2′dNAD+ as a substrate.