Hector Juarez-Salinas

[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.

A new highly sensitive and selective chemical assay for poly(ADP-ribose)

Abstract A new method for the measurement of poly(ADP-ribose) is described. Poly(ADP-ribose) was separated from the bulk of cellular RNA and DNA by quantitative adsorption to dihydroxyboryl-Sepharose. The polymer was digested with venom phosphodiesterase and bacterial alkaline phosphatase to yield the unique nucleoside 2′ → 1″ ribosyladenosine from internal residues. This nucleoside was converted by reaction with chloroacetaldehyde to a highly fluorescent 1- N 6 -etheno derivative which was separated from interfering substances by reversed phase high-pressure liquid chromatography and picomole amounts were quantified by fluorescence. Control experiments are presented which demonstrate that the assay is highly specific for poly(ADP-ribose).

Simultaneous determination of linear and branched residues in poly(ADP-ribose)

Methodology for the routine and simultaneous determination of the linear and branched residues of poly(ADP-ribose) is described. The main features of the procedure consist of the isolation of poly(ADP-ribose) by affinity chromatography; enzymatic digestion of the polymer to the unique nucleosides ribosyladenosine and diribosyladenosine which are derived from linear and branched residues, respectively; formation of fluorescent derivatives of ribosyladenosine and diribosyladenosine; and identification and quantification of these compounds by high-pressure liquid chromatography coupled with fluorescence detection. A variation on the methodology which allows the detection and quantification of ribosyladenosine and diribosyladenosine without formation of their fluorescent derivatives is also presented. Analyses of several cell lines for their capacity to synthesize poly(ADP-ribose) with a branched structure showed that the proportion of branched sites was constant (0.7-0.8%) in each of the cell lines.

Alteration of poly(ADP-ribose) metabolism by hyperthermia.

The intracellular levels of poly(ADP-ribose) in cultured mouse cells were increased in response to hyperthermic treatment (43 degrees C). When hyperthermia was combined with other stressful treatments such as with ethanol and/or an alkylating agent, a dramatic synergistic increase in polymer levels was observed. The effect of hyperthermia did not appear to be related to the presence of DNA strand breaks. A possible involvement of poly(ADP-ribose) metabolism in the general cellular response to environmental stress is suggested.

Environmental Stress and the Regulation of Poly(ADP-Ribose) Metabolism

The nuclear metabolism of poly(ADP-ribose) involves at least three enzymatic activities, poly(ADP-ribose) polymerase, poly(ADP-ribose) glycohydrolase, and protein-mono(ADP-ribose) lyase. Although each of these enzymes has been purified and studied, we still have a poor understanding of poly(ADP-ribose) metabolism and its regulation in intact cells. Two features of poly(ADP-ribose) metabolism in intact cells are illustrated in Fig. 1. First, the occurrence of environmentally-induced DNA damage results in a rapid elevation of the intracellular levels of polymer. Current evidence from both in vitro and in vivo studies argues that this alteration is regulated at the level of poly(ADP-ribose) polymerase and that the activating factor is the appearance of DNA strand breaks [1, 2]. A second feature is that polymers are rapidly turning over in vitro. This indicates that individual polymers are required only transiently to fulfill their function or that the coordinated synthesis and degradation of the polymer results in a longer term change in chromatin structure.