Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP‐ribose) polymerase and the identification of a new enzyme inhibitory site

Abstract

The catalytic activity of highly purified poly(ADP-ribose) polymerase was determined at constant NAD+ concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: (i) graphic presentation of the activation of poly(ADP-ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and (ii) by calculating kn values for the deoxyribonucleotides.As determined by method i, auto-mono-ADP-ribosylation of the enzyme protein at 25 nM NAD+ was maximally activated at 1: 1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.41 sDNA/enzyme ratio, sDNA was the most active coenzyme for mono-ADP-ribosylation.At 200 PM NAD+, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono-ADP-ribosylation at a concentration 150-200 -times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii (kn values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA-dT)s exhibiting the weakest binding. The synthetic molecule 6-amino-1,2-benzopyrone (6-aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD+, identifying a new inhibitory site of poly(ADP-ribose) polymerase. Octadeoxyribonucleotide;Poly(ADP-ribose) polymerase; 6-Amino-1,Zbenzopyrone site

Coenzymatic Activity of Randomly Broken or Intact Double-stranded DNAs in Auto and Histone H1 Trans-poly(ADP-ribosylation), Catalyzed by Poly(ADP-ribose) Polymerase (PARP I)

“…18) during DNA replication has been established (20); thus the assumption of their physiologic existence is feasible. Present results lend support to an early observation reported in 1987 (21), which illustrates different modalities of activation of PARP I by octadeoxyribonucleotides of differing base sequence.…”

Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity

“…A 12 /pg portion of ADPRT and 5 jig of the synthetic DNA analogue (the octameric duplex C; cf. Hakam et al, 1987) were preincubated for 2 min in 100 mM-triethanolamine/HCl buffer, pH 8.0, then treated with glutaraldehyde (2.4 mM) for 10 min at room temperature in a volume of 20 ,l. At the end of the incubation 5,1u of aqueous hydrazine sulphate solution (to achieve 20 mm final concentration) was added to quench the unused glutaraldehyde. In control tests hydrazine sulphate was added before the glutaraldehyde.…”

Apparent Role of Adenosine Diphosphoribosyl Transferase in the Development of Mytilus edulis and the Inhibition of Differentiation by Ligands of the Enzyme Protein

“…The samples were frozen and thawed three times (by submerging in solid CO,/acetone mixture) and were sonicated with 6 x 10 bursts using a Branson sonifier (50% output) at 4OC, with 1 min cooling between each sonication cycle. Finally, the samples were centrifuged at 10,OOOg for 5 min and the supernatants assayed for ADPRT activity as described earlier, with 100 nM NAD as substrate (5,16).…”

“…Coenzymic DNA (2-to 4-kb doublestranded DNA) was the byproduct of enzyme isolation (1 7) and was further purified by phenol extraction. Benzamide was purchased from Sigma (St. Louis, MO) and 6-amino-1,2-benzopyrone was synthesized as described previously ( 16).…”