Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.     

Nonenzymic adenosine 5'-diphosphate ribosylation of poly(adenosine diphosphate ribose)

Poly(adenosine 5'-diphosphate ribose) [poly(ADP-ribose]) is spontaneously ADP-ribosylated when it is incubated with nicotinamide adenine dinucleotide, especially in 0.5 M NaCl and at an alkaline pH. The ADP-ribose residues are monomeric and are attached to the middle of polymer chains. The linkage is similar to, and may be identical with, that of the branch points that are created in cells. RNA is also spontaneously ADP-ribosylated, but not DNA.     

Noncovalent interactions of poly(adenosine diphosphate ribose) with histones.

Covalent linkage of ADP-ribose polymers to proteins is generally considered essential for the posttranslational modification of protein function by poly(ADP-ribosyl)ation. Here we demonstrate an alternative way by which ADP-ribose polymers may modify protein function. Using a highly stringent binding assay in combination with DNA sequencing gels, we found that ADP-ribose polymers bind noncovalently to a specific group of chromatin proteins, i.e., histones H1, H2A, H2B, H3, and H4 and protamine. This binding resisted strong acids, chaotropes, detergents, and high salt concentrations but was readily reversible by DNA. When the interactions of variously sized linear and branched polymer molecules with individual histone species were tested, the hierarchies of binding were branched polymers greater than long, linear polymers greater than short, linear polymers and H1 greater than H2A greater than H2B = H3 greater than H4. For histone H1, the target of polymer binding was the carboxy-terminal domain, which is also the domain most effective in inducing higher order structure of chromatin. Thus, noncovalent interactions may be involved in the modification of histone functions in chromatin.     

A peplomycin-supersensitive cell line lacking activation of poly(adenosine diphosphate ribose) synthetase by peplomycin

In peplomycin-supersensitive Chinese hamster lung cells, the increase in poly(ADP-ribose) synthesizing activity following peplomycin treatment was significantly reduced as compared with the parental lung cells, suggesting that peplomycin-supersensitive lung cells may have some deficiency in DNA repair. On the contrary, peplomycin-supersensitive ovary cells, which undergo increased DNA damage induced by peplomycin, showed normally increased poly(ADP-ribose) polymerizing activity compared with the parental ovary cells. Relationship between poly(ADP-ribose) polymerase and peplomycin sensitivity was discussed.     

Bovine thymus poly(adenosine diphosphate ribose) polymerase. Physical properties and binding to DNA

Purified bovine thymus poly(adenosine diphosphate ribose) polymerase is a monomeric protein with a single polypeptide chain having a molecular weight of approximately 130,000, determined by sodium dodecyl sulfate-gel electrophoresis, analytical ultracentrifugation, and gel filtration. A high frictional ratio (1.81) indicated that the molecule has an elongated shape, or a high solvation, or both. The enzyme is a basic protein (pI 9.8), and amino acid analysis showed a relatively high lysine content. The enzyme activity is dependent on double-stranded DNA and is solely correlated with single- or double-stranded breaks on the DNA. Filter binding assay technique showed that the enzyme-activating efficiency of DNA correlated sufficiently with its enzyme-binding efficiency. Thus, a very high enzyme-activating efficiency of a DNA fraction (active DNA) which was separated from the crude enzyme fraction is mainly due to its high enzyme-binding efficiency. It was also shown that single-stranded DNA and heparin had a strong inhibitory effect on the binding of the enzyme to double-stranded DNA, whereas competitive inhibitors did not affect the binding, We interpret these results to indicate that the binding of the enzyme to double-stranded DNA is a prerequisite step to its catalytic activity and has a dual function: (a) to position the enzyme on specific binding sites such as single- or double-stranded breaks on the DNA, and (b) to induce an active conformation of the enzyme.     

Synthesis of poly(adenosine diphosphate ribose) in synchronized Chinese hamster cells.

Chinese hamster ovary cells were synchronized by mitotic selection and used to study the relation of poly(adenosine diphosphate ribose) synthesis to DNA synthesis and the different phases of the cell cycle. DNA synthesis was measured in cells rendered permeable to exogenously supplied nucleotides. Poly(ADPR) synthesis was also measured in permeable cells in the presence of both minimum and maximum DNA damage. The maximum DNA damage was produced by treating the cells with saturating concentrations of DNase. As anticipated, the DNA synthesis complex showed its maximum activity during S phase and showed 4–5-fold less activity during the other phases of the cell cycle. The basal level of poly(ADPR) synthesis was elevated during G1, fell to its lowest level during S phase, then increased during G2 and rose to its highest level during G1. The DNase responsive activity of poly(ADPR) synthesis was relatively constant thru the cell cycle but showed a peak at the end of S phase; then the activity decreased during the subsequent G2-M period.     

In vivo ischemia and storage injury cause alterations in the activity of poly(adenosine diphosphate ribose) synthetase

The objective of this study was to determine if DNA damage caused by ischemic insult (blood depletion) causes an alteration in the activity of endogenous mouse kidney poly(ADP-ribose) synthetase. The results show that kidneys made nonviable by warm (37 degrees C) in vitro ischemia (organ storage to study the effects of blood loss at normal body temperature) and in vivo ischemia (surgical depletion of the blood supply by arterial clamping) exhibit decreased levels of enzyme activity. Kidneys made nonviable by cold (0 degrees C) storage injury (organ storage as utilized for transplantation), however, possess elevated levels of enzyme activity. The DNA isolated from ischemic kidneys was shown to have a stimulatory effect upon exogenous calf thymus poly(ADP-ribose) synthetase. Also, electron microscopy analysis of DNA from ischemic kidneys showed that cold storage injury leads to the formation of large (average size = 500 bases) single-stranded regions. The results suggest that the activities of both endogenous and exogenous poly(ADP-ribose) synthetase are related to the nature of DNA damage resulting from ischemic insult.     

Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures

Two hybridomas producing monoclonal antibodies to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] were established. One antibody, 10H (IgG3, kappa), bound to most of the poly(ADP-Rib) preparation, which consisted of molecules of various sizes of more than 20 ADP-Rib residues. The binding of this antibody was inhibited by not only poly-(ADP-Rib) but also a monomer unit of poly(ADP-Rib), Ado(P)-Rib-P. The sites protected by antibody 10H were isolated and analyzed by hydrolysis with alkaline phosphomonoesterase and then snake venom phosphodiesterase. The sites contained the same amounts of monomer units and branched portions [Ado(P)-Rib(P)-Rib-P] as the original poly(ADP-Rib) molecules but a lower average number of branched portions per molecule than in the original molecules. The other antibody, 16B (IgM, lambda), reacted with only 50% of the radioactive poly(ADP-Rib), and its binding was not inhibited by a monomer unit. This antibody protected 25% of all the poly(ADP-Rib) molecules from hydrolysis by snake venom phosphodiesterase. The protected sites contained twice as many branched portions per molecule as the original poly(ADP-Rib) molecules. These results show that the two monoclonal antibodies recognize different structures of poly-(ADP-Rib); 10H antibody recognizes the linear structure with ribose-ribose linkages, and 16B antibody may recognize specific structures, including the branched portions of poly-(ADP-Rib).     

Demonstration of high molecular weight poly (adenosine diphosphate ribose).

An electrophoretic system was established that resolves poly(adenosine diphosphate ribose), enzymatically synthesized polymer from NAD+, by size difference of one residue on polyacrylamide gel. The existence of a polymer of at least 65 residues was demonstrated by band counting in this system. The polymer showed a heterogeneous size distribution on the electrophoregram, and the molecular weight of the largest polymer was deduced to be more than 4.5 X 10(5) daltons. The discrepancy between the size, estimated by electrophoresis, and the chain length, determined by the ratio of total radioactivity to that derived from the terminus, suggests that the polymer has a branched structure.     

A new method for the assay of poly(adenosine diphosphate ribose) glycohydrolase activity.

Poly(adenosine diphosphate ribulose) [poly(ADP-Rib)] glycohydrolase activity was determined by measuring the amount of ADP-Rib hydrolyzed from polymers of ADP-Rib as substrate. In principle, the method consists of incubating oligomers or polymers of [¹⁴C]ADP-Rib with testis glycohydrolase. The reaction was stopped by the addition of a suspension of Dowex 1X-2 formate in H₂O (1:3, $\text{v}\text{v}$) which adsorbed monomers and oligomers of ADP-Rib. The adsorbed [¹⁴C]ADP-Rib was selectively extracted from the resin with 6 m formic acid. The amount of [¹⁴C]ADP-Rib was estimated by measuring the radioactivity in aliquots of formic acid extract. Oligomers or polymers of ADP-Rib can be utilized as substrates since the reaction rates were the same with either compound.A method to determine phosphodiesterase and glycohydrolase activities was established. These two enzymic activities were distinguished by treating the products of the reactions with alkaline phosphatase and by differential extraction of the adsorbed reaction products on Dowex with 0.5 m and 6 m formic acid.     

Evidence for two variants of poly(adenosine diphosphate ribose) glycohydrolase in rat testis.

The specific activity of rat poly(adenosine diphosphate ribose) glycohydrolase was higher in the testis than in the liver, brain, spleen or kidney. The enzyme was found primarily in the soluble fraction of the testis. When the soluble enzyme was chromatographed on phosphocellulose, the activity eluted in two peaks, at 0.22 and 0.34 m KCl, respectively, referred to in the present study as enzyme A and B. Enzyme A has an optimal pH of 7.25 and was stimulated by 150 mm KCl. The optimal pH of enyzme B was 6.5, but it was not stimulated by KCl. For maximal activity both enzymes required 10 mm 2-mercaptoethanol, and they were strongly inhibited by 100 μmp-chloromercuribenzoate. The K_m values of enzyme A and B for poly(adenosine diphosphate ribose) were 1.52 and 0.70 μm, respectively. Ribose 5′-phosphate, guanosine 3′,5′-monophosphate, adenosine 3′,5′-monophosphate and adenosine diphosphate ribose inhibited both enzymes. The two latter nucleotides behave as noncompetitive inhibitors. Denatured DNA and the homopolypurines poly(G), poly(I) and poly(A) were very potent inhibitors of both glycohydrolases. The mode of hydrolysis of poly(adenosine diphosphate ribose) by glycohydrolases A and B was exoglycosidic, yielding adenosine diphosphate ribose as the final product.     

Poly(adenosine diphosphate ribose) glycohydrolase in Physarum polycephalum.

Poly(adenosine diphosphate ribose) glycohydrolase, which has thus far only been found in mammalian tissues, was found for the first time in the primitive eukaryotic slime mold Physarum polycephalum. The hydrolytic product of poly(adenosine diphosphate ribose) with this enzyme was identified as adenosine diphosphate ribose by paper and thin-layer chromatography. It is likely that the enzyme caused exoglycosidic hydrolysis. The optimal pH of this enzyme was 6.0, and the K_m value was 4.3 μm, as adenosine diphosphate ribose residues of polymer. Adenosine diphosphate ribose, ADP and ATP at a concentration of 0.1mm strongly inhibited the enzyme activity. 3′,5′-Cyclic AMP was inhibitory at a concentration of 1mm. The molecular weight of this enzyme was estimated to be 57,000.