Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.
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PMID:Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study. 245 68

A non-histone acceptor protein for hen liver nuclear ADP-ribosyltransferase was purified to an apparently homogeneous state through salt extraction and chromatography on hydroxyapatite, phenyl-Sepharose, carboxy-methyl-cellulose, Sephadex G-75, phenyl 5-PW, mono S and Radial PAK C18. This protein was termed p33. The ADP-ribosylation of p33 was enhanced more than 60-fold by double-stranded DNA. Single-stranded DNA, RNA and poly(L-glutamate), but not deoxyribonucleotide, were partially effective. DNA-dependent ADP-ribosylation was also observed when whole histones were used as acceptor. DNA required for the maximal ADP-ribosylation depended on the dose of the acceptor protein; the optimal mass ratio of DNA to the acceptor protein was 1:1 with both p33 and whole histones. DNA decreased the Km for NAD and concomitantly increased the Vmax value, but did not alter the Km for p33. These results are consistent with the idea that p33 may participate in chromatin processes such as replication or transcription, through modification by nuclear ADP-ribosyltransferase.
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PMID:DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in hen liver nuclei. 249 38

Enzymes have been identified in animal tissues that catalyze the mono(ADP-ribosyl)ation of arginine and proteins. Since these NAD:arginine ADP-ribosyltransferases under physiological conditions do not appear to catalyze the degradation of the product ADP-ribose-arginine, the possibility was investigated that a different family of enzymes exists that cleaves the ADP-ribose-arginine linkage. An enzyme was identified in and partially purified from turkey erythrocytes that catalyzed the degradation of ADP-ribose-[14C]arginine synthesized by a salt-activated NAD:arginine ADP-ribosyl-transferase, resulting in the release of a radiolabeled compound that was characterized chromatographically and by amino acid analysis as arginine. This putative arginine product was converted in a reaction dependent on NAD and the NAD:arginine ADP-ribosyltransferase to a compound exhibiting properties characteristic of ADP-ribose-arginine. Action of cleavage enzyme on [adenine-U-14C]ADP-ribose-arginine resulted in the release of a radiolabeled compound that behaved chromatographically like [adenine-U-14C]ADP-ribose. Since degradation of ADP-ribose-arginine appears to generate an arginine moiety that is a substrate for the NAD:arginine ADP-ribosyltransferase, it appears that ADP-ribosylation may be a reversible modification of proteins.
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PMID:Reversibility of arginine-specific mono(ADP-ribosyl)ation: identification in erythrocytes of an ADP-ribose-L-arginine cleavage enzyme. 299 36

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.
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PMID:Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix. 300 Jul 77

We have examined a variety of conditions for solubilizing and electrophoresing cell proteins in order to define optimum conditions for studying proteins modified by ADP-ribosylation. We have identified conditions in which proteins can be quantitatively extracted from cells in an undegraded form with the protein-ADPribose linkages intact. Effective measures include boiling cells briefly (4 min) in the presence of 2% SDS and 2 M urea at pH 6.8. Both SDS and urea were present in the 6-18% gradient polyacrylamide gel matrix used for electrophoresis. Under these conditions good resolution of proteins of a wide molecular-weight range is obtained. This system has been used to compare protein ADP-ribosylation in non-transformed and polyma virus-transformed baby hamster kidney (BHK) fibroblasts, since the latter cells have a greater NAD+ ADP-ribosyltransferase activity (measured in isolated nuclei and permeabilized cells). Addition of DNAase to permeabilized BHK cells over the range 10-150 micrograms led to a progressively greater activation of transferase compared with controls. When PyY cells were used, however, maximum activation was achieved with only 10 micrograms of DNAase, further additions producing a successively smaller activation relative to control cells without added nuclease. There were also differences between these cells in response to salt. Addition of NaCl (to about 0.3 M) to BHK cells resulted in various extents of transferase activation, whereas any addition of NaCl to the incubate of permeabilized PyY cells decreased transferase activity. These different enzyme activities between this transformed and non-transformed cell line are for the most part not reflected in the protein modification profiles seen on autoradiograms of acrylamide gels after electrophoresis 32P-labelled proteins. A variety of proteins are modified and their molecular weights depend on the NA concentration in the permeabilized cell incubation. At 0.5 microM NAD+ there were two major acceptors with Mr values of 14 kDa and 30 kDa, and at 100 microM NAD+, three major acceptors, with Mr values of 19 kDa. 45 kDa and greater than 170 kDa. NAD concentrations of between 1 microM and 100 microM had no further effect on protein ADP-ribosylation profiles, except for the protein(s) of Mr greater than 170 kDa, pointing to a critical difference around 0.5-1.0 microM substrate. In some experiments, however, a difference was observed in the intensity of radioactivity in two bands. This may represent two different proteins, or a single protein modified to different extents.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A gel-electrophoretic analysis of protein ADP-ribosylation in polyoma virus-transformed and non-transformed BHK-21/C13 fibroblasts. 300 86

An ADP-ribosyltransferase from turkey erythrocytes, which catalyzes the mono(ADP-ribosylation) of guanidino compounds such as arginine and of many purified and crude cellular proteins, appears to exist both in high-activity, histone-independent and low-activity, histone-dependent forms. At low salt concentrations, the activity of the transferase with agmatine as acceptor was less than 10% that observed in the presence of 200 mM NaCl. In the absence of salts, ADP-ribosylation of agmatine was stimulated greater than 10-fold by histones, and activity approached that observed with high salt concentration; under these conditions, the histones did not serve as ADP-ribose acceptors themselves. Histone also activated the highly purified ADP-ribosyltransferase from human erythrocytes. Enzyme activity was increased in the presence of salt and was then relatively independent of histones. DNA was not required for the stimulation of ADP-ribosylation by histone; incubation of the transferase and histone with DNase did not significantly decrease enzymatic activity. Additional DNA in the assay decreased the effect of histone. The erythrocyte ADP-ribosyltransferase from diverse species thus appears to exist in two forms: one is dependent on histones for activity and one which, in the presence of salt, has high intrinsic activity and is independent of histone. The fact that the active forms of the transferase generated in the presence of salt or histone have similar catalytic activity suggests that these forms of transferase may be identical. It would appear that the enzymatic activity of transferase from different species may be controlled by histones.
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PMID:Histone-dependent and histone-independent forms of an ADP-ribosyltransferase from human and turkey erythrocytes. 627 74

Chromatin-bound ADP-ribosyltransferase from adult hen liver nuclei was purified to a homogeneous state through salt extraction, gel filtration, hydroxyapatite, phenyl-Sepharose, Cm-cellulose, and DNA-Sepharose. The ADP-ribosyltransferase has a pH optimum at 9.0 and does not require DNA for reaction. The purified enzyme has a molecular weight of 27,500 +/- 500. Agmatine sulfate, arginine methyl ester, histones, and casein proved to be effective acceptors for the ADP-ribose molecule. Among histones, H3 was most active, followed by H2a, H4, and H2b, in that order, the lowest activity seen with H1. With all the acceptors tested, the rate of nicotinamide release was in excess of the ADP-ribosylation. However, changes in the ratio of nicotinamide release to ADP-ribosylation seemed to depend on concentrations of the acceptor used. ADP-ribose-whole histones X adducts formed by ADP-ribosyltransferase served as initiators for poly(ADP-ribose) synthesis when these adducts were incubated in the presence of NAD, DNA, Mg2+, and the purified poly(ADP-ribose) synthetase, in which poly(ADP-ribose) formation can occur.
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PMID:ADP-ribosyltransferase from hen liver nuclei. Purification and characterization. 631 19

Two NAD: arginine ADP-ribosyltransferases (transferase "A" and "B") were identified in turkey erythrocytes and purified to homogeneity. Both transferases in the presence of NAD catalyzed the ADP-ribosylation of arginine, other low molecular weight guanidino compounds and proteins. ADP-ribosyltransferase A was activated by chaotropic salt or histone. Activation was associated with the disaggregation of an inactive, rapidly sedimenting, high molecular weight species to a protomeric form of approximately 28,000 daltons; this protomer in equilibrium aggregate transition was rapidly reversible. In the presence of salt, the Km's for NAD and arginine methyl ester were 15 microM and 1.3 mM, respectively; the turnover number for the purified enzyme was approximately 9,900 mol X min-1 X mol enzyme-1. ADP-ribosyltransferase B exhibited a substrate specificity clearly distinct from that of transferase A. Transferase B had a Mr of 32,000, slightly larger than that of the transferase A protomer. The activity of transferase B was unaffected by histone and inhibited by chaotropic salts; its Km's for NAD and arginine methyl ester of 36 microM and 3 mM, respectively, were similar to those obtained with transferase A. These studies are consistent with the presence of two different NAD: arginine ADP-ribosyltransferases in turkey erythrocytes exhibiting distinct kinetic, regulatory, and physical properties.
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PMID:Characterization of NAD: arginine ADP-ribosyltransferases in animal tissues. 641 12

The activity of an NAD:arginine ADP-ribosyltransferase was stimulated 4-6-fold by lysolecithin; lysolecithins containing long-chain fatty acids such as stearoyl (C18) and palmitoyl (C16) were more effective than those with shorter chains: C14 greater than C12 greater than C10 congruent to C8. The analogue lacking a fatty acid at C-1, alpha-glycerophosphocholine, was inactive as were choline, lysophosphatidic acid, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lecithin, phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine. Activation of the transferase was, however, also observed with certain nonionic (e.g., Triton X-100) and zwitterionic [3-[ ( cholamidopropyl ) dimethylammonio ]-1-propanesulfonate] detergents. The transferase was shown previously to be stimulated by chaotropic salts or histones; in the presence of maximally effective concentrations of lysolecithin, salt, and histone, the activity was similar to that observed in the presence of histone or salt alone. Maximal activation by lysolecithin and detergents was less than that observed with either salt or histone. It appears that activation by lysolecithin shows significant differences from that observed previously with histones or salt and can be mimicked by certain nonionic and zwitterionic detergents.
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PMID:Activation of an erythrocyte NAD:arginine ADP-ribosyltransferase by lysolecithin and nonionic and zwitterionic detergents. 642 4

A cDNA clone pCZ1, with a 1.1 kb insert, was isolated from a NaCl-adapted tobacco cell cDNA library that encodes an apparently full-length 29 kDa protein (251 amino acids) with a calculated pI of 5.7. The encoded peptide had a high amino acid sequence identity with bovine 14-3-3 protein which was originally found as an abundant protein in the animal central nervous system. Recently, proteins with sequence identity to 14-3-3 protein have also been found in plants, insects and yeast, and appear to have diverse physiological functions. Similar to the bovine brain 14-3-3 protein, the recombinant pCZ1 protein stimulated ADP-ribosylation of protein substrate by ADP-ribosyltransferase from the plant and animal pathogenic bacterium Pseudomonas aeruginosa. This recombinant protein also inhibited protein kinase C activity in vitro. Southern blot analyses indicated that most likely five genes encoding 14-3-3-like proteins are present in tobacco. The pCZ1 cDNA insert hybridized to a single mRNA of 1.1 kb from cultured tobacco cells. The level of this mRNA transcript in tobacco cells was downregulated upon adaptation to NaCl but was unaffected by short-term treatment with NaCl, ABA or ethylene. In tobacco plants, expression of transcript that hybridized to pCZ1 was tissue specific, and was most abundant in roots and flower parts. Monoclonal antibody raised against GF14 protein, a maize protein with substantial sequence identity with 14-3-3 protein detected two bands on SDS-PAGE of total proteins from unadapted tobacco cells and only a single band from cells adapted to NaCl. The GF14 antibody was also used to illustrate that the G-box element of a salt-induced gene is associated with a 14-3-3-type protein.
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PMID:A NaCl-regulated plant gene encoding a brain protein homology that activates ADP ribosyltransferase and inhibits protein kinase C. 800 Apr 27


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