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)

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.
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PMID:Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation. 211 Nov 67

A novel ADP-ribosyltransferase is present in the cytosolic fraction of various cells. The kinetic behavior and physical properties of this enzyme's activity are clearly distinguished from other known cytosolic ADP-ribosyltransferases. Agents that release nitric oxide, such as sodium nitroprusside, greatly stimulated this activity, although this effect was dependent on the presence of intact thiol groups. Dithiothreitol, reduced glutathione, or cysteine was needed for activation of the enzyme, while N-ethylmaleimide inhibited enzyme activity. High concentrations of phosphate had a slight stimulatory effect, while high concentrations of sodium chloride and thiocyanate were inhibitory. ATP also inhibited this activity. This cytosolic ADP-ribosyltransferase is clearly distinguished from other known and characterized cytosolic transferases. Its activation by biologically active nitric oxide suggests an important role for this enzymatic activity.
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PMID:Properties of a novel nitric oxide-stimulated ADP-ribosyltransferase. 211 69

In bovine aortic smooth muscle, about 50% of total GTP-binding activity was present in the cytosol fraction. A major GTP-binding protein (G protein) with a Mr value of about 21,000 (21K G) in this fraction was purified to near homogeneity and characterized. 21K G bound maximally about 0.8 mol of [35S]guanosine 5'-(3-O-thio)triphosphate/mol of protein with a Kd value of about 20 nM. 21K G showed GTPase activity with a turnover number of about 0.007 min-1. 21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of 21K G. 21K G and the bovine brain rhoA gene product (rhoA p21) were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography and migrated at the same positions on two-dimensional gel electrophoresis. These results indicate that the major G protein in bovine aortic smooth muscle cytosol is rhoA p21.
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PMID:Identification of a major GTP-binding protein in bovine aortic smooth muscle cytosol as the rhoA gene product. 211 95

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.
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PMID:Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule. 211 1

Membranes purified from cells of Streptomyces griseus strain 52-1 possess an ADP-ribosyltransferase activity. The enzyme transfers the ADP-ribose moiety of NAD to one major membrane protein of Mr 32,000 and 2-3 minor proteins of larger molecular weights. The effects of inhibitors on the ADP-ribosyltransferase activity proves that the reaction is enzymatic and suggests that the enzyme ADP-ribosylates the guanidine group of arginine. The kinetics of liberation of ADP-ribose during alkaline hydrolysis of the modified proteins is consistent with the arginine-ADP-ribose bond. This is the first report of ADP-ribosylation of proteins in a Gram-positive bacterium.
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PMID:ADP-ribosylation of membrane proteins of Streptomyces griseus strain 52-1. 212 Jan 8

Incubation of membranes of human promyelocytic leukemia HL-60 cells with [32P]NAD led to ADP-ribosylation of several proteins including a 38 kDa protein by endogenous ADP-ribosyltransferases. The ADP-ribosylation of the 38 kDa protein was distinctly different from others on the basis of pH dependency and heat stability at 50 degrees C, suggesting that there are at least two endogenous ADP-ribosyltransferases. It was enhanced by CTP, but not affected by ATP, GTP and UTP, whereas it was inhibited by GTP gamma S. [alpha-32P]CTP bound to the 38 kDa protein immobilized on a nitrocellulose sheet, indicating that the 38 kDa protein which bound CTP is strongly ADP-ribosylated by an endogenous ADP-ribosyltransferase.
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PMID:CTP-dependent endogenous ADP-ribosylation of a 38 kDa protein in HL-60 cell membranes. 212 32

The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3. The possible interaction of recombinant rho A proteins expressed in E. coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea. As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea. GDP reduced the light-induced inhibition, while GTP[S] and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.
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PMID:Interaction of recombinant rho A GTP-binding proteins with photoexcited rhodopsin. 212 2

Activated macrophages synthesize and release numerous tumoricidal soluble factors that can be divided into receptor- or nonreceptor-dependent agents. Tumor necrosis factor (TNF) would be an example of the former. In our experimental model the killing of EL4 thymoma cells by syngeneic activated macrophages involves, but not exclusively, TNF. Our results show that approximately 50% of the anti-EL4 activity expressed by macrophages can be specifically inhibited with rabbit anti-mouse TNF antibody. EL4 variants resistant to the lytic activity of TNF were still susceptible to macrophage-mediated lysis. A tumor-promoting phorbol ester, TPA, rendered TNF-sensitive and -insensitive EL4 cells resistant to M phi-mediated lysis. However, TPA down-regulated TNF-specific binding sites on both TNF-sensitive and -resistant cell surface membranes, suggesting that resistance to TNF involves postligand:receptor events. Tumor cell G-protein involvement (ADP-ribosylation), as a result of TNF-TNF receptor interactions, was investigated. The results showed that pertussis toxin was cytotoxic against TNF-sensitive and -resistant EL4 cells but not against TPA-treated target cells. Inhibitors of ADP-ribosyltransferase inhibited pertussis toxin cytotoxicity and macrophage-mediated lysis but did not interfere with recombinant TNF lytic activity.
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PMID:TPA induction of EL4 resistance to macrophage-released TNF: role of ADP-ribosylation in tumoricidal activities of TNF and other factors. 213 20

The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.
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PMID:Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity. 214 19

We have previously shown that there are multiple GTP-binding proteins (G proteins) with Mr values of about 20,000 in bovine brain membranes and identified one G protein with a Mr of 20,000 as the rho gene product. We have also shown that this rho gene product is ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1. In the present studies, we have purified another G protein with a Mr of about 21,000 to near homogeneity from bovine brain membranes by several column chromatographies and identified it as the rhoA gene product. Further analysis of the amino acid sequence of the G protein, which we have purified and identified as the rho gene product previously, has revealed that this G protein is the rhoB gene product. The rhoA gene product binds maximally about 0.9 mol of [35S]guanosine 5'-(3-O-thio) triphosphate (GTP gamma S)/mol of protein with a K d value of about 20 nM. [35S]GTP gamma S-binding to the rhoA gene product is inhibited by pretreatment with N-ethylmaleimide. The rhoA gene product hydrolyzes GTP to liberate Pi with a turnover number of about 0.01 min-1. Moreover, the rhoA gene product is ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type Cl. About 0.3 mol of ADP-ribose is maximally incorporated into 1 mol of the rhoA gene product. The ADP ribosylation of the rhoA gene product does not affect its GTP gamma S-binding or GTPase activity. These properties of the rhoA gene product are similar those of the rhoB gene product described previously. These results together with the earlier observations indicate that there are at least two rho gene products (rhoA, B) among three members of the rho gene family (rhoA, B, C) in bovine brain membranes and that both of them are ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1.
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PMID:Purification and characterization from bovine brain membranes of a GTP-binding protein with a Mr of 21,000, ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1--identification as the rhoA gene product. 215 99


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