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)

The substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography. On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points. The major peak of the substrate was further purified. It was purified about 1,800-fold with a recovery of 2.2% by the above procedures. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000. The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein. A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol. GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP. Thus, the ADP-ribosylation substrate is a GTP-binding protein. This protein, designated Gb (b for botulinum), is widely distributed in various tissues. It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.
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PMID:Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein. 313 28

Ligand binding to the platelet-derived growth factor (PDGF) receptor initiates a complex and diverging cascade of signaling pathways. GTP-binding proteins with intrinsic GTPase activity (G-proteins) frequently link cell surface receptors to intracellular signaling pathways, but no close associations of the PDGF receptor and any small G-proteins, nor any such associations activated by ligand binding to the receptor have been previously reported. We demonstrate that a small GTP-binding protein binds specifically to the murine and human PDGF type-beta receptor. In response to PDGF-BB stimulation, there is an increase in the amount of labeled small G-protein associated with the PDGF type-beta receptor. The GTP-binding protein did not undergo ligand-induced association with a mutant receptor protein that was unable to bind ATP. Proteolytic cleavage analysis, together with two-dimensional separation techniques, identified the small G-protein specifically associating with the PDGF type-beta receptor after ligand binding as a member of the Rho family. This was confirmed by demonstration that the small G-protein coimmunoprecipitated by the anti-PDGF receptor antibody was a substrate for the ADP-ribosyltransferase C3 exoenzyme. Thus, the PDGF type-beta receptor may form a complex with one or more small G-proteins upon binding PDGF-BB, and the Rho small G-protein is likely to be an important component of the proteins making up the multimeric signaling complex of the PDGF type-beta receptor.
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PMID:A small GTP-binding protein, Rho, associates with the platelet-derived growth factor type-beta receptor upon ligand binding. 761 21

Phospholipase D (PLD) is believed to play an important role in cell signal transduction: PLD catalyzes the hydrolysis primarily of phosphatidylcholine (PC) to produce phosphatidic acid that may serve as a lipid second messenger. Although the mechanism of PLD activation has not yet been fully understood, a member of the low molecular weight GTP-binding protein (small G protein) superfamily, ADP-ribosylation factor (ARF), has been identified as a PLD-activating factor. In addition to ARF, we found that RhoA, another member of the small G proteins, activated rat brain PLD, and that ARF and RhoA synergistically stimulated the enzyme activity. When proteins of bovine brain cytosol were subjected to anion exchange column chromatography and then reconstituted with rat brain PLD partially purified from the membranes, fractions eluted at 60 mM NaCl, where ARF was not detected, activated the enzyme in a guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This PLD-stimulating activity seemed to be attributed to a small G protein RhoA. Evidence provided includes the findings that: (1) the partially purified preparation of the PLD-activating factor by subsequent column chromatographies contained a 22 kDa substrate for botulinum C3 exoenzyme ADP-ribosyltransferase; (2) the 22 kDa protein strongly reacted with anti-RhoA antibody; (3) the treatment of the partially purified PLD-activating factor with C3 exoenzyme and NAD together, but not individually, significantly inhibited the PLD-stimulating activity; and (4) recombinant isoprenylated RhoA activated the PLD. On the contrary, recombinant nonisoprenylated RhoA failed to activate the PLD. Interestingly, the partially purified PLD-activating factor and ARF synergistically activated rat brain PLD, and recombinant isoprenylated RhoA could substitute for the partially purified preparation. These results conclude that rat brain PLD is regulated by RhoA in concert with ARF, and that the post-translational modification of RhoA is essential for its function as the PLD activator.
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PMID:Regulation of phospholipase D by low molecular weight GTP-binding proteins. 890 66

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins and members of the Ras superfamily, originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin and more recently recognized as critical participants in vesicular trafficking pathways and phospholipase D activation. ARD1 is a 64-kDa protein with an 18-kDa carboxyl-terminal ARF domain (p3) and a 46-kDa amino-terminal extension (p5) that is widely expressed in mammalian tissues. Using recombinant proteins, we showed that p5, the amino-terminal domain of ARD1, stimulates the GTPase activity of p3, the ARF domain, and appears to be the GTPase-activating protein (GAP) component of this bifunctional protein, whereas in other members of the Ras superfamily a separate GAP molecule interacts with the effector region of the GTP-binding protein. p5 stimulated the GTPase activity of p3 but not of ARF1, which differs from p3 in several amino acids in the effector domain. After substitution of 7 amino acids from p3 in the appropriate position in ARF1, the chimeric protein ARF1(39-45p3) bound to p5, which increased its GTPase activity. Specifically, after Gly40 and Thr45 in the putative effector domain of ARF1 were replaced with the equivalent Asp and Pro, respectively, from p3, functional interaction of the chimeric ARF1 with p5 was increased. Thus, Asp25 and Pro30 of the ARF domain (p3) of ARD1 are involved in its functional and physical interaction with the GTPase-activating (p5) domain of ARD1. After deletion of the amino-terminal 15 amino acids from ARF1(39-45p3), its interaction with p5 was essentially equivalent to that of p3, suggesting that the amino terminus of ARF1(39-45p3) may interfere with binding to p5. These results are consistent with the conclusion that the GAP domain of ARD1 interacts with the effector region of the ARF domain and thereby stimulates GTP hydrolysis.
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PMID:Interaction of the GTP-binding and GTPase-activating domains of ARD1 involves the effector region of the ADP-ribosylation factor domain. 902 91

A monoclonal antibody, named C302, was prepared and characterized against botulinum ADP-ribosyltransferase C3 exoenzyme that inactivates RhoA GTP-binding protein, resulting in the neurite outgrowth of human neuroblastoma GOTO cells. C302 bound not to the smaller fragments derived from the protease-treated C3 exoenzyme but to the intact C3 exoenzyme. It seems that the C302 epitope may depend on the three-dimensional structure of C3 exoenzyme molecule. C302 depressed the enzymatic and biological actions of C3 exoenzyme. The dose-dependent depression pattern of C302 on the enzyme activity was similar to that to the biological one. C302 turned the neurite-bearing shape of the C3 exoenzyme-treated GOTO cells into the intact shape. By using of C302 mAb and C3 exoenzyme, the research concerning GTP-binding proteins would be improved.
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PMID:Characterization of a neutralizing monoclonal antibody against botulinum ADP-ribosyltransferase, C3 exoenzyme. 1239 99


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