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Biomedical subjects

I Just

Publications and source records attributed to I Just.

At least 91 records · Page 5Linked to original sources
PubMed1992

ADP-ribosylation of gelsolin-actin complexes by clostridial toxins.

ADP-ribosylation of the 1:1 (G-A) and 1:2 (G-A-A) gelsolin-actin complexes by Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin was studied. Iota toxin ADP-ribosylated actin in the G-A complex from human platelets as effectively as skeletal muscle actin. The Km for NAD (4 microM) was identical for both substrates. C2 toxin ADP-ribosylated actin in the G-A complex with lower efficacy than nonmuscle actin from platelet cytosol. In the G-A-A complex both actin molecules were ADP-ribosylated by iota toxin. The G-A complex bound ADP-ribosylated actin (Ar) to form the G-A-Ar complex in which the weakly bound actin is ADP-ribosylated. Vice versa, ADP-ribosylated 1:1 gelsolin-actin complex (G-Ar) was able to bind unmodified actin to yield the G-Ar-A complex. ADP-ribosylation did not change the nucleation activity of either the G-Ar complex or the G-Ar-A complex. When monomeric actin was added to the G-A-Ar complex, polymerization of actin was delayed by about 10 min. According to a quantitative kinetic analysis, the delay of polymerization corresponded to the rate of dissociation of ADP-ribosylated actin from the G-A-Ar complex. This suggests that the nucleation activity of the G-A-A complex is inhibited by ADP-ribosylation of the weakly bound actin and that the inhibition can be removed by dissociation of ADP-ribosylated actin from the G-A-Ar complex.

ADP Ribose Transferases↗
PubMed1991

Interaction of mastoparan with the low molecular mass GTP-binding proteins rho/rac.

Mastoparan, which has been shown to active G proteins, inhibits the ADP-ribosylation of 20 kDa human platelet membrane proteins catalyzed by Clostridium botulinum exoenzyme C3 half-maximally and maximally (90%) at 20 and 100 microM concentrations, respectively. Inhibition of ADP-ribosylation was enhanced by GTP-gamma S. Mastoparan increased GTP hydrolysis by porcine brain rho protein and stimulated GTP binding in a concentration dependent manner. The data suggest that mastoparan not only interacts with heterotrimeric G proteins but also with low molecular mass GTP-binding proteins of the rho/rac family.

ADP Ribose Transferases↗
PubMed1991

Suppression of cytoskeletal rearrangement in activated human neutrophils by botulinum C2 toxin. Impact on cellular signal transduction.

Botulinum C2 toxin, a binary toxin which selectively ADP-ribosylates nonmuscle G-actin, was used to evaluate the role of cytoskeletal rearrangement in ligand-evoked signal transduction and secretory processes in human neutrophils (polymorphonuclear leukocyte). Preincubation with the combined toxin components reduced the basal F-actin content and nearly completely suppressed the actin assembly initiated by the peptide and lipid chemoattractants formyl-methionyl-leucyl-phenylalanine, platelet activating factor, and leukotriene B4. Superoxide production and elastase secretion were increased markedly under these conditions. Concomitantly, ligand-elicited phosphoinositide hydrolysis was augmented with particular increase in inositol monophosphate. This was paralleled by a severalfold amplification of diacylglycerol formation and sustained elevation of cytosolic calcium. The toxin-effected amplification of postreceptor events and secretory responses was most pronounced in response to formyl-methionyl-leucyl-phenylalanine greater than platelet activating factor greater than leukotriene B4. All metabolic and secretory effects in C2 toxin-pretreated cells were sensitive to pertussis toxin inhibition. In conjunction with the recent finding of unchanged formyl-methionyl-leucyl-phenylalanine receptor binding and dissociation dynamics under influence of C2 (Norgauer, J., Just, I., Aktories, K., and Sklar, L. A. (1989) J. Cell Biol. 109, 1133-1140), the present investigation suggests amplification of postreceptor events as a major mechanism underlying C2 toxin-related increase in polymorphonuclear leukocyte secretory responses. Cytoskeletal rearrangement, putatively linked to phosphoinositide turnover and calcium transients, thus appears to be operative in temporal and/or spatial limitation of chemoattractant-evoked cellular signal transduction.

Actins↗
PubMed1991

Autoregulatory control of actin synthesis in cultured rat hepatocytes.

ADP-ribosylation of actin by Clostridium botulinum C2 toxin resulted in a depolymerization of filamentous F-actin and an increase of monomeric G-actin in cultured hepatocytes. Simultaneously the de novo synthesis of actin was largely reduced, while the synthesis of albumin and of other proteins was not significantly impaired. The specific decrease of actin mRNA to 30% of the control indicates a down-regulation of actin synthesis at a pretranslational level. On the other hand, treatment with the mycotoxin phalloidin resulted in an increase of F-actin and a decrease of monomeric G-actin. Under this condition the de novo synthesis of actin was specifically enhanced and the level of actin mRNA was increased to 600% of the control. The data suggest an autoregulatory control of the actin synthesis.

Actins↗
PubMed1991

ADP-ribosylation and de-ADP-ribosylation of the rho protein by Clostridium botulinum exoenzyme C3. Regulation by EDTA, guanine nucleotides and pH.

Pretreatment of rho protein purified from pig brain cytosol with EDTA (3 mM) for 10 min at 30 degrees C inhibited its ADP-ribosylation by Clostridium botulinum C3 ADP-ribosyltransferase by more than 90%. The EDTA effect was not caused by alteration of C3. GDP or GDP beta S present during the pretreatment period completely prevented the decrease in ADP-ribosylation with half-maximal and maximal effects at 3 and 300 microM, respectively. GTP or GTP gamma S were less efficacious in preventing the decrease in ADP-ribosylation, but were more potent (half-maximal and maximal effects at 0.1 and 3 microM, respectively). [32P]ADP-ribose incorporated in pig brain rho by C3 was de-ADP-ribosylated by the enzyme in the presence of nicotinamide and at low pH. Concomitantly, [32P]NAD was formed. The pH optima for ADP-ribosylation and de-ADP-ribosylation were pH 7.5 and 5.5, respectively. De-ADP-ribosylation was most efficient with nicotinamide, less effective with 3-acetylpyridine and not observed with 3-aminopyridine, 4-aminopyridine, 4-acetylpyridine and isonicotinic acid. As observed for the ADP-ribosylation, the de-ADP-ribosylation by C3 was maximal with the GDP-bound form of rho and blocked after EDTA treatment.

ADP Ribose Transferases↗
PubMed1991

[ADP ribosylation of actin--a cytotoxic principle of bacterial toxins].

The ADP-ribosylation of actin is the pathobiochemical mechanism by which various clostridial toxins affect the eukaryotic target cell. The toxins are binary in structure and consist of a binding component and an enzyme component with ADP-ribosyltransferase activity. Probably endocytosis-mediated the binding component transfers the clostridial ADP-ribosyltransferase into the target cell, whereupon G-actin but not F-actin is ADP-ribosylated. The ADP-ribosylated actin is incapable of polymerization but binds to the barbed ends of actin filaments to inhibit polymerization of non-modified actin. Thereby the ADP-ribosylation of actin destroys the cellular architecture of the microfilament network. The ADP-ribosylating toxins are novel tools to study the physiological functions of actin.

Actins↗
PubMed1991

Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADP-ribosyltransferase.

The effects of Clostridium botulinum C3 ADP-ribosyltransferase and of Clostridium botulinum C2 toxin were studied on the cytoskeleton of rat hepatoma FAO and human glioma U333 cells. After treatment of these cells for 24 to 48 h with C3 (3-30 micrograms/ml), the actin microfilaments disappeared, and the intermediate filament network was found to collapse, while microtubules remained intact. Similar alterations of the cytoskeletal filaments without affecting microtubules were induced by the actin-ADP-ribosylating C2 toxin. In FAO cells, C3 caused the rounding up of cells. Concomitantly, cytosolic 22 to 24 kDa proteins were ADP-ribosylated in a guanine nucleotide-dependent manner. Rounding up of cells and ADP-ribosylation of proteins in intact cells were observed at similar concentration of the transferase. These data suggest a role of the protein substrates of C3 in the regulation of the cytoskeletal integrity.

ADP Ribose Transferases↗
PubMed1990

Morphological alterations of Xenopus oocytes induced by valine-14 p21rho depend on isoprenylation and are inhibited by Clostridium botulinum C3 ADP-ribosyltransferase.

Microinjection of the constitutively active recombinant Val-14 p21rho A into Xenopus oocytes induced dramatic morphological changes with redistribution of pigments from the animal pole resulting in spotted oocytes. The effects induced by Val-14 p21rho A were regulated by progesterone in a dose-dependent manner whereas prior ADP-ribosylation of the rho protein blocked its activity. About 30 min after microinjection, p21 rho was associated with the plasma membrane. The membrane association of p21rho and its biological activity were inhibited by lovastatin, an inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase. The findings suggest that membrane attachment and biological activity of p21rho depend on isoprenylation of the GTP-binding protein.

ADP Ribose Transferases↗
PubMed1990

ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin.

The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin.

ADP Ribose Transferases↗
PubMed1990

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin.

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.

ADP Ribose Transferases↗
PubMed1990

Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin.

Clostridium perfringens iota toxin belongs to a novel family of actin-ADP-ribosylating toxins. The effects of ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin on cytochalasin D-stimulated actin ATPase activity was studied. Cytochalasin D stimulated actin-catalysed ATP hydrolysis maximally by about 30-fold. ADP-ribosylation of actin completely inhibited cytochalasin D-stimulated ATP hydrolysis. Inhibition of ATPase activity occurred at actin concentrations below the critical concentration (0.1 microM), at low concentrations of Mg2+ (50 microM) and even in the actin-DNAase I complex, indicating that ADP-ribosylation of actin blocks the ATPase activity of monomeric actin and that the inhibitory effect is not due to inhibition of the polymerization of actin.

ADP Ribose Transferases↗
PubMed1990

Microinjection of recombinant p21rho induces rapid changes in cell morphology.

The rho proteins, p21rho, are ubiquitously expressed guanine nucleotide binding proteins with approximately 30% amino acid homology to p21ras, but their biochemical function is unknown. We show here that microinjection of constitutively activated recombinant rho protein (Val14rho) into subconfluent cells induces dramatic changes in cell morphology: 15-30 min after injection cells adopt a distinct and novel phenotype with a contracted cell body and finger-like processes still adherent to the substratum. Ribosylation of Val14rho with the ADP-ribosyltransferase C3 from clostridium botulinum, before microinjection, renders the protein biologically inactive, but it has no effect on either its intrinsic biochemical properties or on its interaction with the GTPase activating protein, rho GAP. Micro-injection of ribosylated normal rho, on the other hand, has a similar effect of injection of C3 transferase and induces complete rounding up of cells. We also report striking biochemical changes in actin filament organization when contact-inhibited quiescent 3T3 cells are injected with Val14rho protein. The effects induced by activation or inactivation of p21rho described here, suggest that the biological function of this protein is to control some aspect of cytoskeletal organization.

Adenosine Diphosphate Ribose↗
PubMed1990

Characterization of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin.

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin belong to a novel family of actin ADP-ribosylating toxins. ADP-ribosylation of actin inhibits actin polymerization and G-actin-associated ATPase activity. The ADP-form of actin is ADP-ribosylated at a higher rate than actin with bound ATP. ADP-ribosylation of actin is reversible, a reaction, which is accompanied by reconstitution of actin ATPase activity.

Actins↗
PubMed1989

Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments.

The effect of nonmuscle actin ADP-ribosylated by botulinum C2 toxin on the polymerization of nonmuscle actin was investigated in order to clarify whether nonmuscle actin is converted into a capping protein by ADP-ribosylation. ADP-ribosylated actin was found to decrease the rate of polymerization of actin filaments which are free at both ends. ADP-ribosylated actin turned out to have no effect on the rate or extent of polymerization at the pointed ends of actin filaments the barbed ends of which were capped by gelsolin. The monomer concentration reached at the final stage of polymerization was similar to the critical concentration of the pointed ends of actin filaments. The results suggest that nonmuscle actin ADP-ribosylated by botulinum C2 toxin acts as a capping protein which binds to the barbed ends to inhibit polymerization.

Actin Depolymerizing Factors↗
PubMed1989

Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product.

The 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 was purified from porcine brain cytosol by acetone precipitation, CM-Sephadex, octyl-Sepharose and TSK phenyl-5PW HPLC chromatography to apparent homogeneity. ADP-ribosylation of the protein was increased by guanine nucleotides (GTP, GDP, GTP gamma S, each 100 microM) but not by GMP, ATP or ATP gamma S. The purified 22 kDa protein bound maximally 0.9 mol [35S]GTP gamma S and hydrolyzed GTP with the rate 0.007 mol per mol protein. Amino acid sequences were obtained from two tryptic peptides, selected from an in situ digestion of Immobilon electrotransferred, gel purified ADP-ribosylated substrate. The two sequences obtained, cover 23 residues from the corresponding sequences in human rho.

ADP Ribose Transferases↗
PubMed1989

The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3.

The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.

ADP Ribose Transferases↗