Clostridium botulinum C3 Exoenzyme and Studies on Rho Proteins

6.1 Introduction

The Rho family of small GTP-binding proteins functions to regulate the assembly of distinct actin structures in cells; Rho regulates stress fiber assembly, Rae regulates lamellipodia protrusion and Cdc42 stimulates protrusion of the plasma membrane to form filopodia (Ridley and Hall, 1992; Ridley et al., 1992; Nobes and Hall, 1995; Kozma et al., 1995). All three regulate attachment of cells to the extracellular matrix via adhesive integrin structures (Nobes and Hall, 1995).

We and others have used the bacterial exoenzyme C3 ADP-ribosyltransferase from Clostridium botulinum to analyze the signaling pathways controlled by the Rho GTPase. The introduction of C3 transferase into a variety of cell types causes them to lose their actin stress fibers, round up, and eventually detach from the underlying substrate (Rubin etal., 1988; Chardin etal., 1989; Paterson etal., 1990). The targets of C3 in cells are the three isoforms of the Rho protein, RhoA, RhoB and RhoC (Narumiya et al., 1988); the enzyme catalyzes the transfer of an ADP-ribose group from NAD+ to an asparagine residue at codon 41 of Rho (Aktories et al., 1989; Sekine et al., 1989) and renders the protein inactive (Paterson et al., 1990). Other Rho family members such as Rae and Cdc42 are essentially not substrates for C3 in vitro (Ridley et al., 1992; Just et al., 1992), and microinjection of C3 into cells does not affect the activity of either Rae or Cdc42 (Ridley et al., 1992; Nobes and Hall, 1995). The introduction of C3 transferase into cells has, therefore, been a valuable tool for assessing the specific cellular roles of the Rho GTPase.

6.2 Purification of Recombinant C3 Transferase from Escherichia coli

High levels of recombinant C3 transferase have been expressed in E. coli using the glutathione S-transferase (GST) gene fusion vector, pGEX-2T (Pharmacia LKB Biotechnology, Inc). The GST-C3 expression vector was constructed in the laboratory of Dr. Larry Feig (Tufts Univer-

sity School of Medicine, Boston, USA) as described in (Dillon and Feig, 1995), and introduced into the E. coli strain JM101 and stored as a glycerol stock at -70 °C.

During cloning, additional amino acids were introduced at the amino terminus of the protein; consequently, after cleavage of the GST fusion protein, the recombinant C3 is 26 amino acids longer than the enzyme purified from Clostridia. The N-terminal extension comprises 19 amino acids from the pUC19 polylinker and the last 7 amino acids of the signal peptide sequence of C3 in C. botulinum (Dillon and Feig, 1995). We have never compared directly, in the same microinjection experiments, the relative activities of recombinant C3 and that purified from C. botulinum. However, both preparations have been microinjected separately in this laboratory and we are unaware of any significant differences between them.

6.2.1 Purification of Recombinant GST-C3

We routinely grow 1 litre of E. coli containing the GST-C3 protein expression plasmid, and this yields approximately 2-3 mg of recombinant C3 protein.

1. L-broth (100 ml) containing 50 ^g/ml ampicillin is inoculated with bacteria containing the GST-C3 construct and incubated overnight at 37 °C in a bacterial shaker.

2. The following morning the culture is diluted 1:10 into fresh L-broth/ampicillin (room temperature) and incubated further with shaking in two 2-litre flasks (500 ml culture/flask) at 37 °C for 1 h.

3. GST-C3 fusion protein expression is induced by adding isopropyl-|3-D-thiogalactopyranoside (IPTG; Calbiochem) to 0.2 mM (1 ml of 0.1 M stock made in water and stored at -20 °C), and the culture is incubated with shaking for a further 3h.

4. The cells are pelleted by centrifugation (4000 r.p.m for lOmin at 4 °C) and resuspended (on ice) in 3 ml cold lysis buffer (50 mM Tris-HCI, pH 7.6, 50 mM NaCI, 5mM MgCI2, 1 mM dithiothreitol [DTT], and 1 mM phenylmethylsulfonyl fluoride [PMSF]).

5. Resuspended bacteria are lysed by sonication on ice using a small probe on an MSE Soniprep 150 sonicator at an amplitude of 12|xm (six bursts for 10s each), and the bacterial debris removed by centrifugation (10 000 r.p.m for 10 min at 4 °C).

6. Glutathione-agarose beads (1ml of a 1:1 suspension, Sigma G4510) are first washed with three changes of lysis buffer (5 ml), and the bacterial supernatant (about 4 ml) is added to the beads. The slurry is incubated for 30 min on a rotating wheel at 4 °C.

7. The beads, with bound GST-C3 fusion protein, are pelleted in a bench top centrifuge at 4000 r.p.m for 1 min and the supernatant removed and discarded. The beads are washed with 10 ml of cold lysis buffer (with 1 mM DTT but without PMSF) three times to remove unbound proteins.

6.2.2 Recovery of Cleaved C3 Transferase

1. The washed beads are transferred to a 1.5 ml microcentrifuge tube, resuspended in 0.5 ml of thrombin digestion buffer (50 mM Tris-HCI, pH 8.0, 150 mM NaCI, 2.5 mM CaCI2, 2mM MgCI2, 1 mM DTT) containing 5 units of bovine thrombin (Sigma T6634), and incubated on a rotating wheel overnight at 4 °C.

2. After thrombin digestion, the beads are pelleted in a microcentrifuge (1 min at 4000 r.p.m) and the supernatant is removed.

3. Any remaining protein associated with the beads is recovered with 0.5 ml of high salt buffer (50 mM Tris-HCI, pH 7.6, 150 mM NaCI, 5 mM MgCI2, 1 mM DTT) for a further 2 min at 4 °C, and the two supernatants combined. The efficiency of thrombin cleavage of GST-C3 approaches 90% (Fig. 1).

4. Thrombin is removed by adding 20 [xl of a 1:1 suspension of p-aminobenzamidine-agarose beads (Sigma A7155) to the supernatant and incubating for a further 30 min at 4 °C on a rotating wheel.

5. Finally, the beads with bound thrombin are pelleted in a microcentrifuge for 1 min at 4000 r.p.m.

30 21

Fig. 1. Purification of thrombin-cleaved C3 transferase. Samples loaded: GST-C3 protein bound to beads before thrombin cleavage (lane 1); protein remaining on beads after thrombin cleavage (mainly GST) (lane 2); C3 protein eluted from beads (lane 3)

30 21

6.2.3 Dialysis and Storage do not over-concentrate C3

I The supernatant (1 ml) containing C3 protein is first dialyzed against two changes of buffer (15 mM Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM MgCI2 and 0.1 mM DTT) at 4 °C for 1 h each. The protein is concentrated to approximately 500 ¡xl in an Amicon Centricon filter unit by centrifugation in a fixed angle rotor at 7000 r.p.m.

It is important not to over-concentrate C3, since it tends to precipitate at high concentration (>5mg/ml). Solutions of recombinant C3 prepared in this way generally contain 3-5mg/ml, and are suitable for microinjection. Small aliquots (20|xl) of the C3 transferase are stored at -20 °C. A working stock, of approximately 0.7-1 mg/ml in dialysis buffer, can be stored at 4 °C and dilutions made from this working stock at the time of microinjection.

6.2.4 Determination of Protein Concentration and Assay of Activity

The concentration of each C3 protein preparation is determined with a Biorad protein assay kit, using BSA as a standard. The relative activities of C3 transferase preparations can be determined by ADP-ribosylation of recombinant Rho protein or by microinjection. For the ribosylation assay, 10 ng of recombinant RhoA protein are incubated for 1 h at 37 °C in 50 [i\ of ADP-ribosylation buffer (50 mM Tris-HCI, pH 7.4, 50 mM NaCI, 5mM MgCI2, 0.3 mM GTP, 1 mM DTT, 1 nCi [32P]NAD) with varying amounts of C3 protein, ranging from 1 to 200 ng. Gel electrophoresis sample buffer is added, the samples are boiled, and proteins resolved by SDS-polyacrylamide gel electrophoresis (13.5 %). Protein is first visualized with Coomassie blue, and then the gel is destained with many washes to remove any unincorporated [32P]NAD. ADP-ribosylated rho is visualized by autoradiography.

Each new preparation of C3 protein is also assayed by microinjection into quiescent serum-starved Swiss 3T3 cells, followed by treatment with lysophosphatidic acid, LPA (lOOng/ml for 15 or 30min; Sigma L7260) to stimulate Rho and stress fibre assembly (see Section 6.6). The lowest concentration of C3 that still blocks the LPA effect provides a measure of the relative activity of different batches of C3.

6.3 Preparation of Swiss 3T3 Cells

Swiss 3T3 cells are maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), 0.11 g/l sodium pyruvate, 4.5 g/l glucose, lOOU/ml penicillin and 100^g/ml streptomycin at 37 °C with 10 % C02. Cells are grown to 90 % confluence in 80cm2 flasks and passaged (1:7) twice a week. We culture Swiss 3T3 cells up to passage 13 (our stock cells are described as passage 5) after which we discard and thaw a new stock of low passage (passage 5) cells. We find that when Swiss 3T3 cells are cultured for longer than passage 13 they show an increasing tendency not to become contact inhibited and quiescent; such populations retain use low passage cells polymerized actin structures (mainly stress fibres) on serum starvation (see Section 6.6).

We inject Swiss 3T3 cells in three different states: confluent, quiescent; subconfluent with serum; and subconfluent without serum.

1. Confluent, quiescent: The cells are prepared by seeding at high density (5 x 104 cells) onto 13 mm acid washed, baked glass coverslips in 15 mm diameter tissue culture wells (Nunc) containing lml DMEM/10% FCS. Prior to washing, glass coverslips are marked at their approximate midpoint with a cross, using a diamond pen, to facilitate localization of injected cells after staining. After 6-10 days, when the cells are quiescent, the medium is removed and replaced (without washing) with 1 ml DMEM (from powder stock; Gibco cat. no. 52100-021) supplemented with 2g/l NaHCOs for 16 h.

2. Subconfluent, with serum: Cells are plated onto coverslips at a density of 6 x 103 cells in DMEM/10 % FCS and allowed to attach for 2-3 h before microinjection.

3. Serum-starved, subconfluent cells: Cells are seeded at a density of 3x 105 cells into 80 cm2 flasks and grown to confluence. The cells are left without medium change to become quiescent (normally 7-10 days after seeding) before serum starving overnight in DMEM with 2g/l NaHC03. After washing with PBS, cells are trypsinized briefly, resuspended in serum-free medium containing 0.5mg/ml soybean trypsin inhibitor (Sigma T9003) pelleted, and resuspended in serum-free medium. Cells are plated on coverslips coated with fibronectin (50pg/ml, [Bio Products Laboratory]) in serum-free medium. These cells contain few polymerized actin structures, and few if any clusters of vinculin in focal adhesions. Coverslips for microinjection are transferred to 60 mm dishes containing 4 ml DMEM (with or without 10 % FCS as appropriate) and are replaced in their original tissue culture wells after microinjection.

6.4 Microinjection of C3 Transferase

Routinely, 10 x stock solutions of recombinant C3 protein (0.7-1 mg/ ml) are stored at 4 °C. Long term storage of more concentrated stocks (4-5mg/ml) is at -20 °C. Appropriate dilutions of C3 transferase for microinjection are made up fresh on the day of injection in 150 mM NaCI, 50 mM Tris pH 7.5, 5mM MgCI2, on ice. So as to be able to identify injected cells later, C3 protein is co-injected with either rat immunoglobulin (final concentration 0.5mg/ml [Pierce cat. no. 31885]) or FITC- or Texas Red-lysinated dextrans (final concentration 2mg/ml [Molecular Probes D1820 and D1863]). This mixture is centri-

optimization fuged for 5 min at 15, 000 g at 4 °C to spin down any large particles which may block the microinjection needle.

Micropipettes for injection are pulled from glass capillaries (Clarke Electromedical Instruments GC120F-10) with a programmable pipette puller (Campden Instruments Model no. 773). The coil temperature, pull force and the time and distance of pull are optimized to obtain of micropipettes micropipettes of approximately 0.5 pirn tip diameter. We microinject cells using a Zeiss/Eppendorf microinjection workstation. With this set-up, the cells are maintained at 37 °C with an atmosphere of 10 % C02 during microinjection. Cells are injected in the manual mode using an Eppendorf micromanipulator (model 5170) and an Eppendorf microinjector (model 5242), and the injection pressure is adjusted to give a constant flow rate of injection material. C3 protein is injected into the cytoplasm of cells. It has been estimated that between 1 and 2x10"" ml is injected per fibroblast cell (Graessmann and Graessmann, 1983). With practice it is possible to inject routinely between 100 and 200 cells per coverslip in a 10-15 minute period. The cells on the coverslips are then returned to the incubator for varying lengths of time as appropriate.

6.5 Fixation and Staining of Cells

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1. Cells injected with C3 transferase are rinsed in PBS before being fixed for 10 min at room temperature in a freshly prepared solution of 4 % paraformaldehyde in PBS.

2. Cells are permeabilized with 0.2 % Triton X-100/PBS at room temperature for 5 min.

3. After reducing free aldehyde groups by treatment with sodium borohydride (1 mg/ml in PBS for 10 min), cells are stained for filamentous actin by incubating for 30 min with 0.1 [xg/ml tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma P1951) in PBS.

4. In the case of co-injection with rat IgG, the cells are incubated at the same time with a 1:300 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rat immunoglobulin antibody (Sigma F7631).

5. Cells can also be labeled for components of focal adhesions using antibodies against, for example, vinculin (Sigma V4505), paxillin (Zymed cat. no. 03-6100), FAK (2A7 clone, a kind gift of Tom Parsons) ortalin (a kind gift of Keith Burridge). Incubation in the presence of appropriate primary antibodies is for 60 min at room temperature. Antibody incubations are carried out by placing coverslips face up on inverted tube caps attached to 10 cm petri dishes by double sided adhesive tape.

6. The coverslips are rinsed by sequential dipping into beakers containing PBS.

7. The coverslips are transferred to the appropriate second antibody mixture containing FITC-conjugated goat anti-mouse (in the case of vinculin, paxillin and FAK staining [Pierce cat. no. 31544]), or goat anti-rabbit (for talin staining [Pierce cat. no. 31670]) and rhodamine-labeled rabbit anti-rat IgG (for detection of injected cells [Sigma T5778]). To visualise both actin and vinculin, along with the injection marker, three color immunofluorescence is used. In this case a Cascade Blue-conjugated anti-rat IgG (Molecular Probes C2794) is used to detect the injected cells.

8. After the final wash, the coverslips are drained of excess liquid and are mounted by inverting onto 5^il Mowiol mountant (Cal-biochem) containing p-phenylenediamine (1 mg/ml) as an antifading agent.

9. The coverslips are observed with a Zeiss Axiophot microscope using Zeiss x 40 (NA 1.3), x 63 (NA 1.4) and x 100 (NA 1.3) oil immersion objectives. Fluorescence images are recorded on Kodak T-MAX 400 ASA film.

6.6 C3 Protein Inhibits LPA-stimulated Stress Fibre Assembly and Focal Adhesion Clustering

Serum-starved confluent quiescent Swiss 3T3 cells display few polymerized actin structures. TRITC-phalloidin staining reveals only a thin ring of polymerized actin at the periphery of the cells (Fig. 2). In addition, unlike growing cells, vinculin and other focal adhesion components are not clustered into focal adhesion structures. The addition to these cells of serum (the major active constituent of which is lysophos-phatidic acid, LPA) induces the rapid assembly of actin stress fibres and the clustering of vinculin and talin to form new focal adhesions (Fig. 2; Ridley and Hall, 1992). This response to LPA is blocked completely if cells are first microinjected with C3 transferase (Fig. 3; Ridley and Hall, 1992). Normally we microinject C3 protein at a concentration between 75 and 125(xg/ml lO-20min prior to stimulation of the cells with LPA (100 ng/ml [Sigma L7260]) or 1 % FCS. Each new preparation of C3 protein is titrated by microinjecting serially diluted sam-pies of the C3 protein, for example 200, 100, 75, 50 and 20|j,g/ml >

before stimulating cells with LPA for 15-30 min. The cells are then fixed test each new C3 and stained for filamentous actin with TRITC-labeled phalloidin, or for preparation focal adhesion components as described above (Fig. 3). Since focal adhesions are generally located at the periphery of the cell, it is often difficult to determine whether or not a C3 injected cell has focal adhesions in a confluent monolayer where cells are in close apposition to each other. In this case we sometimes stain for vinculin in serum-starved subconfluent cells prepared as described earlier.

Fig. 2. LPA-induced actin reorganization. Filamentous actin in untreated control Swiss 3T3 cells serum-starved for 16 h (a) or stimulated with LPA (lOOng/ml) for 30 min (b)

It should be noted that microinjection of C3 into quiescent serum-starved Swiss 3T3 cells is not without effect. Despite the cells having few if any actin stress fibres, or visible vinculin staining in focal adhesions, C3 causes cells to round up and to become detached from the underlying substrate. This indicates that there is, even in serum-free conditions, a low basal activity of Rho, perhaps maintained by a tyrosine kinase/phosphotyrosine phosphatase cycle upstream of Rho (Nobes et a/., 1995). Interestingly, the cell rounding and detachment induced by injection of C3 into quiescent cells is inhibited by co-injection of activated Rac or by addition of PDGF (5ng/ml), which

Fig. 3. C3 transferase inhibits LPA-induced stress fiber assembly. C3 was injected into confluent serum-starved Swiss 3T3 cells at concentrations of 200|xg/ml (a, b), 100ng/ml (c, d) and 20(xg/ml (e, f). Cells were stimulated after 15min with LPA (lOOng/ml), fixed 30min later and stained for filamentous actin with TRITC-conjugated phalloidin (a, c, e). FITC-conjugated dextran was co-injected with C3 transferase as a marker of injected cells (b, d, f). Note injection of C3 transferase at 200|xg/ml caused rapid rounding and detachment of the cells, and stress fibre assembly was not completely inhibited in cells injected with 20ng/ml C3

Fig. 3. C3 transferase inhibits LPA-induced stress fiber assembly. C3 was injected into confluent serum-starved Swiss 3T3 cells at concentrations of 200|xg/ml (a, b), 100ng/ml (c, d) and 20(xg/ml (e, f). Cells were stimulated after 15min with LPA (lOOng/ml), fixed 30min later and stained for filamentous actin with TRITC-conjugated phalloidin (a, c, e). FITC-conjugated dextran was co-injected with C3 transferase as a marker of injected cells (b, d, f). Note injection of C3 transferase at 200|xg/ml caused rapid rounding and detachment of the cells, and stress fibre assembly was not completely inhibited in cells injected with 20ng/ml C3

activates endogenous Rac, to the culture medium. This is due to the formation of focal complexes induced by Rac (Nobes and Hall, 1995; Hotchin and Hall, 1995).

Fig. 4. Active Rho is required for the maintanence of focal adhesion plaques. C3 (100 ng/ml) was injected into Swiss 3T3 cells growing in the presence of 10 % FCS. Cells were fixed after 5 min (b), 10 min (c) and 20 min (d) and labeled to show vincu-lin distribution, (a) Vinculin labeling in a control uninjected cell

6.7 Timecourse of Focal Adhesion Breakdown -Rho Is Also Required for Maintenance of Focal Adhesions

In addition to being required for the formation of focal adhesions, Rho is required for their maintenance. Swiss 3T3 cells newly attached and spread on glass coverslips in the presence of serum contain large focal adhesions (Fig. 4). Injection of C3 protein into such cells results in loss of staining for vinculin and other focal adhesion components. If cells are fixed at different times after microinjection of C3, and then stained for vinculin, it is possible to estimate how rapidly focal adhesions are broken down after inhibition of Rho. Within 15-20 min of C3 injection, vinculin is no longer clustered to focal adhesions suggesting a half life of around 10 min in the absence of rho (Fig. 4). This indicates that focal adhesions are dynamic structures and are likely to be turning over rapidly under normal conditions.

6.8 Alternative Delivery of C3 Transferase

C3 transferase can also be introduced into cells simply by adding it to the external culture medium, and it appears to enter cells by pinocyto-sis (Weigers et al., 1991; Morii and Narumiya, 1995). A range of C3 concentrations are used depending on cell type, but generally [ig/ml concentrations of C3 are required. It is recommended that cells should be incubated with 1, 3, 10, 30, and 100^g/ml for 24 to 48 h (Morii and Narumiya, 1995). Once Rho in cells has been ADP-ribosylated, it is no longer a substrate for C3, and the extent of ADP-ribosylation can be estimated by incubation of cell homogenates with C3 and [32P]NAD in vitro (Morii and Narumiya, 1995).

More effective uptake of C3 has been achieved by making a fusion protein of C3 transferase with the subunit of diphtheria toxin involved in binding and transport across membranes (Aullo etal., 1993). Unfortunately this delivery system does not work for all cell types since mouse and rat cells do not bind diphtheria toxin. Vero cells appear to be the cells most sensitive to this construct (Aullo et al., 1993; Boquet etal., 1995).

Intracellular C3 transferase expression has also been used to test whether endogenous Rho is required for regulation of transcriptional activation by SRF (Hill et al., 1995). Cells can be transfected with expression vectors containing C3.

6.9 Conclusion

C3 transferase is a specific inhibitor of the Rho GTPase and has been used in lymphocytes, neutrophils, neuronal cells, epithelial cells and platelets as well as fibroblasts. Although all members of the Rho family of GTPases contain an asparagine residue at codon 41, only Rho seems to be a substrate for C3 transferase. However, many members of the Rho family have yet to be characterized, and it is possible therefore that other substrates for C3 transferase will be identified.

6.10 Reagents and Chemicals




FITC goat anti-rabbit IgG












Biorad protein assay kit






Cascade blue anti-rat IgG




DMEM (powder)












fetal calf serum (FCS)



FITC goat anti-mouse IgG



FITC goat anti-rabbit IgG



FITC goat anti-rat IgG



FITC lysinated dextran

Molecular probes


Glutathione-agarose beads






lysophosphatidic acid



Mowiol mountant



p-aminobenzamidine agarose beads



p-phenylenediamine (anti-fade)












rat IgG



Rhodamine rabbit anti-rat IgG



sodium borohydride



sodium pyruvate



Soybean trypsin inhibitor



Texas red lysinated dextran

Molecular probes


thrombin (bovine)






Triton X-100





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Ridley, AJ, Paterson, HF, Johnston, CL et al. (1992): The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. In Cell 70:401 -10.

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Sekine, A, Fujiwara, M and Narumiya, S (1989): Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. In J. Biol. Chem. 264:8602-5.

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