[Show abstract][Hide abstract] ABSTRACT: S100 proteins bind to numerous target proteins, as well as other S100 proteins and activate signaling cascades. S100 proteins can be modified by various post‑translational modifications, such as phosphorylation, methylation and acetylation. In addition, oxidation is important for modulating their activities. Previous studies have shown that S100A1 interacts with S100A4 in vitro and in vivo. Due to this potential cross‑talk among the S100 proteins, the aim of the present study was to examine whether S100A4 modulates the activity of S100A1. S100A4 was readily oxidized and formed disulfide‑linked dimers and oligomers. Although non‑oxidized S100A4 bound to protein phosphatase 5 (PP5), the Cu‑oxidized S100A4 failed to bind PP5. Instead, the Cu‑oxidized S100A4 directly interacted with S100A1 and prevented PP5 activation. Hydrogen peroxide induced S100A4 oxidation in MKN‑45 gastric adenocarcinoma cells and decreased S100A1‑PP5 interaction, resulted in the inhibition of PP5 activation by S100A1. These data indicate that oxidized S100A4 regulates PP5 activity in a unique manner under oxidative stress conditions.
International Journal of Molecular Medicine 09/2014; 34(6). DOI:10.3892/ijmm.2014.1947 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FKBP38 (FK506-binding protein 38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, regulates signaling pathways such as cell survival, apoptosis, proliferation, and metastasis. However, the mechanisms that regulate the activity of FKBP38 are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR-proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin-light chain, Tom70, FKBP52, CyP40, C-terminus of Hsc70-interacting protein (CHIP) and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR-proteins with their target proteins. Therefore, we have hypothesized that Ca2+/S100 proteins can interact with FKBP38 and regulate its function. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, S100B, and S100P specifically interact with FKBP38 and inhibit the interaction of FKBP38 with Bcl-2 and Hsp90. Overexpression of permanently active S100P in Huh-7 cells inhibited the interaction of FKBP38 with Bcl-2, resulting in the suppression of Bcl-2 stability. The association of the S100 proteins with FKBP38 provides a Ca2+-dependent regulatory mechanism of the FKBP38 mediated signaling pathways.
[Show abstract][Hide abstract] ABSTRACT: Suramin is an activator of ryanodine receptors and competitively binds to the calmodulin-binding site. In addition, S100A1 and calmodulin compete for the same binding site on ryanodine receptors. We therefore studied the effects of suramin on protein phosphatase 5 (PP5) and S100-activated PP5. In the absence of S100 proteins, suramin bound to the tetratricopeptide repeat (TPR) domain of PP5 and activated the enzyme in a dose-dependent manner. In the presence of S100A2/Ca(2+), lower concentrations of suramin dose-dependently inhibited PP5 activity as an S100 antagonist, whereas higher concentrations of suramin reactivated PP5. Although the C-terminal fragment of heat shock protein 90 (HspC90) also weakly activated PP5, the binding site of suramin and HspC90 may be different, and addition of suramin showed no clear effect on the phosphatase activity of PP5. Similar biphasic effects of suramin were observed with S100A1-, S100B- or S100P-activated PP5. However, the inhibitory effects of lower concentrations of suramin on S100A6-activated PP5 are weak and high concentrations of suramin further activated PP5. SPR and the cross-linking study showed inhibition of the interaction between S100 protein and PP5 by suramin. Our results revealed that suramin is a novel PP5 activator and modulates S100-activated PP5 activity by competitively binding to the TPR domain.
[Show abstract][Hide abstract] ABSTRACT: FKBP8/FKBP38 is a unique FK506-binding protein with a C-terminal membrane anchor and localizes at the outer membranes of mitochondria and the endoplasmic reticulum. Similar to some immunophilins, such as FKBP51, FKBP52 and Cyclophilin 40, FKBP8/FKBP38 contain a putative Calmodulin-binding domain and a tetratricopeptide-repeat (TPR) domain for the binding of Hsp90. Both Hsp90 and the non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) interact specifically with FKBP8/FKBP38 through its TPR domain, and the ternary complex formation plays a critical role in HCV RNA replication. The goal of this study is to evaluate that the host factor inhibits the ternary complex formation and the replication of HCV in vitro and in vivo.
S100 proteins, FKBP38, FKBP8, HCV NS5A, Hsp90, and calmodulin were expressed in E.coli and purified. In vitro binding studies were performed by GST pull-down, S-tag pull-down and surface plasmon resonance analyses. The effect of S100 proteins on HCV replication was analysed by Western blotting using an HCV NS3 antibody following transfection of S100 proteins into the HCV replicon harbouring cell line (sO cells).
In vitro binding studies showed that S100A1, S100A2, S100A6, S100B and S100P directly interacted with FKBP8/FKBP38 in a Ca2+-dependent manner and inhibited the FKBP8/FKBP38–Hsp90 and FKBP8/FKBP38–NS5A interactions. Furthermore, overexpression of S100A1, S100A2 and S100A6 in sO cells resulted in the efficient inhibition of HCV replication.
The association of the S100 proteins with FKBP8/FKBP38 provides a novel Ca2+-dependent regulatory role in HCV replication through the NS5A–host protein interaction.
Liver international: official journal of the International Association for the Study of the Liver 03/2013; 33(7). DOI:10.1111/liv.12151 · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The U box E3 ubiquitin ligase CHIP binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca(2+)/S100 proteins directly associate with the TPR-proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin-light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR-proteins with their target proteins. Therefore, we have hypothesized that Ca(2+)/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca(2+)/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca(2+)/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpressions of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca(2+)-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.
Journal of Biological Chemistry 01/2013; DOI:10.1074/jbc.M112.436758 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We propose a novel surface plasmon resonance (SPR) sensor chip with a microfabricated slit array. The microslit excludes micrometre-size objects larger than its slit size from the SPR sensing area, so that it functions as an in situ filter. We demonstrated the sensing of microparticles of different diameters using the chip, and the results show a successful size-exclusion effect. As a demonstration of the biological application, we performed the detection of aggregation and disaggregation of biological particles using sugar-chain-immobilized gold nanoparticles as a test sample.
The Analyst 03/2012; 137(9):2192-8. DOI:10.1039/c2an16010b · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.
[Show abstract][Hide abstract] ABSTRACT: In this study, we propose a novel surface plasmon resonance (SPR) chip on which the microslit array was fabricated. The microslit excludes micrometer-size objects that are larger than its slit size from sensing field, so that it acts as a filter. In order to confirm the filtereffect, we demonstrated the sensing of microparticles of different diameters using the SPR chip. As the demonstration of the biotechnology application, we performed the discrimination of aggregation of bio-molecules using SGNP (Sugar chain imobilized Gold Nano Particle) as a model sample.
[Show abstract][Hide abstract] ABSTRACT: Although the precise intracellular roles of S100 proteins are not fully understood, these proteins are thought to be involved in Ca(2+)-dependent diverse signal transduction pathways. In this report, we identified importin α as a novel target of S100A6. Importin α contains armadillo repeats, essential for binding to nuclear localization signals. Based on the results from GST pull-down assay, gel-shift assay, and co-immunoprecipitation, we demonstrated that S100A6 specifically interacts with the armadillo repeats of importin α in a Ca(2+)-dependent manner, resulting in inhibition of the nuclear localization signal (NLS)-importin α complex formation in vitro and in vivo. These results indicate S100A6 may regulate the nuclear transport of NLS-cargos in response to increasing concentrations of intracellular Ca(2+).
[Show abstract][Hide abstract] ABSTRACT: S100 proteins are a subfamily of the EF-hand type calcium sensing proteins, the exact biological functions of which have not been clarified yet. In this work, we have identified Cyclophilin 40 (CyP40) and FKBP52 (called immunophilins) as novel targets of S100 proteins. These immunophilins contain a tetratricopeptide repeat (TPR) domain for Hsp90 binding. Using glutathione-S transferase pull-down assays and immunoprecipitation, we have demonstrated that S100A1 and S100A2 specifically interact with the TPR domains of FKBP52 and CyP40 in a Ca(2+)-dependent manner, and lead to inhibition of the CyP40-Hsp90 and FKBP52-Hsp90 interactions. These findings have suggested that the Ca(2+)/S100 proteins are TPR-targeting regulators of the immunophilins-Hsp90 complex formations.
[Show abstract][Hide abstract] ABSTRACT: S100A2 and S100A6 interact with several target proteins in a Ca2+-regulated manner. However, the exact intracellular roles of the S100 proteins are unclear. In this study we identified Hsp70/Hsp90-organizing protein (Hop) and kinesin light chain (KLC) as novel targets of S100A2 and S100A6. Hop directly associates with Hsp70 and Hsp90 through the tetratricopeptide (TPR) domains and regulates Hop-Hsp70 and Hop-Hsp90 complex formation. We have found that S100A2 and S100A6 bind to the TPR domain of Hop, resulting in inhibition of the Hop-Hsp70 and Hop-Hsp90 interactions in vitro. Although endogenous Hsp70 and Hsp90 interact with Hop in resting Cos-7 cells, but not with S100A6, stimulation of these cells with ionomycin caused a Hop-S100A6 interaction, resulting in the dissociation of Hsp70 and Hsp90 from Hop. Similarly, glutathione S-transferase pulldown and co-immunoprecipitation experiments revealed that S100A6 binds to the TPR domain of KLC, resulting in inhibition of the KLC-c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1) interaction in vitro. The transiently expressed JIP-1 interacts with KLC in resting Cos-7 cells but not with S100A6. Stimulation of these cells with ionomycin also caused a KLC-S100A6 interaction, resulting in dissociation of JIP-1 from KLC. These results strongly suggest that the S100 proteins modulate Hsp70-Hop-Hsp90 multichaperone complex formation and KLC-cargo interaction via Ca2+-dependent S100 protein-TPR protein complex formation in vivo as well as in vitro. Moreover, we have shown that S100A2 and S100A6 interact with another TPR protein Tom70 and regulate the Tom70-ligand interaction in vitro. Thus, our findings suggest a new intracellular Ca2+-signaling pathway via S100 proteins-TPR motif interactions.
[Show abstract][Hide abstract] ABSTRACT: In this study, we proposed a new sensing method using a special SPR chip on which microstructures are fabricated, and performed a principle confirmation of the sensing feasibility using this chip. We examined a fabrication method of micro structures on the SPR chip that has a filter effect by the pillar structure, which can filter the target substance reaching to the detection area of SPR sensing. In order to confirm the filter effect of the micro structure on the SPR chip, we performed the principle confirmation experiment of the SPR sensing system using the fine particles. Next, in order to adapt to the biological molecules measurement, we performed the experiment using yeast cells and demonstrated the filter effect of this chip with the micro structure.
Proceedings of SPIE - The International Society for Optical Engineering 10/2007; DOI:10.1117/12.754374 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Clostridium perfringens S40, spore germination-specific enzymes are synthesized during sporulation. Previous reports have demonstrated that two cortex-lytic enzymes, SleC and SleM, and a component of germination-specific protease, CspC, are located outside the cortex as an integral part of the dormant spore. In the present study, we examined the time and compartment of these enzymes' gene expression using reverse transcription-PCR (RT-PCR) and fluorescence microscopy on green fluorescence protein (GFP)-fused proteins. These results suggested that CspABC, SleC, and SleM are synthesized in the mother cell compartment of sporulating cells, probably at stages II approximately III of sporulation, and that the expression of cspABC genes is tricistronic.
[Show abstract][Hide abstract] ABSTRACT: Epsilon-toxin (ET) of Clostridium perfringens, which causes fatal enterotoxemia in ungulates, was previously shown to bind to and form a heptameric pore within the detergent-resistant membranes (DRMs) of MDCK cells. Depletion of cholesterol has also been shown to decrease the cytotoxicity of ET and its heptamerization. In this study, we investigated the effects of changes in sphingolipids, other DRM components of MDCK cells, on the cells' susceptibility to ET. Treatment with fumonisin B1 and PDMP, inhibitors of sphingolipid and glycosphingolipid syntheses, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside G(M1) dramatically decreased the ET binding, heptamerization and cytotoxicity. These effects were shown not to be due to ET binding to G(M1) or to denaturation of ET. We also found that the ET cytotoxicity towards MDCK cells decreased with an increase in culture time. In accordance with the resistance observed for prolonged cultured cells, G(M3), a major ganglioside component, increased and sialidase treatment increased their susceptibility. These results suggest that membrane-anchored sialic acid of G(M3) within DRMs inhibits ET binding, leading to prevention of the heptamerization of ET and cell death. It is also suggested that sialidase produced by this organism aids the targeting of ET to MDCK cells.
Microbiology and Immunology 02/2005; 49(3):245-53. DOI:10.1111/j.1348-0421.2005.tb03726.x · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.
European Journal of Biochemistry 10/2004; 271(18):3765-75. DOI:10.1111/j.1432-1033.2004.04318.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A "large" sialidase isozyme (NanI) from Clostridium perfringens is a representative microbial sialidase with broad substrate specificity, being used for the analysis of sialoglycoconjugates. It is also a possible virulence factor. However, purification of the native enzyme in a large quantity is not practical due to its low productivity. To obtain the enzyme in a satisfactory yield, a gene encoding the NanI was transcriptionally fused to the fdx gene promoter (P(fdx)) in a shuttle-vector, pFF, and transformed into C. perfringens 13. The resultant strain released the enzyme into the culture medium, as the original strain does. The enzyme activity increased during the first 6 h of culture and thereafter remained at maximal levels. The maximal activity was approximately 3000-fold compared with that of the original strain, and 15-fold compared with that of recombinant Escherichia coli, which possesses extra copies of the tRNA gene for selected rare codons. This suggests the usefulness of a P(fdx)-based plasmid for expressing AT-rich genes in C. perfringens. The enzyme was successfully purified by two-step procedure with a specific activity of 2860 U/mg using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and a yield of 1.69 mg of NanI per 100 ml of culture. The method described here can facilitate purification of NanI in enough quality and quantity to analyze the role of sialoglycoconjugates in cells and the pathogenic importance of NanI sialidase.
Protein Expression and Purification 08/2004; 36(1):70-5. DOI:10.1016/j.pep.2004.03.004 · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the -35 region. The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C. perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively. DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions -69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C. perfringens transformant. Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc. Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter. Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes. These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.
[Show abstract][Hide abstract] ABSTRACT: Clostridium perfringens epsilon-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled epsilon-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-beta-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When epsilon-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.
[Show abstract][Hide abstract] ABSTRACT: A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.
Journal of Bacteriology 07/2001; 183(12):3742-51. DOI:10.1128/JB.183.12.3742-3751.2001 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of Clostridium perfringens epsilon-protoxin by tryptic digestion is accompanied by removal of the 13 N-terminal and 22 C-terminal amino acid residues. In this study, we examined the toxicity of four constructs: an epsilon-protoxin derivative (PD), in which a factor Xa cleavage site was generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (DeltaN-PD); PD without the 23 C-terminal residues (DeltaC-PD); and PD without either the N- or C-terminal residues (DeltaNC-PD). A mouse lethality test showed that DeltaN-PD was inactive, as is PD, whereas DeltaC-PD and DeltaNC-PD were equally active. DeltaC-PD and DeltaNC-PD, but not the other constructs formed a large SDS-resistant complex in rat synaptosomal membranes as demonstrated by SDS-polyacrylamide gel electrophoresis. When DeltaNC-PD and DeltaC-PD, both labeled with (32)P and mixed in various ratios, were incubated with membranes, eight distinct high molecular weight bands corresponding to six heteropolymers and two homopolymers were detected on a SDS-polyacrylamide gel, indicating the active toxin forms a heptameric complex. These results indicate that C-terminal processing is responsible for activation of the toxin and that it is essential for its heptamerization within the membrane.