Permeabilization toxin activity

Based on the results of a-LTX mutagenesis, strong correlation exists between pore formation and stimulation of Ca2+-dependent exocytosis from neuroendocrine cells. However, in some experiments with chromaffin cells, a-LTX action does not involve Ca2+ entry (Michelena et al. 1997). In addition, a-LTX sensitizes chromaffin cells to Ca2+ even when the cells are permeabilized and toxin pores should have no effect this involves protein kinase C (PKC) activation (Bittner and Holz 2000). Furthermore, the ability of a-LTXN4C to induce Ca2+-dependent exocytosis without forming pores implicates a stimulating mechanism other than pore formation. 

L chain or Di-chain-toxin If the biological poisoning pathway is being used, the toxins must be in their di-chain form. Isolated L chains cannot enter the cell and are thus ineffective when added to intact cells or isolated nerve terminals. However, isolated L chains block exocytosis if they are introduced directly into the cytoplasm, e.g., by microinjection (Penner ef al., 1986 Mochida ef al., 1989 Hunt ef al., 1994), by permeabilization of the cells prior to toxin exposure (Bittner and Holz, 1988 Ahnert-Hilger efal., 1989a,b, and this volume. Chapter 18 Dayanithi ef al., 1992), or by expression of recombinant L chains (Mochida efal., 1990, Sweeney efal., 1995). These techniques allow the inhibition of exocytosis in neurosecretory cells that are toxin-resistant due to a lack of receptors. Likewise, only the free L chains (reduced di-chain toxin or isolated L chain) are active towards the respective substrates in vifro (Schiavo ef al., 1992 Link ef al., 1992 Schiavo efal., 1993c Link efal., 1994). 

In addition to the buffers needed for cell permeabilization prepare Solution LC supplemented with the required amount of TeTx. If the whole toxin (heavy plus light chains) is used, the catalytic subunit must be separated from the heavy chain by dithiothreitol (DTT) treatment. We usually incubate a 200-fold concentrated toxin in LC supplemented with DTT (final concentration 10 mM) for 30 min at 37°C. If the light chain is used this activation step is unnecessary. 

Structural homologies between PFTs and other toxins have not been identified. However, the process of membrane permeabilization may be operative in many cases where proteins have to escape from an intracellular compartment. Well known examples are diphtheria toxin, the neurotoxins and anthrax toxin. Specific domains in many intracel-lularly active toxins have in fact been shown to produce pores in artificial lipid bilayers, and membrane permeabilization is thought to form the basis for translocation of the active moieties from the late endo-some to the cytoplasm (reviewed in Montecucco et ai, 1994). The molecular mechanism of this translocation remains obscure. In the... 

Of the 25 animal phyla, almost half are worms. Thus, it is not at all surprising that some worms contain toxins. The nemertines are a phylum of over 800 known species which resemble flatworms but are active predators on crustaceans and other worms. This phylum is exceptionally toxic among the various worm phyla. The Heteronemertine side possesses peptide toxins which appear to be only defensive, as these animals have no means of injecting a venom. The peptides include neurotoxins, which enhance excitability of nerve membranes, and cytolysins, which permeabilize and destroy cell membranes. Members of the Hoplonemertine class inject a venom into their prey using a mineralized stylet located in their proboscis, which is also used to immobilize the prey. Their toxins are alkaloids similar to nicotine which in minute amounts paralyze crustaceans and annelid worms and primarily activate nicotinic acetylcholine receptors. Another well-known worm toxin is nereistoxin, a disulfide-containing alkaloid which also binds to nicotinic... 

In triton skinned and in a-toxin-permeabilized smooth muscle preparations, the Ca + sensitivity of force production is also decreased by cGMP (Pfitzer et al., 1984,1986 Nishimura efflZ., 1992). This may be due to an up-regulation of MLCP (Pfitzer etal., 1986). Clear evidence that modulation of the activity of MLCP would affect Ca + sensitivity of tension development was in fact obtained in triton skinned smooth muscle when it was shown that inhibition of MLCP by the black sponge toxin, okadaic acid, increased Ca + sensitivity (Takai et al., 1987 Bialojan et al., 1988). On the other hand, incubation of triton skinned chicken gizzard fibers with a purified phosphatase decreased Ca + sensitivity (Bialojan et al., 1987). 

Exogenous application of GTP analogues or contractile agonists increased the [Ca +J sensitivity of phosphorylation in smooth muscle permeabilized with staphylococcal oi-toxin (Nishimura et al, 1988 Kitazawaeffl/., 1991a). Exogenous application of either histamine or AIF4 (a nonspecific activator of G proteins) to depolarized intact tissues also increased the [Ca +Ji sensitivity of phosphorylation (Rembold,... 

FIGU R E 7 A similar set of substrates is tyrosine phosphorylated during activation of either intact taenia coli, cultured VSMC, or staphylococcal a-toxin-permeabilized deal longitudinal smooth muscle. In these experiments, tyrosine.-phosphorylated substrates were detected by immunoblotting with antiphosphotyrosine antibodies and enhanced chemiluminescence technology rather than the less sensitive I-labeled protein A technology used in Fig. 2. Stimulation of guinea pig taenia coli with either 10 jcM carbachol (Carb) or 1.5 mM vanadate (Van) resulted in pronounced tyrosine phosphorylation of at least nine substrates with apparent masses of 42-45, 50, 70, 80-85, 95,100, 110, 116, and 205 kDa. In like fashion, stimulation of canine femoral VSMC with 100 jjlM phenylephrine (PE) resulted in enhanced tyrosine phosphorylation of a similar set of substrates (however, note that qualitative differences were evident with respect to some substrates, such as the one of 205 kDa). Similarly, the same substrates appeared to be tyrosine phosphorylated when permeabilized ileal smooth muscle was contracted with Ca + (pCa 4.5). From Di Salvo et al. (1994), Fig. 5, p. 1438. 

Other results suggest a lower rate of phosphate turnover on LC20. Kitazawa et al. (1991) measured phosphatase activity in a-toxin-permeabilized portal vein from the rate of dephosphorylation in rigor solution with inhibited myosin light chain kinase (MLCK) activity. Their value was 0.017 s i at 15°C, which, corrected for temperature (Mitsui et al., 1994), is about nine times smaller than the value of Butler et al. Reconciliation of these findings will require additional studies. However, the conditions of the experiments were quite different and it should also be pointed out that in the experiments of Butler et al. (1994), only 76% of the light chain phosphate turned over at the fast rate, al-... 

As pointed out by Butler et al. (1994), the a-toxin-permeabilized preparation used by Kitazawa et al. might contain endogenous phosphatase inhibitors that have been lost in the Triton-skinned preparation. This could at least partially explain the observed difference in phosphatase rate, and is an intriguing possibility relating to the demonstration that receptor agonists are able to increase Ca2+ sensitivity in neuroeffector-coupled permeabilized preparations by a mechanism involving inhibited myosin light chain phosphatase (MLCP) activity (Kitazawa et al., 1991 Kubota et al.,... 

Trinkle-Mulcahy L, Ichikawa K, Hartshome DJ, Siegman MJ, Butler TM (1995) Thio-phosphorylation of the 130-kDa subunit associatesd with a decreased activity of myosin light chain phosphatase in a-toxin permeabilized smooth muscle. J Biol Chem 270 18191-18194... 

Von Tersch, M.A., S.L. Slatin, C.A. Kulesza, and L.H. English. 1994. Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide. Appl. Environ. Microbiol. 60 3711-3717. 

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