Binding of triton X-100 to diphtheria toxin, crossreacting material 45, and their fragments.

Abstract

Binding of the nonionic detergent [3HrTriton X-100 by diphtheria toxin, by the nontoxic serologically related protein crossreacting material (CRM) 45, and by their respective A and B fragments has been studied. If Fragment A then catalyzes the transfer of the ADP-ribosyl group from NAD+ to elongation factor 2 (EF-2), thereby causing its inactivation (1, 2).Previous studies (3, 4) with mammalian cell cultures have shown that each sensitive cell carries about 4000 specific surface membrane receptors that initially react reversibly with groups located near the COOH-terminus of the toxin B fragment. This initial rapid reaction is followed by a slow irreversible process involving a major conformational change, during which the molecule enters the plasma membrane. Finally, fragment A is split off to reach the cytoplasm while B apparently remains behind in the membrane. If this model is indeed correct, we would expect that fragment B should behave like other membrane proteins and should contain a hydrophobic "domain" (5) which becomes inserted into the lipid bilayer during the entry process.Membrane proteins may be extracted into aqueous solvents that contain a nonionic detergent such as Lubrol or Triton X-100. During this process, protein-bound phospholipid molecules are replaced by molecules of the detergent. By measuring the quantity of detergent that it can bind, it is possible to estimate the fraction of a protein molecule's surface capable of hydrophobic interaction (6, 7). We are now reporting studies on the binding-of Triton X-100 to diphtheria toxin, to its A and B fragments, and to the nontoxic, serologically related tox gene product, crossreacting material (CRM) 45, which lacks the 17,000 dalton COOH-terminal amino acid sequence of the intact toxin molecule (8). Based on our findings, we are proposing a model to describe the process by which the diphtheria toxin A fragment is transported across the plasma membrane. MATERIALS AND METHODSDetergent Preparation. Triton X-100 (polyoxyethylene octyl phenol, averaging 9.6 ethylene oxide units per monomer) was obtained from Rohm & Haas Co., Philadelphia. An aqueous solution of ring-labeled [3H]Triton X-100 (0.93 mg/ml) was a gift from Steven Clarke. The concentration of this stock solution was calculated from the absorbance at 274 nm of dilutions in 1 mM Tris-HCI buffer containing 0.1 M Na2SO4 at pH 7.5, assuming A (1%) = 23.2. The specific activity was 125 cpm/,gg of [3H]Triton X-100 as determined in 3 ml of Aquasol (New England Nuclear) in a Beckman LS230 liquid scintillation counter. The critical micelle concentration (CMG) of Triton X-100 was determined using methyl orange by the method of Benzonana (9) to be 0.13 mg/ml in the 10 mM phosphate buffer at pH 7.2 used for binding studies. Labile tritium was estimated by passing a small volume of the stock [3H]Triton X-100 solution through a Sephadex G-0 Icolumn. Almost 99% of the radioactivity emerged in the void volume.Proteins. Partially purified diphtheria toxin (30-5% nicked) was obtained from Connaugh...

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids

“…Different experimental approaches have indicated that at low pH DT becomes hydrophobic and inserts into the lipid bilayer [21,[24][25][26][27]. As shown in fig.2, at pH 4.5 all toxins induce a very large change in turbidity that may be related to liposome aggregation and/or fusion.…”

Ricin A‐chain induces fusion of small unilamellar vesicles at neutral pH

“…Whether or not the phenomenon by itself is involved in the translocation process has not been elucidated. Diphtheria toxin, which requires an acidic environment for internalization [14,15], was found to induce fusion of DPPC vesicles only at acidic pH [ 161. Recently, from several experimental results [5,7,14], it' was proposed that the acidic environment might not be required for ricin internalization.…”

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery

“…In a second step, DT is endocytosed in clathrincoated vesicles (Moya et a/., 1985;Morris et a/., 1985). In an endosomal compartment, the translocation domain (T: molecular mass 20 kDa) at the amino-terminal end of DTB (Boquet et al, 1976) facilitates the transport of the catalytic domain (C or DTA: molecular mass 21 kDa) to the cytosol. In the third step, the C domain ADP-ribosylates and subsequently inactivates elongation factor 2 (EF2) (Collier, 1967;Honjo et a/., 1968;Gill et a/., 1969), leading to cell death.…”