Diphtheria toxin fragment

Miskimins, W.K., and Shimizu, N. (1979) Synthesis of cytotoxic insulin cross-linked to diphtheria toxin fragment a capable of recognizing insulin receptors. Biochem. Biophys. Res. Comm. 91, 143. 

Oeltmann, T.N. (1985) Synthesis and in vitro activity of a hormone-diphtheria toxin fragment A hybrid. Biochem. Biophys. Res. Comm. 133, 430. 

Oeltmann, T.N., and Forbes, J.T. (1981) Inhibition of mouse spleen cell function by diphtheria toxin fragment A coupled to anti-mouse Thy-1.2 and by ricin A chain coupled to anti-mouse IgM. Arch. Biochem. Biophys. 209, 362. 

A. General description Denileukin dif-titox is a recombinant, DNA-derived, interleukin-2 receptor specific ligand, cytotoxic fusion protein consisting of diphtheria toxin fragments A and B fused to interleukin-2. It is produced by expression of a recombinant fusion protein in Escherichia coli that contains nucleotide sequences for human interleukin-2, and sequences for the enzymatically active fragment A of diphtheria toxin and the membrane-translocating portion of diph-... 

Cytotoxic fusion protein composed of diphtheria toxin fragments A and B and 11-2... 

The structurally related antibiotics kirromycin and pulvomycin both act upon EF-Tu of most bacteria (and chloroplasts) although not upon its eucaryal (EF-la) counterpart [158,159]. The steroid antibiotic fusidic acid interacts systematically with both the eucaryal (EF-2) and the bacterial (EF-G) translocating factors, including chloroplasts of higher plants. Diphtheria toxin (fragment A) discriminates between bacterial-mitochondrial and eucaryal translocating factors by selectively and irreversibly impairing the eucaryal EF-2 factors [160,161]. 

Zhao J-M, London E (1990) Conformation and model membrane interactions of diphtheria toxin fragment A. J Biol Cbem 263 15369-15377. 

Denileukin diftitox, a rDNA-derived cytotoxic protein composed of the amino acid sequences for diphtheria toxin fragments A and B (Met 1-Thr 387)-His followed by the sequences for interleukin-2 (IL-2 Ala 1-Thr 133) and has a MW of 58 kDa. 

Yamaizumi, M., Mekada, E., Uchida, T., and Okada, Y. (1978) One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15,245-250. 

Denileukin, a recombinant-DNA-derived cytotoxic protein fused to diphtheria toxin fragments A and B, is designed to direct the cytocidal action of diphtheria toxin to cells that express the IL-2 receptor. Denileukin is indicated in treatment of cutaneous T-cell lymphoma. 

Denileukin diftitox or DAB3jg-IL-2, (ontak) is a fusion protein composed of diphtheria toxin fragments A and B and the receptor-binding portion of IL-2. DAB3g,-IL-2 is indicated for advanced cutaneous T-cell lymphoma in patients with >20% of T cells expressing the surface marker CD25. The IL-2 portion of the fusion protein binds the CD25 marker on the T cell and promotes destruction of the T cell by the cytocidal action of diphtheria toxin. 

Ontak Fusion protein Diphtheria toxin fragment Active (CD25) T-cell limphoma FDAapproved - ... 

During the characterization of EF-2 preparation from pyBHK cells, it was noted that there was a transfer of the [ C]-adenosine moiety from NAD to a trichloroacetic acid precipitable form during incubation in a Tris-HQ buffered reaction mixture containing no toxin, only the EF-2 preparation and [adenosine- C]-NAD [7]. The transfer of label was time dependent, reaching a maximum level at 80 to 160 min of incubation. Adding diphtheria toxin fragment A to the reaction resulted in a more rapid transfer of label to an acid precipitable form, but the maximum amount of label transferred in the two reactions was similar. The ability to transfer [ C]-adenosine from NAD to an acid precipitable form suggested that the EF-2 preparations contained a novel transferase activity. This activity was probably a contaminant in the EF-2 preparations since the majority, but not all, of the EF-2 preparations examined contained the transferase activity. 

Table 1. Reconstitution of a reticulocyte cell-free protein synthesizing system by addition of pyBHK EF-2 or pyBHK EF-2 ADP-ribosylated by pyBHK ADP-ribosyltransferase or by diphtheria toxin fragment A...

NT = not tested Pseudomonas toxin A antisera does not neutralize diphtheria toxin fragment A [10]... 

Fig. 1. Separation of pyBHK EF-2 from the cellular ADP-ribosyltransferase by immunoabsorbant chromatography. A 50 jug sample of an EF-2 preparation containing the cellular transferase activity was applied to a 250 jul column of Sepharose 4B resin coupled to Pseudomonas toxin A antibody. Unbound sample was washed from the resin with phosphate-buffered saline containing 1 vaM DTT (PBS). Protein bound to the immunoabsorbant was dissociated and eluted in 1 M propionic add containing 1 mAf DTT. Acid-containing fractions were immediately neutralized with 2 M Tris-Base. A portion of each fraction was assayed for EF-2 ( — ) by the transfer of [ C]-adenosine from NAD to the EF-2 in the presence of diphtheria toxin fragment A [6]. A second portion of each fraction was assayed for pyBHK ADP-ribosyltransferase activity (o—o) by the transfer of [ C]-adenosine from NAD to EF-2 in our standard cellular transferase reaction mixture [7] which was supplemented with 4 jug of a pyBHK EF-2 preparation which lacked its own endogenous transferase activity. Acid precipitable radioactivity was detected using a liquid scintillation counter...

An ADP-ribosyltransferase was found in EF-2 preparations from pyBHK cells and beef liver. This eukaryotic cellular enzyme transfers [ C]-adenosine from NAD to EF-2. Digestion of the [ C]-adenosine labeled EF-2 product of the cellular transferase reaction with snake venom phosphodiesterase yielded [ Cj-AMP, indicating the enzyme is a mono(ADP-ribosyl)transferase. The forward ADP-ribosylation reaction catalyzed by the pyBHK or beef liver enzyme is reversed by diphtheria toxin fragment A, yield-... 

Photochemical Transfer of the Nicotinamide Moiety of NAD to a Specific Residue in the Catalytic Center of Diphtheria Toxin Fragment A... 

We have described an efficient UV-induced reaction in which the nicotinamide moiety of NAD is transferred to the y-methylene group of Glu-148 of diphtheria toxin fragment A, with concomitant decarboxylation of that group. Efficient photolabeling of Pseudomonas exotoxin A has also been observed under the same conditions [3], and we are in the process of determining the site of attachment and the nature of the photoproduct formed with this toxin. 

Michel A, Dirkx J (1977) Occurrence of tryptophan in the enzymically active site of diphtheria toxin fragment A. Biochim Biophys Acta 491 286-295... 

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