Characterization of a cloned temperature-sensitive construct of the diphtheria toxin A domain.

Our goal was to determine how the DTM mutant construct of the A domain of diphtheria toxin (DTx) causes temperature-sensitive effects in Drosophila and yeast [Bellen, H. J., D'Evelyn, D.

Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin.

Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks.

Characterization of the deoxyribonuclease and ADP-ribosyltransferase activities of CRM45, a truncated homologue of diphtheria toxin.

CRM45 is a mutant form of diphtheria toxin (DTx) that lacks a 17-kDa carboxyl-terminal segment of the receptor-binding B subunit (DTB). The missing segment is a discrete structural domain of DTB that normally rests against the NAD binding pocket of the enzymically-active A subunit (DTA). Proteolytic cleavage and disulfide bridge reduction in the DTA-DTB linker region of DTx are required for optimal ADP-ribosylation of elongation factor 2 (EF-2).

Structural changes of tumor necrosis factor alpha associated with membrane insertion and channel formation.

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation.

Conformation and ion channel activity of lymphotoxin at neutral and low pH.

Lymphotoxin (LT or TNF-beta), a T cell-derived lymphokine with partial homology to TNF-alpha, was found to bind to dimyristoylphosphatidylcholine vesicles in a pH-dependent manner: binding increased with decreasing pH. Binding was not limited to surface association with phospholipid head groups because studies with a photoreactive membrane-restricted probe revealed protein penetration of the hydrocarbon core of the bilayer. Intramembranous photolabeling of the trimeric form of LT demonstrated maintenance of quaternary structure upon bilayer insertion.

Formation of ion-permeable channels by tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF, cachectin), a protein secreted by activated macrophages, participates in inflammatory responses and in infectious and neoplastic disease states. The mechanisms by which TNF exerts cytotoxic, hormonal, and other specific effects are obscure. Structural studies of the TNF trimer have revealed a central pore-like region.

Localization of diphtheria toxin nuclease activity to fragment A.

We describe a series of experiments that aimed to establish whether nuclease activity is actually associated with diphtheria toxin (DTx) and its A subunit (DTA), as we originally reported (M. P. Chang, R.

Comparison of the intoxication pathways of tumor necrosis factor and diphtheria toxin.

The mechanism by which tumor necrosis factor alpha (TNF) initiates tumor cell destruction is unknown. Having established that a brief drop in extracellular pH enhances the killing activity of TNF, our next objective was to explore whether TNF-induced cell death is dependent on endosomal acidification. Diphtheria toxin (DTx), a well-characterized acid-dependent cytotoxin, served as an indicator of the effectiveness of each treatment condition.

Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity.

The cytotoxic mechanism of diphtheria toxin (DTx) is associated with its ability to inhibit protein synthesis by ADP-ribosylation of elongation factor 2. Although DTx intoxication leads to internucleosomal DNA cleavage and cell lysis, these events do not occur when protein synthesis is inhibited by alternative treatments (e.g.

Characterization of the deoxyribonuclease activity of diphtheria toxin.

Having discovered that the A domain of diphtheria toxin exhibits intrinsic nuclease activity (Chang, M. P., Baldwin, R.

Second cytotoxic pathway of diphtheria toxin suggested by nuclease activity.

Diphtheria toxin (DTx) provokes extensive internucleosomal degradation of DNA before cell lysis. The possibility that DNA cleavage stems from direct chromosomal attack by intracellular toxin molecules was tested by in vitro assays for a DTx-associated nuclease activity. DTx incubated with DNA in solution or in a DNA-gel assay showed Ca2+- and Mg2+-stimulated nuclease activity.

Internucleosomal DNA cleavage precedes diphtheria toxin-induced cytolysis. Evidence that cell lysis is not a simple consequence of translation inhibition.

Diphtheria toxin (DTx) is an extremely potent inhibitor of protein synthesis. Cell death has been generally accepted as a straightforward effect of translation inhibition. Using human U937 cells, we found that DTx intoxication leads to cytolysis; indeed, release of 51Cr- and 75Se-labeled proteins could be detected within 7 h.

The acid-triggered entry pathway of Pseudomonas exotoxin A.

In this study we examined the pH requirements and reversibility of early events in the Pseudomonas toxin entry pathway, namely, membrane binding, insertion, and translocation. At pH 7.4, toxin binding to vesicles and insertion into the bilayer are very inefficient.

An endosomal model for acid triggering of diphtheria toxin translocation.

An endosomal model system was developed for studying the effects of pH on vesicle-entrapped diphtheria toxin. The "endosomes" were prepared from dioleoylphosphatidylcholine (1 mg), diphtheria toxin (0.25 mg), and lysozyme (2.

Capacity of tumor necrosis factor to bind and penetrate membranes is pH-dependent.

Studies with human U937 cells as targets established that a 15-min exposure to rTNF at pH 5.3 caused a significant increase in TNF-mediated cytolysis when compared to cells exposed to TNF at pH 7.4.

Effect of low pH on the conformation of Pseudomonas exotoxin A.

Previously we examined factors involved in the entry mechanism of Pseudomonas exotoxin A (PTx) at the level of lipid-protein interactions (Farahbakhsh, Z. T., Baldwin, R.

Pseudomonas exotoxin A. Membrane binding, insertion, and traversal.

Using vesicle targets composed of phosphatidylcholine and cholesterol (1:1 molar ratio), we found that Pseudomonas aeruginosa exotoxin A (PTx) binding and insertion are not only dependent on pH (Zalman, L.S., and Wisnieski, B.

Characterization of the insertion of Pseudomonas exotoxin A into membranes.

Pseudomonas aeruginosa exotoxin A (PTx) is an extremely potent inhibitor of protein synthesis, similar to diphtheria toxin in its mode of action. It is synthesized in precursor form and secreted as an Mr 66,583 protein lacking a 25-amino acid leader sequence. While the primary sequence and the nature of the enzyme activity that leads to inactivation of elongation factor 2 are known, the mechanism of PTx internalization remains obscure.

Mechanism of insertion of diphtheria toxin: peptide entry and pore size determinations.

Diphtheria toxin ( DTx ) is an extremely potent inhibitor of protein synthesis. It is secreted as a linear polypeptide, which is cleaved to produce disulfide-linked A and B fragments. Fragment A, the inhibitor of protein synthesis, requires fragment B, the recognition subunit, for entry into intact cells.

Characterization of a glycoprotein fusogen isolated from Sendai virus.

After isolation from Sendai virus, the glycoproteins HN and F retained their ability to induce hemagglutination and both heterologous and homologous cell-cell fusion. Both methods for demonstrating cell fusion indicated that the isolated HN and F glycoproteins compared favorably with whole Sendai virus as a fusogen. Conditions affecting the degree of fusion were examined and optimized.

Heat modifiability and detergent solubility of outer membrane proteins of Rhodopseudomonas sphaeroides.

The outer membrane fraction from Rhodopseudomonas sphaeroides was isolated by isopycnic density centrifugation. The purity of this fraction was assayed by several methods. When the outer membrane fraction obtained after French press lysis of cells was compared with the outer membrane fragments released during spheroplast formation, the polypeptide profiles were identical.