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.