Role of a luciferin-binding protein in the circadian bioluminescent reaction of Gonyaulax polyedra.

A luciferin-binding protein (LBP), which binds and protects from autoxidation the substrate of the circadian bioluminescent reaction of Gonyaulax polyedra, has been purified to near homogeneity. The purified protein is a dimer with two identical 72-kDa subunits, and an isoelectric point of 6.7.

Properties and cellular localization of a luciferin binding protein in the bioluminescence reaction of Gonyaulax polyedra.

A luciferin binding protein LBP involved in the bioluminescence reaction of Gonyaulax polyedra was purified and used for antibody production. Luciferin bound to LBP is fluorescent and can be used as a marker in living cells, allowing the localization of LBP in cortical organelles to be visualized. In cell sections, the same peripheral localization was observed using anti-LBP and immunofluorescence microscopy.

Monoclonal antibody analysis of diphtheria toxin--II. Inhibition of ADP-ribosyl-transferase activity.

Monoclonal antibodies directed against the enzymatically active A-fragment of diphtheria toxin were used to investigate further the structure-function relationships within fragment A. Of 16 such antibodies, all but two were directed against epitopes located within the carboxy-terminal 30-40 amino acids of fragment A. Interestingly, the antibodies recognize several epitopes in this small region and varied considerably in their effects on toxin functions.

Studies on the molecular epidemiology of diphtheria.

DNA was extracted from toxigenic and nontoxigenic (tox+ and tox-) diphtheria bacilli isolated during a carrier survey that followed recovery of a tox+ Corynebacterium diphtheriae mitis from a baby with membranous tonsillitis. The electrophoretic gel patterns of restriction enzyme digests were indistinguishable from one another. They were, however, readily distinguishable from similar gels of DNAs extracted from diphtheria bacilli associated with outbreaks elsewhere.

Occurrence of diphthamide in archaebacteria.

We examined the nature of the diphtheria toxin fragment A recognition site in the protein synthesis translocating factor present in cell-free preparations from the archaebacteria Thermoplasma acidophilum and Halobacterium halobium. In agreement with earlier work (M. Kessel and F.

Restriction endonuclease map of the nontoxigenic corynephage gamma c and its relationship to the toxigenic corynephage beta c.

Clear-plaque-forming mutant gamma tox- corynephages were isolated independently from nontoxigenic lysogenic Corynebacterium diphtheriae strains C7s(gamma tox-) and C4(gamma tox-). A physical map was constructed by using restriction endonucleases BamHI, EcoRI, HindIII, and KpnI. A comparison of nontoxigenic gamma c with toxigenic corynephage beta c revealed large areas of homology, including common regions for cohesive ends (cos) and attachment sites (att).

Diphtheria toxin and related proteins: effect of route of injection on toxicity and the determination of cytotoxicity for various cultured cells.

The effect of route of injection on the toxicity of intact diphtheria toxin, cross-reacting material (CRM45), and diphtherial fragment A was compared in several animal species. By ordinary routes of injection, neither CRM45 nor fragment A was toxic, even in species for which 0.1 micrograms of toxin/kg of body weight was lethal.

Kinetics of adenosinediphosphoribosylation of elongation factor 2 in cells exposed to diphtheria toxin.

When susceptible cells are exposed to diphtheria toxin (Mr, 62,000) the N-terminal 21,150-dalton A fragment of toxin reaches the cytoplasm, where it catalyzes the transfer of adenosinediphosphoribose from nicotinamide adenine dinucleotide to elongation factor 2 (EF2). Adenosinediphosphoribose-EF2 is inactive, so that protein synthesis is blocked. Using a simple, rapid assay for the amount of adenosinediphosphoribosylatable EF2 in unfractionated lysates of cultured cells we have followed the kinetics of inactivation of EF2 in CV-1 and BHK cells exposed to diphtheria toxin.

Transport of diphtheria toxin A fragment across the plasma membrane.

The 60,000-dalton diphtheria toxin molecule is synthesized and released from the bacteria as a single polypeptide chain which may be subdivided into three functional regions of approximately equal length. There is an enzymically active 21,150-dalton A fragment extending from the N-terminal glycine residue to the first of the two disulfide bridges. This hydrophilic, negatively charged polypeptide must cross the plasma membrane of the target cell and reach the cytoplasm in order to inactivate EF-2 by ADP-ribosylation and thereby block protein synthesis.

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

Binding of the nonionic detergent [3H]Triton 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 first denatured in 0.1% sodium dodecyl sulfate, all of the proteins with the exception of fragment A bind increasing amounts of Triton X-100, reaching a maximum of more than 40 mol bound per mol of protein when the detergent concentration exceeds its critical micelle concentration.

Interaction of diphtheria toxin with mammalian cell membranes.

Uptake of 125I-labeled diphtheria toxin and serologically related proteins by a sensitive human HeLa cell line and by a resistant mouse L929 cell line has been studied. The evidence suggests that there is an initial rapid reaction between a recognition site present on the toxin Fragment B and specific plasma membrane receptors on the sensitive cell (there are approximately 4000/HeLa cell). This initial interaction is followed by a slow irreversible process during which there is a major conformational alteration of the toxin molecule causing the enzymically active 22,000-dalton Fragment A to become exposed to the cytosol.

On the alleged high sensitivity of mouse Ehrlich-Lettre ascites tumor cells to diphtheria toxin.

It was recently reported by Iglewski and Rittenberg in THESE PROCEEDINGS (71, 2707-2710, 1974) that low doses of purified diphtheria toxin inhibit protein synthesis in mouse Ehrlich-Lettre ascites carcinoma cells cultured in vitro. These observations could not be confirmed by us nor could the authors' further claim that toxin can cause regression of well-established ascites tumors in preimmunized mice be confirmed. Although temporary regression of such tumors can be demonstrated in unimmunized mice following intraperitoneal injection of diphtheria toxin, the amounts of toxin required are high and approach the lethal dose.

Synthesis of diphtheria tox-gene products in Escherichia coli extracts.

In a protein-synthesizing system extracted from E. coli, purified DNA from corynephages betac(tox+) and beta45c was used to direct the in vitro synthesis of diphtheria toxin and of the related nontoxic protein, CRM45, as well as of other beta-phage proteins. When betac(tox+)-DNA or betac-DNA was added to a similar system extracted from the nonlysogenic Corynebacterium diphtheriae strain, C7(s)(-)(tox-), neither toxin nor the CRM45 protein was produced, although other beta-phage proteins were synthesized in amounts equivalent to those produced in the E.