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. LBP is a major component of the cells, comprising about 1% of the total protein during the night phase, but drops to only about 0.1% during the day. The luciferin is protected from autoxidation by binding to LBP, and one luciferin is bound per dimer at alkaline pH (Ka approximately 5 x 10(7) M-1). The protein undergoes a conformational change with release of luciferin at pH values below 7, concurrent with an activation of Gonyaulax luciferase. LBP thus has a dual role in the circadian bioluminescent system.

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. The amount of LBP is ten-fold greater from cells from in night phase compared to those from in day phase, as determined both by immunoblots of cell extracts, and in vivo fluorescence. These changes correlate with the circadian changes in bioluminescence of living cells.

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. With regard to their effects on the enzymatic activity of fragment A, three types of antibodies were found: (1) antibodies which bind fragment A but fail to inhibit its ADP-ribosyltransferase activity, (2) antibodies which completely inhibit enzyme activity, and (3) antibodies which interact with fragment A to yield antigen-antibody complexes of diminished activity. The results are consistent with location of the catalytic center of fragment A within its carboxy-terminal ca 4000 dalton region.

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. Hybridisation of a labelled nick-translated corynephage-beta c-DNA probe to nitrocellulose blots of these gels occurred only to blots from tox+ strains. Other hybridisation studies showed that all of seven strains, each isolated from a diphtheria case or carrier in a different part of the world, carried a prophage with DNA closely related to phage beta tox+. When an individual carrying a tox+ diphtheria bacillus arrives in an immunised community, spread of the tox gene to other individuals may be via phage conversion of tox- C diphtheriae already prevalent among the nasopharyngeal bacterial flora of the local populace, rather than by colonisation with the tox+ strain itself.

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. Klink, Nature (London) 287:250-251, 1980), we found that extracts from these organisms contain a protein factor which is a substrate for the ADP-ribosylation reaction catalyzed by diphtheria toxin fragment A. However, the rate of the reaction was approximately 1,000 times slower than that typically observed with eucaryotic elongation factor 2. We also demonstrated the presence of diphthine (the deamidated form of diphthamide, i.e., 2-[3-carboxyamide-3-(trimethylammonio)propyl]histidine) in acid hydrolysates of H. halobium protein in amounts comparable to those found in hydrolysates of similar preparations from eucaryotic cells (Saccharomyces cerevisiae and HeLa). Diphthine could not be detected in hydrolysates of protein from the eubacterium Escherichia coli. Whereas both archaebacterial and eucaryotic elongation factors contain diphthamide, they differ importantly in other respects.

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). Localization of the att sites on the beta prophage and correlation of the physical and genetic maps defined the orientation of the diphtheria tox operon. Diphtheria tox sequence homologies were mapped on gamma c by hybridizing 32P-labeled diphtheria tox mRNA to restriction fragments of gamma c DNA. Two regions of heterogeneity between phages beta c and gamma c were localized and these regions accounted for the 3-kilobase larger molecular size of gamma c compared with beta c. One change occurs near the tox promoter and may explain the nontoxigenic phenotype of corynephage gamma tox-.

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. After intracerebral injection, however, small amounts of CRM45 led to paralysis and death, even in mice and rats--species that are resistant to toxin administered intravenously. High doses of fragment A were nontoxic even by the intracerebral route. The cytotoxic dose od CRM45 was approximately 10(-7) M for a variety of cell lines derived from toxin-sensitive or toxin-resistant species. Cultured at Schwann's cells, however, were more sensitive CRM45 than other cell lines tested and 50-100 times more sensitive to toxin than cells cultured from other adult rat tissues. Fragment A has virtually no cytotoxicity for any mammalian cell line tested.

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. With both cell lines a lag was observed between the addition of toxin to the cells and the adenosinediphosphoribosylation of EF2. The lag decreased with increasing toxin concentration until a limiting value of about 12 min was reached. The rate of adenosinediphosphoribosylation of EF2 after the lag was 10 to 20 times more rapid in CV-1 cells than in BHK cells exposed to the same toxin concentration. The concentration of fragment A active in the cytoplasm of toxin-treated cells was estimated from the rate of adenosinediphosphoribosylation observed. Comparison of these estimates with data from studies of binding of 125I-toxin to cells suggests that the fragment A of only a minor fraction of toxin molecules bound to cell surface receptors reaches the cytoplasm and participates in the inactivation of EF2. A model summarizing our current views on the process by which fragment A enters cells is presented.

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. There is a C-terminal postiviely charged polypeptide sequence of 10,000--20,000 daltons which interacts with specific receptors present on the membranes of sensitive cells and which includes the second cystine disulfide. Between these two hydrophilic regions there is an hydrophobic zone which, when "unmasked," is capable of binding about 44 molecules of the nonionic detergent Triton X-100 and readily becomes inserted into membrane vesicles. It is suggested that the entry process involves an initial reversible interaction with membrane receptors, followed by an irreversible process in which the C-terminal region is released by a proteolytic cleavage, thus permitting the hydrophobic portion of the molecule to enter the lipid bilayer and form a channel through which the A fragment is drawn in an extended form to reach the cytoplasm.

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. No measurable amount of Triton X-100 is bound by native toxin or its A fragment of any concentration of the detergent. Undenatured CRM45 or its B45 fragment, on the other hand, readily became inserted into Triton X-100 micelles when the detergent reaches its critical micelle concentration. The results show that the toxin molecule contains a hydrophobic domain located on the portion of the B fragment that is linked to A. This region is masked in native toxin. Based on these findings, a model is proposed to describe how fragment B facilitates the transport of the enzymically active hydrophilic fragment A across the plasma membrane to reach the cytoplasm.

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. We suggest that it is at this point that cleavage of the NH2-terminal disulfide bond occurs leading to release of Fragment A into the cytoplasm. The toxin Fragment B remains attached to the membrane, probably formed in a complex with receptor, and blocks entry of additional toxin molecules through the same site. Specific membrane receptors are lacking from mouse cells. Both HeLa cells and L929 cells internalize toxin, related nontoxic proteins, and inert molecules such as inulin nonspecifically into endocytotoc vesicles. At 30 degrees the bulk internalization of extracellular fluid is about 1.2% of their cell volume per h for both cell lines. Fragment A does not traverse the plasma membrane by a mechanism that depends on endocytosis. The interaction of diphtheria toxin with the sensitive cell membrane is discussed in relation to other protein toxins and certain glycopeptide tropic hormones in which relatively large, hydrophilic polypeptide fragments or subunits are presumed to traverse the target cell plasma membrane and reach the cytoplasm in biologically active form.