Biomedical subjects

A M Pappenheimer

Publications and source records attributed to A M Pappenheimer.

At least 19 recordsLinked to original sources

PubMed1989

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.

Animals↗

PubMed1984

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.

Animals↗

PubMed1983

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.

Autoradiography↗

PubMed1982

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-.

Bacteriophages↗

PubMed1979

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.

Animals↗

PubMed1977

Diphtheria toxin.

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Adenosine Diphosphate Sugars↗

PubMed1977

Interaction of diphtheria toxin with mammalian cell membranes.

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Cell Membrane↗

PubMed1976

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.

Binding Sites↗

PubMed1976

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.

Binding Sites↗

PubMed1975

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. About the same amount of CRM45, a tox gene product serologically related to diphtheria toxin but only 1/10,000th as toxic for guinea pigs, will also cause temporary regression in tumor-bearing mice.

Animals↗

PubMed1974

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. coli system from the same amount of beta-phage DNA. Preliminary experiments suggest that both toxinogenic and nontoxinogenic strains of the diphtheria bacillus contain a factor that specificially blocks expression of the tox gene. Synthesis of toxin and the CRM45 protein in the E. coli system could not be inhibited by relatively high concentrations of inorganic iron, but could be inhibited by extracts from the C7(s)(-)(tox-) strain of C. diphtheriae.

Amino Acids↗

PubMed1974