Frieder Lutz

Binding of 3H-desmethylphalloin to isolated plasma membranes from rat liver

Summary3H-desmethylphalloin (DMPh), a phallotoxin chemically very similar to phalloidin, binds specifically to rat liver plasma membranes in vitro. Already after 30 sec at 37°C, >90% of maximal binding had taken place. At low 3H-DMPh concentrations, isolated plasma membranes bind 20 times more of the cyclopeptide per mg protein as compared with microsomes or mitochondria. Scatchard plot argues for the presence of two different binding sites in the plasma membranes. The dissociation constants of them at 37°C are 2.2·10−8 moles/l and 1.2·10−6 moles/l, respectively. 3H-DMPh bound on membranes could be exchanged by nonlabeled phalloidin but not by antamanide or tetraethylammonium ions.

Interaction of Pseudomonas aeruginosa cytotoxin with plasma membranes from Ehrlich ascites tumor cells

SummaryBiologically active125I-cytotoxin fromPseudomonas aeruginosa binds to plasma membranes from Ehrlich ascites tumor cells in a saturable manner. The Scatchard plot indicated a single binding site with a capacity of 260 pmoles/mg of membrane protein and aKD of 2×10−8 M. Specific binding was dependent on temperature, pH and ionic strength. Thus constant levels of bound125I-cytotoxin were attained either within 30 min at 30°C or within 3 h at 4°C. Binding was 30-fold higher at 4°C vs 30°C and 2-6-fold higher at pH 5.3 vs pH 8.3. Binding was not effected by 50 mM sugar or sialic acid. 300 mM sucrose, however, instead of phosphate buffer, reduced binding by 50%. Pretreatment of plasma membranes with trypsin or papain led to a significant decrease in125I-cytotoxin binding. A pretreatment with phospholipase C or D had no effect, whereas phospholipase A2 induced a decrease by 34%. The collected data suggest that the binding site for125I-cytotoxin within the plasma membrane from Ehrlich ascites tumor cells is a membrane protein.Correlation of125I-cytotoxin binding and membrane action of the unlabelled cytotoxin can be observed through (a) increased lowering of the cellular K+ and Na+ gradient by decrease of medium pH, (b) decreased toxicity after substitution of ions by sugar, and (c) increased breakdown of cellular cationic gradient after temperature shift from 4°C to 37°C.

The influence of sodium dehydrocholate on the uptake of3H-desmethylphalloin by the perfused rat liver

SummarySodium dehydrocholate diminished the uptake of3H-desmethylphalloin by the perfused rat liver. Only about 1% of the polypeptide was eliminated by bile.

Site-specific integration of the phage ?CTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage ϕCTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ϕCTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.

Pore-forming Pseudomonas aeruginosa cytotoxin

Pseudomonas aeruginosa procytotoxin protein is processed C-terminally during bacterial autolysis to generate the active 29-kDa cytotoxin molecule. Binding to target cell membranes is dependent upon Cys23 and Cys215 and a domain flanked to Cys215. On rabbit erythrocytes, cytotoxin binds to a 28-kDa peptide of a glycoprotein, its N-terminus shows high homology to channel integral membrane protein CHIP28. At concentrations of more than 3 x 10(-9) M, cytotoxin increases plasma membrane permeability of most eucaryotic cells investigated. The role of cytotoxin in the formation of pores with a diameter of 2 nm on mammalian cells is discussed. The cytotoxin effects are coordinated with other pseudomonal products and the resultant concept of pathogenesis is presented.

Action of a cytotoxin from Pseudomonas aeruginosaon human leukemic cell lines: Increase in cell permeability to Ca2+ and Mn2+ and lack of stimulation of inositol lipid turnover

Quin2 loaded human leukemic, JURKAT and K562 cells, were exposed to various doses of Pseudomonas aeruginosa cytotoxin. This cytotoxin induced an increase in quin2 fluorescence indicating an increase in the cytoplasmic free Ca2+ concentration. The rate of the fluorescence increase and the lag time before the response were dependent on the doses of the cytotoxin. Addition of MnCl2 to the cytotoxin‐treated cells induced a decrease in the quin2 fluorescence at rates dependent on the doses of the cytotoxin. The cytotoxin did not stimulate the inositol lipid turnover in JURKAT cells, which was determined by the accumulation of [3H]inositol phosphates in myo‐[2‐3H]inositol‐prelabeled cells in the presence of LiCl. These results indicate that the cytotoxin increases cell permeability to both Ca2+ and Mn2+ by direct breakdown of the permeability barrier of the plasma membrane.Quin2 loaded human leukemic, JURKAT and K562 cells, were exposed to various doses of Pseudomonas aeruginosa cytotoxin. This cytotoxin induced an increase in quin2 fluorescence indicating an increase in the cytoplasmic free Ca2+ concentration. The rate of the fluorescence increase and the lag time before the response were dependent on the doses of the cytotoxin. Addition of MnCl2 to the cytotoxin-treated cells induced a decrease in the quin2 fluorescence at rates dependent on the doses of the cytotoxin. The cytotoxin did not stimulate the inositol lipid turnover in JURKAT cells, which was determined by the accumulation of [3H]inositol phosphates in myo-[2-3H]inositol-prelabeled cells in the presence of LiCl. These results indicate that the cytotoxin increases cell permeability to both Ca2+ and Mn2+ by direct breakdown of the permeability barrier of the plasma membrane.

Nucleotide sequence of attP and cos sites of phage CTX and expression of cytotoxin in Pseudomonas aeruginosa PA158

SummaryThe gene for Pseudomonas aeruginosa cytotoxin (CTX) has been found to be part of a temperate phage with a total size of 35.5 kb. We have investigated several DNA fragments of this phage for CTX production. For phage integration, the phage genome cohesive (cos) ends covalently associate with host DNA of strain PA158. The cos ends and the CTX gene are found on a 3.4 kb EcoRI fragment B and are included in the 11 kb HindIII fragment A and the 8.5 kb BamHI fragment B of the phage DNA. The cos ends are 20 nucleotides long and are located at 338–357 nucleotides upstream of the CTX transcriptional initiation site. The phage attachment (attP) site is also present on the 3.4 kb EcoRI fragment B. The attP site consists of 34 by and is located at 974–1007 nucleotides upstream of the CTX gene start site. Replication of the vegetative form of the phage is increased at 37° C compared to that at 30° C, while cytotoxin production in infected cells is similar at 30° C and 37° C. It can be concluded, therefore, that the integrated form of the CTX gene is responsible for CTX production. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed ten proteins in purified phage preparations; however, CTX could not be detected on Western blots using an enzyme-linked immunofluorescence assay.

Relationship of primary structure to functional properties of the cytotoxin protein from Pseudomonas aeruginosa

SummaryDeletion mutants of plasmid pSN3 carrying the Pseudomonas aeruginosa cytotoxin gene were prepared and expression in Escherichia coli of proteins with different molecular weights has been proved by Western blot. In addition, through PCR amplification of the cytotoxin gene and ligation into the vector, a single nucleotide change leading to an amino acid exchange from histidine to arginine has been constructed. The activities of the mutants were tested by binding of 125I-cytotoxin to rabbit erythrocyte ghosts and swelling of human granulocytes, showing that the cytotoxin activity is dependent on at least three different domains. Amino acids 12–20 from the C-terminus might be very important for proteolytic activation of protoxin to toxin.

Release from the phalloidin poisoned rat liver of a cytotoxic principle producing liver cell swelling, potassium release and hemolysis.

Summary0728 021.Double perfusion experiments with isolated rat livers confirm our earlier hypothesis that phalloidin activates a cytotoxic principle in the poisoned liver.2.After absorption of the toadstool cyclopeptide, the cytotoxic material is released from the liver, even after removal of the remaining cyclopeptide from the perfusion medium.3.The cytotoxic material leads to swelling of the perfused livers, provokes the release of potassium and organic compounds, and causes hemolysis in the perfusate.4.The potassium releasing activity of the perfusate is thermolabile at 50° C and is precipitated by ammonium sulphate. Its activity is inhibited by dithiothreitol. Low molecular substances released from poisoned livers are unlikely to play an important part as autotoxic substances.

Isolierung histaminliberierender und permeationsfördernder Polypeptide aus lysosomalen Granula polymorphkerniger Leukocyten des zirkulierenden Rinderblutes

SummaryIsolated lysosomal granules were prepared using the method of Hegner and lysed by Triton X 100. A crude peptide was isolated from concentrated extracts by precipitation with picric acid. Seven homogeneous peptides were separated by preparative polyvinylchloride block electrophoresis. The IEPs of these products were respectively about 2; 4.5; 6.5; 9.8; 11.2 and >12. The amino acid composition of two basic peptides (IEPs>12) was similar to arginine rich products found earlier in exudate leucocytes by Spitznagel and Zeya. MWs of 10,500 and 5,200 were estimated by gel filtration. These two arginine rich polypeptides were potent histamine liberators (0.5–1.0 μg/ml released more than 50% histamine from isolated peritoneal mast cells of the rat).Another basic polypeptide (MW=16,000–19,000, IEP=11.2) increased the permeability of venules in rabbit skin after i.c. injection of 0.2 μg. This peptide was found to be less potent in releasing histamine. Our observations were in agreement with those of Seegers and Janoff; Kelleret al. In addition, not only lysosomes from leucocytes of exudates contain biologically active peptides, but also granules from PMN leucocytes of the circulating blood which store remarkable amounts of active basic products.ZusammenfassungAus Granulocyten des peripheren Rinderblutes wurde eine lysosomenreiche Fraktion dargestellt, aus der durch Extraktion, Pikratfällung und Gelelektrophorese sieben teils saure, teils basische Polypeptide gewonnen wurden.Zwei Fraktionen mit hohem Arginingehalt sind hochwirksame Histaminliberatoren. Eine andere basische Fraktion ist an isolierten Mastzellen weniger wirksam, erhöht aber die Gefäßdurchlässigkeit in der Kaninchenhaut weit stärker als die vorgenannten Peptide. Unsere Ergebnisse stehen in Einklang mit Befunden anderer Autoren, die an Exsudatleukocyten kleiner Laboratoriumstiere gewonnen wurden. Der wichtigste Schluß aus der vorliegenden Studie ist, daß auch der Granulocyt des zirkulierenden Blutes bereits die bei der Emigration bedeutsamen Polypeptide enthält.