The effect of toluene on the structure and permeability of the outer and cytoplasmic membranes of Escherichia coli.

The effect of toluene on Escherichia coli has been examined. In the presence of Mg2+, toluene removes very little protein, phospholipid, or lipopolysacharide from E. coli. In the absence of Mg2+, or in the presence of EDTA, toluene removes considerably more cell material, including several specific cytoplasmic proteins such as malate dehydrogenase (EC 1.1.1.37). In contrast, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutamate dehydrogenase (EC 1.4.1.4) are not released at all under the same conditions. Cells treated with toluene in the presence of Mg2+ remain relatively impermeable to pyridne nucleotides, while cells treated with toluene in the presence of EDTA become permeable to these compounds. Freeze-fracture electron microscopy shows that toluene causes considerable damage to the cytoplasmic membrane, while the outer membrane remains relatively intact. These results indicate that the permeability characteristics of toluene-treated cells depend at least partly on the state of the outer membrane after the toluene treatment.     

Preparation of Cells Permeable to Macromolecules by Treatment with Toluene: Studies of Transfer Ribonucleic Acid Nucleotidyltransferase

Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene. The conditions of toluene treatment necessary for obtaining maximal activity were defined. Toluene treatment was most efficient when carried out for 5 min at 37 C at pH 9.0 on log-phase cells. No activity could be detected if cells were treated at 0 C, or in the presence of MgCl₂, or if the cells were in the stationary phase of growth. However, inclusion of lysozyme and ethylenediaminetetraacetic acid during the toluene treatment did render stationary phase cells permeable. The properties of tRNA nucleotidyltransferase from toluene-treated cells were essentially identical to those of purified enzyme with regard to pH optimum, specificity for nucleoside triphosphates and tRNA, and apparent K_m values for substrates. In addition to tRNA nucleotidyltransferase, a variety of other enzymes which incorporate adenosine 5′-triphosphate into acid-precipitable material could also be detected in toluene-treated cells. Centrifugation of cells treated with toluene revealed that tRNA nucleotidyltransferase leaked out of cells, whereas other activities remained associated with the cell pellets. Chromatography of the material extracted from toluene-treated cells on Sephadex G-100 indicated that toluene treatment selectively extracts lower molecular weight proteins. The usefulness of such a procedure as an initial step in purification of such enzymes, and its application to tRNA nucleotidyltransferase, is discussed.     

The effect of toluene on the structure and permeability of the outer and cytoplasmic membranes of escherichia coli

The effect of toluene on Escherichia coli has been examined. In the presence of Mg²⁺, toluene removes very little protein, phospholipid, or lipopolysaccharide from E. coli. In the absence of Mg²⁺, or in the presence of EDTA, toluene removes considerably more cell material, including several specific cytoplasmic proteins such as malate dehydrogenase (EC 1.1.1.37). In contrast, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutamate dehydrogenase (EC 1.4.1.4) are not released at all under the same conditions.Cells treated with toluene in the presence of Mg²⁺ remain relatively impermeable to pyridine nucleotides, while cells treated with toluene in the presence of EDTA become permeable to these compounds. Freeze-fracture electron microscopy shows that toluene causes considerable damage to the cytoplasmic membrane, while the outer membrane remains relatively intact. These results indicate that the permeability characteristics of toluene-treated cells depend at least partly on the state of the outer membrane after the toluene treatment.     

Polypeptide synthesis in toluene-treated lymphocytes

Normal human lymphocytes stimulated by phytohemagglutinin P, and other mammalian cells were rendered permeable to macromolecules such as poly(U) and proteins, by treatment with a low concentration of toluene. Under this condition, poly(U) translation was more efficient in the permeabilized cells than in 10000 X g extracts. Such a process occurs inside the treated cells as demonstrated by the fact that [14C]uridine-labelled ribosomes remain associated with the toluene-treated lymphocytes even after incubation at 37 degrees C. A nuclease from Staphylococcus aureus was able to penetrate the permeabilized cells and to break the polysome-bound endogenous messenger RNA. However, the protein-synthesizing machinery inside the toluene-treated lymphocytes was unaffected by the nuclease, as demonstrated by the unimpairment of polyphenylalanine synthesis when poly(U) was added after the preincubation with the enzyme. These results suggest that the toluene treatment can be considered as an important tool for the study of the synthesis of macromolecules and its regulation in eukaryotic cells.     

Basis for the observed fluctuation of carboxypeptidase II activity during the cell cycle in BUG 6, a temperature-sensitive division mutant of Escherichia coli.

Diaminopimelyl-d-alanyl carboxypeptidase (carboxypeptidase II) is most active at the time of division, whether measured in toluene-treated cells of Escherichia coli K-12 strain D11-1, fractionated by size, or in toluene-treated cells of the temperature-sensitive division mutant, BUG 6 (B. D. Beck and J. T. Park, 1976). The present investigation has now shown that, under conditions that permit division, the increased carboxypeptidase II activity in toluenetreated cells of BUG 6 is probably not due to protein synthesis. Although dividing cells are more permeable than nondividing cells, permeability differences are not sufficient to account for the changes in carboxypeptidase II activity. Thus, in the toluene-treated nondividing cells, carboxypeptidase II is present, but its activity is masked, which suggests the presence of an inhibitor. Another striking difference between nondividing and dividing cells is that carboxypeptidase II is much more readily released from dividing cells by both tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid and toluene treatment. Carboxypeptidase II was partially purified and found to be an 86,000-molecular-weight protein consisting of two 43,000-molecular-weight polypeptides. Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid treatment of nondividing cells releases less than 10% of the carboxypeptidase II and other periplasmic proteins that are releasable from dividing cells.     

High-yield extraction and purification of glutathione reductase from baker's yeast.

Glutathione reductase was extracted from toluene-treated baker's yeast cells by a two-stage buffer autolysis method. The yeast cells were treated with toluene for 1 h at 40 degrees C. After removal of the toluene, the cells were then allowed to autolysis in buffer for 72 h at 4 degrees C. The cells were collected and resuspended in buffer. A second stage autolysis was carried out for another 96 h at 4 degrees C. The enzyme was purified to 786-fold from the second stage cell autolysate by using two steps of affinity chromatography with triazine dyes (Yellow H-E4G and Yellow H-E6G) coupled to Sepharose CL-4B. By using this simplified method, 1.44 mg (165 units/mg) of glutathione reductase was obtained from 65 g (wet weight) of yeast cells, equivalent to 80% enzyme recovery.     

Theophylline reversal of alkylator-induced replicon initiation inhibition: no correlation with DDP-induced cytotoxicity

The synergistic cytotoxic activity exhibited by bifunctional alkylating agents in the presence of methylxanthines has been associated with methylxanthine-induced reversal of alkylator-induced DNA replicon initiation inhibition. This has also been seen with methylxanthines and ionizing irradiation. Methylxanthines do not appear exacerbate drug or ionizing radiation-induced damage. We report here a situation in which methylxanthine-induced reversal of DNA replicon initiation inhibition is not associated with increased cytotoxicity of the alkylator. Murine L1210 leukemia cells were assayed for cytotoxicity following treatment with either L-PAM or cis-DDP in the presence or absence of theophylline. Theophylline increased the cytotoxicity seen after L-PAM treatment but failed to increase the cis-DDP induced cytotoxicity. Analysis of pulse-labeled DNA on alkaline sucrose gradients revealed the expected decrease in DNA replicon initiation in L1210 cells treated with either L-PAM or cis-DDP. Theophylline had no effect on replicon initiation in untreated cells. Theophylline reversed the replicon initiation inhibition in cells treated with either L-PAM or cis-DDP. The reason for the apparent lack of added toxicity of the replicon initiation inhibition reversal in L1210 cells treated with theophylline and DDP is unknown.     

High-Yield Extraction and Purification of Glutathione Reductase from Baker's Yeast

Abstract

Glutathione reductase was extracted from toluene-treated baker's yeast cells by a two-stage buffer autolysis method. The yeast cells were treated with toluene for 1 h at 40[ddot]C. After removal of the toluene, the cells were then allowed to autolysis in buffer for 72 h at 4[ddot]C. The cells were collected and resus-pended in buffer. A second stage autolysis was carried out for another 96 h at 4[ddot]C. The enzyme was purified to 786-fold from the second stage cell autolysate by using two steps of affinity chromatography with triazine dyes (Yellow H-E4G and Yellow H-E6G) coupled to Sepharose CL-4B. By using this simplified method, 1.44 mg (165 units/mg) of glutathione reductase was obtained from 65 g (wet weight) of yeast cells, equivalent to 80% enzyme recovery.     

Involvement of thylakoid membranes in supramolecular organisation of Calvin cycle enzymes in Anacystis nidulans

The cells of unicellular photosynthetic cyanobacterium Anacystis nidulans were permeated with lysozyme, toluene, toluene-triton, toluene-triton-lysozyme. Transmission electron microscopy of semi-thin sections (500 nm) using TEM at 160 kV showed that cells permeated with only lysozyme or toluene showed the typical concentric arrangement of thylakoid membranes. However, when toluene-treated cells were further treated with triton and lysozyme the thylakoid membranes were disrupted. Sequential reactions of Calvin cycle were studied in the differentially permeated cells in vivo, using various intermediates such as 3-PGA, GA-3-P, FDP, SDP, R-5-P, RuBP and cofactors like ATP, NADPH depending on the requirement. RuBP and R-5-P + ATP dependent activities could be observed in all types of permeated cells. Sequential reactions of the entire Calvin cycle using 3-PGA could be detected in the cells that had retained the internal organisation of the thylakoid membranes after permeation and were lost on disruption of this organisation. Light dependent CO2 fixation could be detected only in the cells permeated with lysozyme. This activity was abolished in the cells after treatment with toluene. The results suggested that the integrity of thylakoid membranes may be essential for the organisation of sequential enzymes of the Calvin cycle in vivo and facilitate their functioning.     

Isolation and Characterization of dnaX and dnaY Temperature-Sensitive Mutants of ESCHERICHIA COLI

Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage P1, dnaX and Y have been mapped at minutes 10.4--10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dna Xts36 and Yts10 are recessive to wild-type alleles present on episomes. F13 carries both dnaX+ and Y+; the shorter F210 carries dnaY+, but not X+. Lambda tranducing phages that carry dnaX+ or Y+ have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the Clarke and Carbon (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the lambda transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5--The dnaXts36 mutant, after a shift to 42 degrees, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to 44 degrees, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42 degrees and was partially inhibited even at 30 degrees.--When the dnaYts10 mutant was shifted to 42 degrees, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44 degrees, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42 degrees or by "preincubation" of cells at 42 degrees before toluene treatment.--The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.     

Synthesis of yeast wall glucan.

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.     

Measurement of matrix enzyme activity in isolated mitochondria made permeable with toluene.

Mitochondria isolated from rat liver and heart were made permeable to normally nonpentrating substrates and cofactors by treatment with toluene. The optimal conditions for preparing stable, permeable mitochondria were 2% toluene for 2 min at 4 °C in a buffered, isotonic medium containing 8.5% polyethylene glycol (Mr 6000–7500). Without polyethylene glycol, the toluene-treated mitochondria were unstable and released their matrix enzymes. The treated mitochondria were particularly unstable in dilute suspension under normal assay conditions of their enzyme activities. The levels of matrix enzyme activities unmasked by toluene treatment of mitochondria were very close to those of sonicated mitochondria under identical assay conditions. Mitochondria made permeable with toluene lost only small amounts of their protein and retained a major fraction of the nucleotides and coenzymes. Electron microscopic examination of toluenetreated mitochondria indicated that they were relatively intact with swollen and vesiculated cristae membranes. Such preparations will allow the study of mitochondrial enzymes at approximate in vivo concentrations.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Kinetics of toluene-induced leakage of low molecular weight solutes from excised sorghum tissues

The relationship between toluene concentration and the rate of leakage of solutes from toluene-treated roots and leaves of Sorghum bicolor, L. Moench, was studied to determine the effect of toluene on plant cell membranes. A threshold concentration of 0.2% toluene was needed to induce leakage. Maximal leakage rates were obtained with 0.5% toluene. Low molecular weight solutes, such as amino acids, sugars, and inorganic ions, leaked from treated tissue, while macromolecules, such as protein were retained. The rates at which the low molecular weight solutes diffused from treated cells decreased with increasing molecular weight. At 25°C, treatment of roots and leaves with 0.5% toluene resulted in the quasi-quantitative leakage of solutes within 180 minutes. At 1°C, roots and leaves differed in their response to toluene. The rates of leakage from roots at 1°C were much lower and the total amounts much smaller than at 25°C, while in leaves the difference between the two temperatures was very small.     

DNA replication initiation as a key element in thymineless death

Thymine deprivation results in the loss of viability in cells from bacteria to eukaryotes. Numerous studies have identified a variety of molecular processes and cellular responses associated with thymineless death (TLD). It has been observed that TLD occurs in actively growing cells, and DNA damage and DNA recombination structures have been associated with cells undergoing TLD. We measured the loss of viability in thymine-starved cells differing in the number of overlapping replication cycles (n), and we found that the magnitude of TLD correlates with the number of replication forks. By using pulsed field gel electrophoresis (PFGE), we determined the proportion of linear DNA (DSBs) and the amount of DNA remaining in the well after treatment with XbaI (nmDNA) under thymine starvation in the absence or presence of both rifampicin (suppressing TLD) and hydroxyurea (maintaining TLD). Our results indicate that DSBs and nmDNA are induced by thymine starvation, but they do not correlate with the lethality observed in the presence of the drugs. We asked whether TLD was related to chromosomal DNA initiation. DNA labeling experiments and flow cytometric analyses showed that new initiation events were induced under thymine starvation. These new DNA replication initiation events were inhibited in the presence of rifampicin but not in the presence of hydroxyurea, indicating that TLD correlates with the induction of new initiation events in Escherichia coli. In support of this finding, cells carrying a deletion of the datA site, in which DNA initiation is allowed in the presence of rifampicin, underwent TLD in the presence of rifampicin. We propose that thymineless-induced DNA initiation generates a fraction of DNA damage and/or nmDNA at origins that is critical for TLD. Our model provides new elements to be considered when testing mammalian chemotherapies that are based on the inhibition of thymidylate synthetase.     

Structural and Nonstructural Proteins of an Arbovirus

Purified Semliki Forest virus (SFV) contains three structural proteins while its core (nucleocapsid) contains two of these proteins. To identify all of the proteins synthesized under virus direction, cells were infected with SFV in the presence of actinomycin D and guanidine. Cell protein synthesis was markedly and irreversibly inhibited under these conditions; virus growth was reversibly inhibited by guanidine and began when the cells were washed to remove the guanidine. When cells were treated with guanidine for 4 hr after virus infection and then were washed, five major proteins were produced early in infection. Three of these proteins corresponded to virus structural proteins. None of these five proteins was a major protein of uninfected cells or of virus-infected cells which had been incubated with partially purified interferon before infection. Late in infection, three major proteins, the virus structural proteins, were produced.     

<Emphasis Type="SmallCaps">d</Emphasis>-Psicose production from <Emphasis Type="SmallCaps">d</Emphasis>-fructose using an isolated strain, <Emphasis Type="Italic">Sinorhizobium</Emphasis> sp.

Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h.     

Energy requirement for the initiation of colicin action in Escherichia coli

1. Starved cells of a strain of Escherichia coli and its mutant uncA, treated with colicin K, E2 or E3, remained fully rescuable upon trypsin treatment (stage I in colicin action). The transition to stage II in colicin action (cells no longer rescuable by trypsin) was promoted by the addition of either glucose or d-lactate.2. Aerobically glucose-grown cells of the normal strain were irreversibly killed by colicin K, E2 or E3 under anaerobic conditions, while similarly treated cells of its mutant uncA remained fully rescuable. The stage I-stage II transition in colicin action was blocked in normal cells under anaerobic conditions when succinate was the sole carbon source.3. Arsenate alone had little effect on the progression of the stage I-stage II transition in normal cells, treated with colicin K. However, this transition was abolished in the presence of both arsenate and anaerobic conditions.4. The initiation of colicin action could be coupled to the anaerobic electron transfer systems formate dehydrogenase-nitrate reductase and α-glycerophosphate dehydrogenase-fumarate reductase.5. These results indicate that an energized state of the cytoplasmic membrane is required for the initiation of colicin action and that no high-energy phosphorylated compounds are necessary.     

Morphine-induced desensitization and down-regulation at mu-receptors in 7315C pituitary tumor cells

Pituitary 7315c tumor cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 microM, for periods of five or forty-eight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed membrane preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same membrane preparations, the number and affinity of mu-opioid receptors was estimated by measurements of [3H]diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for [3H]diprenorphine. However, after 48 hr of morphine treatment, greater than 25% reductions in receptor number were apparent with morphine pretreatment concentrations of 10 microM or higher. These results suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor number occurred after exposure to high concentrations of morphine for periods longer than 5 hr.