Showing papers on "Lysis published in 1986"

Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores.

Abstract: Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After binding to target erythrocytes, the membrane-bound toxin resembled an integral membrane protein in that it was refractory towards extraction with salt solutions of low ionic strength. Toxin-induced hemolysis could be totally inhibited by addition of 30 mM dextran 4 (mean Mr, 4,000; molecular diameter approximately 3 nm) to the extracellular medium. Uncharged molecules of smaller size (e.g., sucrose, with a molecular diameter of 0.9 nm, or raffinose, with a molecular diameter of 1.2 to 1.3 nm) did not afford such protection. Treatment of erythrocytes suspended in dextran-containing buffer with the toxin induced rapid efflux of cellular K+ and influx of 45Ca2+, as well as influx of [14C]mannitol and [3H]sucrose. [3H]inulin only slowly permeated into toxin-treated cells, and [3H]dextran uptake was virtually nil. Membranes lysed with high doses of E. coli hemolysin exhibited no recognizable ultrastructural lesions when examined by negative-staining electron microscopy. Sucrose density gradient centrifugation of deoxycholate-solubilized target membranes led to recovery of the toxin exclusively in monomer form. Incubation of toxin-treated cells with trypsin caused limited proteolysis with the generation of membrane-bound, toxin-derived polypeptides of Mr approximately 80,000 without destroying the functional pore. We suggest that E. coli hemolysin may damage cell membranes by partial insertion into the lipid bilayer and generation of a discrete, hydrophilic transmembrane pore with an effective diameter of approximately 3 nm. In contrast to the structured pores generated by cytolysins of gram-positive bacteria such as staphylococcal alpha-toxin and streptolysin O, pore formation by E. coli hemolysin may be caused by the insertion of toxin monomers into the target lipid bilayers.

Homologous species restriction in lysis of human erythrocytes: a membrane-derived protein with C8-binding capacity functions as an inhibitor.

Abstract: An intrinsic membrane protein with a m.w. of 65,000 that can bind human C8 has been identified after separation of human erythrocyte membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose sheets. The protein, tentatively designated as the C8-binding protein (C8bp) could be isolated from papain-treated erythrocyte (E) membranes by phenol-water extraction and isoelectric focusing. In a functional assay, with chicken (ch) E as target cells, C8bp inhibited the lysis of ch E C5b67 intermediates by human C8 and C9, whereas the lysis by rabbit C8 and C9 was not affected. Because the decay accelerating factor (DAF) from human erythrocyte membranes also inhibits the activity of C3/C5 convertases in an homologous system, we tested whether or not a DAF activity was present in C8bp. C8bp, however, did not accelerate the decay of the classic C3 convertases. Thus, it appears that C8bp and DAF are two different factors of E membranes with a similar molecular size inhibiting different sites of the activation cascade of complement while they can function synergistically to minimize the self-inflicted damage by complement.

Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels

Abstract: Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.

Purification and restriction endonuclease analysis of plant nuclear DNA

Abstract: Publisher Summary This chapter discusses the methods for the purification and restriction endonuclease analysis of plant nuclear DNA. The rationale of the methods for the isolation of DNA from purified nuclei have the features, such as a pretreatment of the tissue to enhance cell disruption, homogenization in the presence of membrane stabilizing agents, filtration to remove whole cells and large debris, differential lysis of organelles with Triton X-100 in the presence of divalent cations (Mg 2+ or Ca 2+ ), and purification of the nuclei by density gradient centrifugation. Nuclei are separated from other cellular debris by density centrifugation in suspensions of Percoll, colloidal silica coated with polyvinylpyrolidone. Once the nuclei have been purified, extraction of DNA is accomplished by detergent lysis and thorough protease digestion. Failure to completely digest proteins at this point is the most frequent reason for inability to digest the DNA with restriction enzymes. DNA is separated from RNA and any residual protein by repeated CsCl–ethidium bromide gradient centrifugation.

Guanidine Isothiocyanate Preparation of Total RNA

Abstract: This chapter discusses the method of guanidine isothiocyanate preparation of total RNA, which is a versatile and efficient way to extract intact RNA from most tissues and cultured cells, even if the endogenous level of RNase is high. The cells are lysed in guanidine isothiocyanate using a tissue homogenizer. The lysate is layered onto a CsCl gradient and spun in an ultracentrifuge. Proteins remain in the aqueous guanidine portion, DNA bands in the CsCl, and RNA pellets in the bottom of the tube. The RNA is recovered by redissolving the pellet. The recovery of RNA is usually excellent if the capacity of the gradient is not exceeded. The method can also be used to isolate RNA from tissue or to isolate both RNA and DNA from cells. The time taken to follow this method is two days.

Lysis of human solid tumor cells by lymphokine-activated natural killer cells.

Abstract: The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.

Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9.

Abstract: Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.

Gentamicin interaction with Pseudomonas aeruginosa cell envelope.

Abstract: Gentamicin, an aminoglycoside antibiotic known to inhibit protein synthesis, had a detrimental effect on the integrity of the cell wall of Pseudomonas aeruginosa ATCC 9027 (a susceptible strain) as shown by electron microscopy using negative-staining, thin-sectioning, and freeze-fracture techniques. The disruption occurred in a sequential manner, moving from the outer membrane to the inner membrane, and could result in lysis of the cell. During this process the outer membrane lost 34% of its total protein and 30% of its lipopolysaccharide (measured as 2-keto-3-deoxyoctonate) upon exposure to 25 micrograms of gentamicin per ml for 15 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the outer membrane proteins showed altered banding patterns after exposure to gentamicin. Atomic absorption spectrophotometry revealed a decrease in magnesium and calcium content (18 and 38%, respectively) in the cell envelopes after gentamicin treatment. It is proposed that gentamicin displaces essential metal cations within the outer membrane, consequently destabilizing and extracting organic constituents. Small transient holes are thereby produced which make the outer membrane more permeable to the antibiotic and which expose the protoplast to high concentrations of gentamicin. This membrane effect may contribute to the effects of protein synthesis inhibition during the killing process.

Effects of Divalent Cations and of Phospholipase A Activity on Excretion of Cloacin DF13 and Lysis of Host Cells

Abstract: SUMMARY: Induction of cloacin DF13 synthesis in Escherichia coli harbouring plasmid CloDF13 results in the release of cloacin DF13, inhibition of growth and ultimately in lysis of the host cells. Expression of the pCloDF13-encoded protein H is essential for both the release of cloacin DF13 and the lysis of the cells. The divalent cations Mg2+ and Ca2+ interfered with the mitomycin C-induced, protein H-dependent lysis, but hardly affected the release of cloacin DF13. Essentially all of the bacteriocin was released from the cells before a detectable degradation of the peptidoglycan occurred, independent of the presence of mitomycin C. Experiments with phospholipase A mutants revealed that activation of detergent-resistant phospholipase A was essential for the export of cloacin DF13 across the outer membrane and the lysis of induced cells. Transport of cloacin DF13 across the cytoplasmic membrane was mainly dependent on protein H. A revised model for the excretion of cloacin DF13 is presented.

Cell-surface proteins of Streptococcus sanguis associated with cell hydrophobicity and coaggregation properties.

Abstract: Summary: Incubating cells of Streptococcus sanguis with sodium lauroyl sarcosinate, under conditions that did not cause lysis, solubilized material comprising 5-8% of the cell dry weight. The treatment reduced cell hydrophobicity, and reduced the ability of the cells to coaggregate with Actinomyces spp. The extract contained about 20 polypeptides and these were identified as being cell-surface components on the basis of one or more of the following criteria: (a) being degraded when cells were incubated with protease; (b) being labelled when cells were iodinated using a lactoperoxidase-catalysed reaction; (c) reacting with antibodies raised to fixed whole cells. Eight of the polypeptides accounted for more than 70% of the total protein extracted, and one component (molecular mass 16 kDa) was hydrophobic. The cell-surface proteins described are implicated in cell hydrophobicity and coaggregation.

A simple and generally applicable procedure for releasing DNA from bacterial cells.

Abstract: Treatment of Staphylococcus simulans biovar staphylolyticus cells with acetone before digestion with lysozyme made the cells susceptible to lysis by sodium dodecyl sulfate. This technique was found to be useful for releasing DNA from a wide variety of gram-positive and gram-negative organisms.

Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors.

Abstract: Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present in normal growing cells A method was developed to investigate whether there exists an excess of cell wall-synthesizing enzymes (penicillin-binding proteins [PBPs]) which all remain functional or whether a mixed population of functional and nonfunctional enzymes characterize normal cells Surprisingly, cells in which less than 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts, culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis This subset of functional enzymes was biosynthetically new Penicillin-induced lysis occurred contingent on the acylation of this same small fraction of PBPs, the copy number and affinities of which were below the level of detection by current fluorographic assay techniques We propose that PBPs have a short functional half-life and that cell wall synthesis and bacterial lysis reflect the activity of newly synthesized PBPs

Cadmium-Binding Proteins in Pseudomonas putida: Pseudothioneins

Abstract: Pseudomonas putida adapted to growth in 3 mM cadmium. The resistance mechanism involved complexation of cadmium in polyphosphate granules, changes in the structure of the cell membrane and induction of three cysteine-rich, low molecular weight proteins (3500-7000) containing 4 to 7 g-atoms per mole of cadmium, zinc, and copper. Each protein was produced during a different phase of growth, and the smallest protein (3500) was released into the environment when the cells lysed at the end of the exponential phase. The metal binding sites of the major protein were further characterized using a range of physical methods, including 113Cd NMR. The properties of the bacterial pseudothioneins are compared to those of metallothioneins.

Cell wall of pathogenic yeasts and implications for antimycotic therapy.

Abstract: Yeast cell wall is a complex, multilayered structure where amorphous, granular and fibrillar components interact with each other to confer both the specific cell shape and osmotic protection against lysis. Thus it is widely recognized that as is the case with bacteria, yeast cell wall is a major potential target for selective chemotherapeutic drugs. Despite intensive research, very few such drugs have been discovered and none has found substantial application in human diseases to date. Among the different cell wall components, beta-glucan and chitin are the fibrillar materials playing a fundamental role in the overall rigidity and resistance of the wall. Inhibition of the metabolism of these polymers, therefore, should promptly lead to lysis. This indeed occurs and aculeacin, echinocandin and polyoxins are examples of agents producing such an action. Particular attention should be focused on chitin synthesis. Although quantitatively a minor cell wall component, chitin is important in the mechanism of dimorphic transition, especially in Candida albicans, a major human opportunistic pathogen. This transition is associated with increased invasiveness and general virulence of the fungus. Yeast cell wall may also limit the effect of antifungals which owe their action to disturbance of the cytoplasmic membrane or of cell metabolism. Indeed, the cell wall may hinder access to the cell interior both under growing conditions and, particularly, during cell ageing in the stationary phase, when important structural changes occur in the cell wall due to unbalanced wall growth (phenotypic drug resistance).

DNA of human Raji target cells is damaged upon lymphocyte-mediated lysis.

Abstract: Human Raji target cells DNA is degraded by the introduction of single-strand breaks (alkali-sensitive sites) upon lymphocyte-mediated lysis. This type of DNA degradation appears earlier and is more extensive in lymphocyte-than in antibody + complement-mediated lysis of Raji cells, regardless of the species of effector lymphocytes (human or mouse). Mouse P815 target cell DNA is extensively fragmented (yielding 200 base pair fragments) when human or mouse lymphocytes are used to lyse P815. Thus, these observations indicate that both human and mouse target cell DNA are affected during lymphocyte-mediated lysis. Moreover, the pattern of DNA degradation in target cells lysed by effector lymphocytes is characteristic of the target cell species, suggesting that DNA degradation proceeds through the activation of target cell endonuclease(s).

Primary structures of the ColE2-P9 and ColE3-CA38 lysis genes.

Abstract: The lysis genes of plasmids ColE2-P9 and ColE3-CA38 were identified by DNA sequencing and electrophoretic analysis of the products of both wild type and artificially introduced ochre mutant genes. The E2 and E3 lysis genes had identical primary structures and were shown to encode 47 amino acids with a calculated molecular weight of 4,861, which is much smaller than that proposed previously for the ColE3-CA38 lysis protein. They are homologous with ColDF13 gene H, except in their 3'-portions. The nine C-terminal amino acids of the E2 and E3 lysis proteins proved to be non-essential for the lysis phenotype.

Modulation of bacteriolysis by cooperative effects of penicillin-binding proteins 1a and 3 in Escherichia coli.

Abstract: Escherichia coli characteristically lyses upon treatment with most beta-lactam antimicrobial agents. In contrast, an investigational aminothiazole cephem, CGP 31523A, produced a new combination of antibacterial effects: it was highly bactericidal without causing cell wall degradation or lysis. Killing was associated with the formation of vacuolated filaments. Because the compound bound to penicillin-binding proteins (PBPs) 1a and 3, we investigated the role of PBP 3 in modulation of lysis caused by inhibition of PBP 1a. A temperature-sensitive mutant with a nonfunctional PBP 3 lysed when treated with CGP 31523A. The combination of a PBP 1 inhibitor (cefsulodin) and a PBP 3 inhibitor (aztreonam) also caused filamentation and death without lysis of wild-type cells over a narrow concentration range. We conclude that cooperative effects between PBPs in E. coli can lead to a dissociation of bacterial killing and lysis.

Purification of pichia produced lipophilic proteins

Abstract: Method for the selective extraction of desired lipophilic proteins from transformed cells of the genus Pichia by cell lysis in the presence of chaotrophic salts is disclosed. The total protein extracted under the invention cell lysis conditions is reduced while the recovery of desired lipophilic proteins remains relatively constant, thereby producing a cell extract with enhanced concentration of the desired lipophilic protein relative to a control cell extract.

Activity, death and lysis during microbial growth in a chemostat

Abstract: The significance of death and lysis processes during microbial growth in chemostat cultures is discussed. A structured model is developed to describe such processes and the model is partially verified for carbon limited chemostat cultures of Klebsiella pneumoniae. It seems probable that death and lysis are coincident, when non-viable cells are not considered to be dead.

Complement lysis of U937, a nucleated mammalian cell line in the absence of C9: effect of C9 on C5b-8 mediated cell lysis.

Abstract: Previous studies have demonstrated that in general, nucleated cells are more resistant to killing by serum complement than are erythrocytes. During studies aimed at defining the mechanisms of nucleated cell resistance, we found that the human histiocytic cell line U937 was easily lysed by homologous serum. U937 cells were also killed by serum depleted of C9, but not by serum depleted of C8, implying that the C5b-8 complex was sufficient to cause lysis of these cells. Enumeration of complexes on the cell surface demonstrated that approximately 40-fold more complexes were required to lyse U937 cells in the absence of C9 than in the presence of an excess of C9. Examination of the effects of small amounts of C9 on lysis of U937 cells by the C5b-8 complex demonstrated that at very low doses, C9 inhibited C5b-8 mediated lysis. The use of radiolabeled anti-C8 antibody showed that C5b-8 complexes were eliminated from the surface of U937 cells at 37 degrees C, and C9 at the dose causing inhibition of lysis accelerated the elimination of complexes. These results suggest that the increased lytic potential resulting from binding of small amounts of C9 to C5b-8 complexes is outweighed by enhanced elimination of complexes resulting in decreased cell death.

Evasion of the alternative complement pathway by metacyclic trypomastigotes of Trypanosoma cruzi: dependence on the developmentally regulated synthesis of surface protein and N-linked carbohydrate.

Abstract: Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation

Substrate-induced membrane association of phosphatidylserine synthase from Escherichia coli.

Abstract: To better establish the intracellular location of the phosphatidylserine synthase of Escherichia coli and hence better understand how it is regulated in the cell, we compared the size, function, and binding properties of the enzyme made in vitro with the enzyme found in cell lysates and with the purified enzyme. The enzyme made either in vivo or in an active form in vitro was found primarily associated with the ribosomal fraction of the cell and had the same apparent molecular mass as the purified enzyme. These results were unaffected by the presence of protease inhibitors. Addition of unsupplemented E. coli membranes or membranes supplemented with phosphatidylethanolamine did not affect the subcellular distribution of the enzyme in these experiments. However, addition of membranes supplemented with either the lipid substrate, CDP-diacylglycerol, or the lipid product, phosphatidylserine, resulted in membrane association by the enzyme rather than ribosomal association. Addition of membranes supplemented with acidic lipids also brought about membrane association, but this association was primarily ionic since it was disrupted by high salt concentrations. These results strongly suggest that the ribosomal location of this enzyme is not the result of some modification event occurring after cell lysis and that the normal functioning of the enzyme involves membrane association which is primarily induced by the presence of a membrane-associated substrate.

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

Abstract: When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.