Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp

The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.     

UDP-Glucose: Sterol Glucosyltransferase and a Steryl Glucoside Acyltransferase Activity in Amyloplast Membranes from Potato Tubers

Envelope membranes were isolated from potato tuber amyloplastby a discontinuous sucrose density gradient and high speed centrifugation.These membranes catalyzed the transfer of [¹⁴C]glucose fromUDP-[¹⁴C]glucose to endogenous sterol acceptors and, in turn,catalyzed the esterification of steryl glucosides with fattyacids from an endogenous acyl donor. The synthesis of sterylglucosides was stimulated in the presence of Triton. X-100,while formation of acyl steryl glucosides was inhibited by thedetergent. However, in the presence of an added sterol acceptorand Triton X-100, the inhibition of acyl steryl glucoside synthesiswas overcome by the addition of phosphatidylethanolamine. Theenzyme involved in steryl glucoside formation was solubilizedby treatment of the envelope membranes with 0.3% Triton X-100.The solubilized enzyme had an almost absolute requirement forsterol acceptors. Key words: Solanum tuberosum, Sterol glucosylation, Steryl glucoside acylation, Amyloplast membrane     

Amphiphilic and hydrophilic molecular forms of acetylcholinesterase in membranes derived from sarcoplasmic reticulum of skeletal muscle

Native molecular forms of acetylcholinesterase (AChE) present in a microsomal fraction enriched in SR of rabbit skeletal muscle were characterized by sedimentation analysis in sucrose gradients and by digestion with phospholipases and proteinases. The hydrophobic properties of AChE forms were studied by phase-partition of Triton X-114 and Triton X-100-solubilized enzyme and by comparing their migration in sucrose gradient containing either Triton X-100 or Brij 96. We found that in the microsomal preparation two hydrophilic 13.5 S and 10.5 S forms and an amphiphilic 4.5 S form exist. The 13.5 S is an asymmetric molecule which by incubation with collagenase and trypsin is converted into a 'lytic' 10.5 S form. The hydrophobic 4.5 S form is the predominant one in extracts prepared with Triton X-100. Proteolytic digestion of the membranes with trypsin brought into solution a significant portion of the total activity. Incubation of the membranes with phospholipase C failed to solubilize the enzyme. The sedimentation coefficient of the amphiphilic 4.5 S form remained unchanged after partial reduction, thus confirming its monomeric structure. Conversion of the monomeric amphiphilic form into a monomeric hydrophilic molecule was performed by incubating the 4.5 S AChE with trypsin. This conversion was not produced by phospholipase treatment.     

Atrial muscarinic receptors: Effect of triton X-100 on guanine nucleotide and ammonium ion modulation

Membranes isolated from bovine atria were labeled with [³H]quinuclidinyl benzylate (³H-QNB), in control conditions and after 0.02% Triton X-100. This treatment inactivated abcut 20% of muscarinic receptor sites without loss of protein. The remaining 80% sites showed no changes in affinity, as determined by equilibrium or kinetic binding. Competition experiments with carbachol showed no differences in IC₅₀ and Hill number between the control and detergent-membranes, suggesting that the different populations of agonist binding sites are inactivated in equal proportions by the detergent. In binding experiments, done in the presence of carbachol and guanine nucleotides, the detergent treated membranes were slightly more sensitive to the enhancing action of the nucleotide. The inhibition caused by ammonium ions was also more marked in the Triton X-100 treated membranes. The decay of binding with thermal inactivation was faster in the detergent treated membranes and this effect was enhanced in the presence of ammonium ions. These results may be interpreted as an indication that the receptors, remaining after the mild Triton X-100 treatment, are equally sensitive to the inactivation. We suggest that, while maintaining the heterogeneity of sites, the detergent produces a perturbation that could affect the molecular interactions between the receptor and other components of the membrane.     

Localization of adenylate cyclase in Dictyostelium discoideum. I. Preparation and biochemical characterizations of cell fractions and isolated plasma membrane vesicles.

A difference in the organization of adenylate cyclase and 3′5′-cyclic phosphodiesterase in isolated plasma membranes was observed. Observation of this difference was made possible by the development of a new technique for the lysis of Dictyostelium discoideum using the polyene antibiotic amphotericin B. A particulate fraction prepared from the cell lysate contains adenylate cyclase, 3′5′-cyclic phosphodiesterase and 5′-nucleotidase. The yield of adenylate cyclase is 40% higher than in paniculate fractions prepared from cells lysed by sonication or with Triton X-100. Purification of the particulate fraction on discontinuous sucrose gradient completely separates membranes from mitochondria and other cellular material as shown by electron microscopic analysis of different fractions. Biochemical characterization of the purified membrane fraction shows it contains adenylate cyclase, 3′5′-cyclic phosphodiesterase and 5′-nucleotidase activities while electron microscopic analysis shows a vesicular morphology. Additional studies on the purified membranes used Triton X-100, trypsin and phospholipase C to probe the relationship between membrane structural elements and enzymatic activities. The results of these studies show distinct differences in the organization of each enzyme molecule within the membrane.     

Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells

Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and inserted into the wall via exocytosis. The most abundant pectic polysaccharide, homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase homogalacturonan methyltransferase (HGA-MT). The subcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradients. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reticulum, was detected in the Golgi fraction. Homogalacturonan methyltransferase activity was not reduced by protease treatment of intact membranes or membranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was reduced by protease treatment of membranes permeabilized with 0.02% Triton X-100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and the lack of inhibition of HGA-MT activity by protease-treatment of intact membranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi. Received: 2 December 1998 / Accepted: 9 February 1999     

Rapid protein release from Escherichia coli by chemical permeabilization under fermentation conditions

Overall protein release greater than 75% in less than 1 h can be attained by exposing exponentially growing Escherichia coli cells to 0.4 M guanidine plus 0.5% Triton X-100 at 37 degrees C in medium. Cell growth stops immediately upon addition of the chemicals, but the cells are not lysed. Guanidine concentrations lower than 0.2 M, in conjunction with 0.5% Triton X-100, do not release significant intracellular protein, nor do they inhibit cell growth. Under these conditions, the cells undergo an adaptation that confers resistance to protein release by further treatment with guanidine and Triton X-100. Cells treated with 0.2 M guanidine plus 0.5% Triton X-100 display intermediate behavior. Protein release is approximately 35%, and growth is temporarily interrupted by an extended lag phase. Subsequent resumption of cell growth results in resistant cells and no additional protein release. This resistance is shown to be reversible and is most likely due to physiological adaptation rather than genetic mutation.     

On the mechanism of membrane damage by Staphylococcus aureus alpha- toxin

Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.     

The solubilization of the L and M antigens from sheep red cell membranes

The Lp, L1 and M antigens from sheep red cells were solubilized using the non-ionic detergent Triton X-100 in the presence of dithiothreitol. Recovery rates were improved when membranes were sonicated at 4 degrees C in the presence of the detergent; values in the range 16-25% (M) and 9-17% (Lp and L1) were achieved for recovery.     

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.     

T cell receptor can be recruited to a subset of plasma membrane rafts,independently of cell signaling and attendantly to raft clustering

The constitutive/inducible association of the T cell receptor (TCR) with isolated detergent-resistant, lipid raft-derived membranes has been studied in Jurkat T lymphocytes. Membranes resistant to 1% Triton X-100 contained virtually no CD3epsilon, part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3epsilon amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane subset because (i) all three membrane preparations contained a similar amount of the raft marker tyrosine kinase Lck but no detectable amounts of the conventional membrane markers, CD45 phosphatase and transferrin receptor; (ii) a larger amount of particulate membranes were resistant to solubilization with 0.2% Triton X-100 and Brij 58 than to solubilization with 1% Triton X-100; and (iii) higher cholesterol levels were present in membranes resistant to either the lower Triton X-100 concentration or to Brij 58, as compared with those resistant to 1% Triton X-100. The recruitment of CD3 to the raft-derived membrane subset appeared (i) to occur independently of cell signaling events, such as protein-tyrosine phosphorylation and Ca(2+) mobilization/influx, and (ii) to be associated with clustering of plasma membrane rafts induced by multiple cross-linking of either TCR or the raft component, ganglioside GM(1). We suggest that during T cell stimulation a lateral reorganization of rafts into polarized larger domains can determine the recruitment of TCR into these domains, which favors a polarization of the signaling cascade.     

Release of membrane proteins in relation to heat injury of spinach chloroplasts

Thylakoids isolated from spinach leaves ( Spinacia oleracea L. cvs. Monatol and Montako) were exposed to supraoptimal temperatures that inactivated their photochemical reactions. Membrane injury was accompanied by release of a small amount of membrane proteins. When sucrose was present during high-temperature treatment, thylakoids were partially protected and release of membrane proteins was less pronounced than in the absence of sugar. From thylakoids, which were isolated from heat-damaged spinach leaves, less protein was released when heated again after the isolation procedure, indicating that protein release also takes place during heat inactivation in vivo . Sodium dodecyl sulfate gel electropherograms of thylakoids demonstrated that heat inactivation of the lamellae was not accompanied by significant changes in the pattern of the proteins, which remained in the membranes. The same was found when thylakoids were solubilized with Triton X-100 before and after heat damage. It is suggested that the protein release that occurs during heat treatment is a consequence of irreversible alterations in the membrane structure; these changes may be responsible for thermal damage of chloroplast membranes.     

The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The K(m) of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.     

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.     

Low temperature development of winter rye leaves alters the detergent solubilization of thylakoid membranes

Thylakoids isolated from leaves of winter rye (Secale cereale L. cv Puma) grown at either 20 or 5°C were extracted with the nonionic detergents Triton X-100 and octyl glucoside. Less total chlorophyll was extracted from 5°C thylakoids by these detergents under all conditions, including pretreatment with cations. Thylakoids from either 20 or 5°C leaves were solubilized in 0.7% Triton X-100 and centrifuged on sucrose gradients to purify the light harvesting complex (LHCII). Greater yields of LHCII were obtained by cation precipitation of particles derived from 20°C thylakoids than from 5°C thylakoids. When 20 and 5°C thylakoids were phosphorylated and completely solubilized in sodium dodecyl sulfate, no differences were observed in the ³²Pi-labeling characteristics of the membrane polypeptides. However, when phosphorylated thylakoids were extracted with octyl glucoside, extraction of LHCII associated with the 5°C thylakoids was markedly reduced in comparison with the extraction of LHCII from 20°C membranes. Since 20 and 5°C thylakoids exhibited significant differences in the Chl content and Chl a/b ratios of membrane fractions produced after solubilization with either Triton X-100 or octyl glucoside, and since few differences between the proteins of the two membranes could be observed following complete denaturation in sodium dodecyl sulfate, we conclude that the integral structure of the thylakoid membrane is affected during rye leaf development at low temperature.     

Location of an F-pilin pool in the inner membrane.

Polyacrylamide gel analysis of [35S]methionine-labeled membrane preparations from Escherichia coli has revealed the presence of five polypeptides present only in the membranes of cells containing the conjugative plasmid F. In addition to the previously reported product of traT, polypeptides migrating with apparent molecular weights of 100,000, 23,500, 12,000, and 7,000 were resolved. Membrane preparations from F traJ mutants lacked these polypeptides, indicating that all of these proteins are tra gene products. The 7,000-molecular-weight polypeptide comigrated with unlabeled purified F-pilin protein. About 4 to 5% of the total radioactive label in whole membrane preparations was present in this polypeptide, indicating the existence of a substantial pool of membrane-associated F-pilin. The polypeptide could be extracted from whole membrane preparations with Triton X-100 and was found in the inner membrane fraction of membranes separated by sucrose density centrifugation.     

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X-100. The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O. The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X-100, are 0.73 and 0.74 ml . g-1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X-100. The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent. The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X-100 compared to 6.7 nm in the absence of detergent. The apparent molecular weight therefore increases from 242,500 to 466,000 on removal of detergent. In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63,000. The enzyme therefore behaves as a non-covalently linked tetramer in the presence of Triton X-100. Removal of Triton X-100 results in association of tetramers to form octamers.     

Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody

A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes. Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg++ buffer. This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phorphocellulose-purified tubulin dimers at 37 degrees C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores. Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed.     

Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non-ionic detergent Triton X-100

The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X-100 (a non-ionic detergent). This Triton X-100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X-100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein-water-ion interactions within the lens is the rate-limiting step of the observed equilibration of monovalent cations and sucrose molecules.     

Mg2+-ATPase activity in wheat root plasma membrane vesicles: Time-dependence and effect of sucrose and detergents

Purified plasma membrane vesicles were isolated in the presence of 250 mM sucrose from 7-day-old roots of Triticum aestivum L. cv. Drabant by aqueous polymer two-phase partitioning. When added to a low-salt medium containing 9-aminoacridine (9-AA), the vesicles caused a much larger total decrease in 9-AA fluorescence when sucrose was absent than when sucrose was present. A slow component of the decrease was also larger in the absence of sucrose. Triton X-100 reduced the decrease in 9-AA fluorescence upon vesicle addition and abolished completely the slow component of the decrease. There was no correlation between the time-dependence of 9-AA fluorescence and that of the Mg²⁺-ATPase described below. The time course of Mg²⁺-ATPase activity was followed by sampling at short intervals (down to 10 s) and analyzing for P, released. In the absence of detergent, the rates of P, release were linear from zero minutes, whether 250 mM sucrose was present or not, but the rate was 10^?50% higher in the absence of sucrose than in its presence. Sucrose (250 mM) added during a minus-sucrose assay lowered Mg²⁺-ATPase activity within 2 min to the level observed with 250 mM sucrose present from the start. The effect of 25-1 100 mM sucrose was tested and there was little or no effect below KM) mM. Above 100 mM sucrose the rate of P, release decreased drastically; at 1 100 mM sucrose the rate was ca 20% the rate at 25 mM sucrose. The inhibitory effect of sucrose was not alleviated by increased concentrations of Mg²⁺ and/or ATP. nor was it affected by the presence or absence of Triton X-100. We conclude that sucrose somehow inhibits the Mg²⁺-ATPase directly or affects the conformation of the plasma membrane in such a way as to inhibit the enzyme. The presence of detergents increased Mg²⁺-ATPase activity in the order Triton X-100 (4–5-fold) > Zwittergent 3–14 = Na-cholate = octylglucoside > digitonin (2-fold). In all cases optimal activity was observed at detergent concentrations at or below the critical micellar concentration. The detergent concentration curves could be simulated by the sum of a stimulatory and an inhibitory reaction. At the optimal concentration, digitonin gave a linear time-course of P, release, whereas all the other detergents showed a distinct lag of 1–3 min before maximal rates were attained. The problems of using detergents in polarity assays are discussed.