Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6539 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5-9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demonstration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four other solubilized membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.     

Solubilization, purification and characterization of D-alanine dehydrogenase from Pseudomonas aeruginosa and effects of solubilization on its properties

D-alanine dehydrogenase, an inducible, membrane associated enzyme of Pseudomonas aeruginosa was solubilized from envelope preparations by treatment with Triton X-100 and purified 31-fold in the presence of 0.05% Triton X-100 to 60% homogeneity. Gel electrophoresis indicated the presence of a single subunit of approximately 49,000 molecular weight. The enzyme contained FAD, and absorption spectra were typical of an iron-sulfur flavoprotein. Solubilization produced significant changes in some properties of the enzyme: solubilized enzyme showed increased affinity for D-alanine; a broader substrate specificity; and increased temperature sensitivity, compared with the membrane associated form.     

Triton X—100加KI对燕麦根细胞质膜ATP酶的溶解

在酸性条件下,1％ Triton X—100加 0.25mol/L KI能有效地溶解燕麦根细胞质膜ATP酶。溶解的ATP酶水解ATP的最适pH在6.5左右,酶活性受到Na_3VO_4和DES的强烈抑制,而不受Na_2MoO_4和NaN_3的抑制。溶解的酶液经透析后,K~ —ATP酶活性占Mg~(2 ),KCl—ATP酶活性的85％。     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Solubilization and immunochemical identification of mitochondrial glycerol-3-phosphate dehydrogenase

Lysophosphatidylcholine (contrary to Lubrol WX, Triton X-100, digitonine and deoxycholate) solubilizes hamster brown fat mitochondrial glycerol-3-phosphate dehydrogenase without inactivation. Optimal ratio of lysophosphatidylcholine and membrane protein for solubilization of the enzyme was found to be 0.25 mg of lysophosphatidylcholine per mg protein. The activity of solubilized enzyme, however, was not affected by low concentrations of Lubrol WX, Triton X-100, digitonine, Zwittergent TM 314. Deoxycholate exhibited a pronounced inactivating effect. One-dimensional immunoelectrophoresis of the solubilized membrane proteins revealed 10 protein bands, 3-4 of which exhibited the enzyme activity. Two-dimensional immunoelectrophoresis revealed only a single main band of glycerol-3-phosphate dehydrogenase. This technique thus appears to be the best means for the identification of glycerol-3-phosphate dehydrogenase in the mixture of solubilized membrane proteins and for concentration of the enzyme activity in one major precipitating band.     

Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).

NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Summary Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells ofChenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Selective delipidation of the plasma membrane by surfactants : enrichment of sterols and activation of ATPase

The influence of plasma membrane lipid components on the activity of the H⁺-ATPase has been studied by determining the effect of surfactants on membrane lipids and ATPase activity of oat (Avena sativa L.) root plasma membrane vesicles purified by a two-phase partitioning procedure. Triton X-100, at 25 to 1 (weight/weight) Triton to plasma membrane protein, an amount that causes maximal activation of the ATPase in the ATPase assay, extracted 59% of the membrane protein but did not solubilize the bulk of the ATPase. The Triton-insoluble proteins had associated with them, on a micromole per milligram protein basis, only 14% as much phospholipid, but 38% of the glycolipids and sterols, as compared with the native membranes. The Triton insoluble ATPase could still be activated by Triton X-100. When solubilized by lysolecithin, there were still sterols associated with the ATPase fraction. Free sterols were found associated with the ATPase in the same relative proportions, whether treated with surfactants or not. We suggest that surfactants activate the ATPase by altering the hydrophobic environment around the enzyme. We propose that sterols, through their interaction with the ATPase, may be essential for ATPase activity.     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

HCO-3-activated adenosine triphosphatase in intestinal mucosa of the eel.

An HCO-3-activated and SCN--inhibited ATPase (ATP phosphohydrolase, EC 3.6.1.3) found in homogenates of intestinal mucosa of the eel was solubilized by Triton X-100. Optimal HCO-3-concentration and pH for the enzyme were 25 mM and 8.7, respectively. HCO-3-ATPase activity in both homogenate and solubilized preparations increased after seawater adaptation. This adaptive increase in enzyme activity was also observed in the gills and the kidney. The HCO-3-ATPase seems to be related to transport mechanisms, especially for Cl-, in osmoregulatory surfaces of the eel.     

Multiple forms of acetylcholinesterase from rat erythrocytes. Effect of fat-free diet.

Electrophoretic patterns of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from rat erythrocyte were studied. The enzyme was solubilized by the following treatments: a) Triton X-100, b) sodium deoxycholate, or c) ultrasonic irradiation. When the erythrocyte membrane was solubilized by Triton X-100 at concentrations higher than 0.3%, by 10 mM sodium deoxycholate, or by ultrasonic irradiation for more than 5 min, a single band of acetylcholinesterase activity appeared in the gel. Two bands of activity were stained in the gel when the membrane was solubilized by Triton X-100 at concentrations between 0.1--0.2%, or by ultrasound for 5 min. Electrophoretic patterns of acetylcholinesterase from rats fed a fat-sufficient diet were similar to those for the enzyme from animals fed a fat-free diet. The recombination of lipids with the enzyme eluted from the gels confirmed the "phenotypic allosteric desensitization phenomenon".     

The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N.

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 X 10(-7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 X 10(-6) M) than for the other two forms (IC50 = 1.6 X 10(-5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 X 10(-5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 X 10(-4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.     

Membrane-surfactant interactions. The effect of Triton X-100 on sarcoplasmic reticulum vesicles

The effect of Triton X-100 on purified sarcoplasmic reticulum vesicles has been studied by means of chemical, ultrastructural and enzymic techniques. At low detergent/membrane ratios (about 1 Triton X-100 per 60 phospholipid molecules) the only effect observed is an increase in vesicle permeability. Higher surfactant concentrations, up to a 1:1 detergent/phospholipid ratio, produce a large enhancement of ATPase activity. Membrane solubilization occurs as a critical phenomenon when the surfactant/phospholipid molar ratio reaches a value around 1.5:1, corresponding to 2 mumol Triton X-100/mg protein. At this point, the suspension turbidity drops, virtually all the protein and phospholipid is solubilized and every organized structure disappears. Simultaneously, a dramatic increase in the specific activity of the solubilized ATPase is observed. The sudden solubilization of almost all the bilayer components at a given detergent concentration is attributed to the relative simplicity of this membrane system. Solubilization takes place at the same surfactant/membrane ratio, at least between 0.5 and 4 mg membrane protein/ml. The non-solubilized residue seems to consist mainly of delipidized aggregated forms of ATPase.     

Membrane-surfactant interactions The effect of triton X-100 on sarcoplasmic reticulum vesicles

The effect of Triton X-100 on purified sarcoplasmic reticulum vesicles has been studied by means of chemical, ultrastructural and enzymic techniques. At low detergent/membrane ratios (about 1 Triton X-100 per 60 phospholipid molecules) the only effect observed is an increase in vesicle permeability. Higher surfactant concentrations, up to a 1:1 detergent/phospholipid ratio, produce a large enhancement of ATPase activity. Membrane solubilization occurs as a critical phenomenon when the surfactant/phospholipid molar ratio reaches a value around 1.5:1, corresponding to 2 μmol Triton X-100/mg protein. At this point, the suspension turbidity drops, virtually all the protein and phospholipid is solubilized and every organized structure disappears. Simultaneously, a dramatic increase in the specific activity of the solubilized ATPase is observed. The sudden solubilization of almost all the bilayer components at a given detergent concentration is attributed to the relative simplicity of this membrane system. Solubilization takes place at the same surfactant/membrane ratio, at least between 0.5 and 4 mg membrane protein/ml. The non-solubilized residue seems to consist mainly of delipidized aggregated forms of ATPase.     

Partial purification of plant plasma membrane K+, Mg2+-ATPase by ion exchange chromatography

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H⁺-pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two-phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non-ionic detergent (either Triton X-100 or C₁₂E₈). The Mg-ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl-eluted fractions contained a Mg-ATPase which showed the characteristics of Mg-ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg⁻¹ min⁻¹ when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.     

Triton X-100 solubilization of mitochondrial inner and outer membranes

Rat liver mitochondrial inner and outer membranes were subjected to the solubilizing effect of the nonionic detergent Triton X-100 under various conditions. After centrifugation, the supernatants (containing the solubilized fraction) and pellets were characterized chemically and/or ultrastructurally. The detergent seems to act by inducing a phase transition from membrane lamellae to mixed protein-lipid-detergent micelles. Different electron-micro-scopy patterns are shown by the inner membranes after treatment with different amounts of surfactant, whereas the corresponding images from outer membranes vary but slightly. Selective solubilization of various components is observed, especially in the case of the inner membrane. Some membrane lipids (e.g., cardiolipin) are totally solubilized at detergent concentrations when others, such as sphyngomyelin, remain in the membrane. Other inner-membrane components (flavins, cytochromes, coenzymeQ) show different solubilization patterns. This allows the selection of conditions for optimal solubilization of a given membrane component with some degree of selectivity. The influence of Triton X-100 on various mitochondrial inner-membrane enzyme activities was studied. The detergent seems to act especially through disruption of the topology of the functional complexes, although the activity of the individual enzymes appears to be preserved. Relatively simple enzyme activities, such as ATPase, are more or less solubilized according to the detergent concentration, whereas the more complex succinate-cytochromec reductase activity practically disappears even at low Triton X-100 concentrations.     

Selective solubilization of nicotinamide nucleotide transhydrogenase from the mitochondrial inner membrane

Nicotinamide nucleotide transhydrogenase was solubilized from beef heart submitochondrial particles employing Triton X-100 or lysolecithin. Lysolecithin was considerably more efficient and selective and released over 80 % of the transhydrogenase acdtivity from the membrane together with succinate dehydrogenase. Solubilization of NADH dehydrogenase and cytochrome oxidase was more efficiently accomplished with Triton than with lysolecithin. Both detergents released ATPase to various extents. Transhydrogenase remaining bound to particles after treatment with lysolecithin still catalyzed energy-linked transhydrogenation.     

Solubilization,stabilization and isoelectric focusing of human liver neuraminidase activity

Homogenate preparations of human liver have been prepared and over 75% of the particulate neuraminidase activity (which comprises approx. 90% of the total activity) has been solubilized using 0.85% (w/v) Triton X-100 in 25 mM phosphate buffer (pH 6.8). The solubilized neuraminidase activity is extremely labile, but can be stabilized for at least 4 weeks at 2–4°C, using 10 mM N-acetylneuraminic acid. Kinetic characterization of homogenate and solubilized supernatant fluid neuraminidase activities indicated comparable pH optimum curves (maximum activity at pH 4.5–4.7) and apparent K_m values (0.2–0.4 mM) for the synthetic fluorometric substrate $\text{4-}\text{methylbelliferyl}\text{-α-}\text{D}\text{-N-}\text{acetylneuraminic}$ acid. Isoelectric focusing has been performed on human liver homogenates and Triton X-100-solubilized neuraminidase activities, and the presence of several forms (4–6) with isoelectric points (pI values) between 4.4 and 5.2 has been demonstrated in both preparations. The similar kinetic and isoelectric focusing properties of the two preparations suggest that the solubilized enzyme activity is representative of the homogenate activity and that the solubilized enzyme is suitable for purification purposes.     

Immunochemical Analysis of Triton X-100-Insoluble Residues from Micrococcus lysodeikticus Membranes

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.