Selective effects of nonionic detergent and salt solutions in dissolving nuclear envelope protein

Protein has been selectively extracted from isolated chicken erythrocyte nuclear envelope by (1) dilute MgCl₂/Triton X-100 followed by (2) concentrated MgCl₂/Triton X-100 solutions. Certain proteins appear to be selectively dissolved in the first solvent and may occur in the nuclear envelope primarily as lipoproteins. Among the proteins insoluble in the low MgCl₂/Triton X-100 wash, as well as in 500 mM MgCl₂ without Triton previously used in the preparation of the envelope fraction, the quantitatively major polypeptides dissolve in a combination of high MgCl₂ and Triton X-100. Further, much of this dissolved protein precipitates when the MgCl₂ concentration is lowered by dialysis. The insolubility of these proteins appears to result from a combination of ionic and hydrophobic interactions and may explain the resistance of nuclei to various manipulative procedures including nonionic detergent washes. The procedures described provide a route for gently and selectively dissolving representative proteins from the nuclear envelope lipoprotein matrix and from the envelope “residual” protein.     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

The effects of cations on the structure and photochemistry of the photosystem II core complex

We have studied the effects of cations and detergents on the structure (molecular weight) and photochemistry of Triton X-100 Photosystem II subchloroplast particles (TSF-IIa). The effect of Mg²⁺ ions on activity depended on the Triton X-100 content of the preparation. If the residual Triton X-100 was not removed prior to assay, MgCl₂ increased the rate of electron transport, acting at a site on the reducing side of Photosystem II. Lowering the pH also increased the rate of electron transport. If the Triton X-100 was removed from the particles, both MgCl₂ and NaCl caused a decrease in the rate of electron transport. Addition of Triton X-100 caused a reversible decrease in the number of active Photosystem II reaction centers. Both cations and Triton X-100 had a profound effect on the molecular weight of the Photosystem II particles as determined by gel filtration. At 20 °C, addition of 0.05% Triton X-100 decreased the molecular weight from a high value (≥800,000) to 250,000. At 4 °C, addition of 1 mm MgCl₂ or 100 mm NaCl increased the molecular weight of the complex. In the absence of these salts 67% of the protein eluted with a molecular weight of 460,000 (the rest was >800,000-in the void volume). In the presence of these salts all of the material had a molecular weight of ≥800,000. A similar effect was observed when the pH was lowered from 8 to 6. Further work is needed to determine whether there is a correlation between the changes in molecular weight and activity.     

A method for obtaining high recovery of purified subcellular fractions of rat liver homogenate

A method for obtaining highly purified subcellular fractions of rat liver is described. The recovery of each fraction approaches 100%. The method is based on differential centrifugation and the use of appropriate concentrations of Triton X-100 and MgCl₂ at certain specific steps in the fractionation procedure.     

Production of uridine 5′-monophosphate by Corynebacterium ammoniagenes ATCC 6872 using a statistically improved biocatalytic process

Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH₂PO₄ and K₂HPO₄), MgCl₂, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l⁻¹) UMP was accumulated in 24 h from 38.5 mM (6 g l⁻¹) orotic acid. The yield was threefold higher than the original UMP yield before optimization.     

Optimization of D-xylose to xylitol biotransformation by Candida guilliermondii cells permeabilized with Triton X-100

Abstract

The effect of NADP⁺ and glucose-6-phosphate (G6P) on the biotransformation of D-xylose to xylitol by cells of Candida guilliermondii permeabilized with surfactant Triton X-100 was evaluated. The experimental runs were performed with 12 g L^?1 of permeabilized cells and a reaction medium composed of Tris–HCl buffer (0.1 M pH 7), D-xylose (57 g L^?1), and MgCl₂.6H₂O (5 mM). The levels of NADP⁺ (from 0.0 to 1.7 mM) and G6P (from 0.00 to 0.17 M) were varied according a 2²-full factorial composed design. Under optimized conditions (NADP⁺ 0.5 mM and 0.05 M G6P), the xylitol volumetric productivity (Q_P) and yield factor (Y_P/S) predicted were 1.86 ± 0.03 g L^?1 h^{? 1} and 0.64 ± 0.03 g g^?1, respectively. These values were 94% and 19% higher than those obtained with unpermeabilized cells under fermentation conditions (0.97 g L^?1 h^?1 and 0.53 g g^?1, respectively). On the basis of the results, it can be concluded that xylitol production by biotransformation with cells of C. guilliermondii permeabilized with Triton X-100 is a promising alternative to the fermentative process.     

Biosorption of Copper (II) by the Microorganisms Isolated from the Crude-Oil-Contaminated Soil

The objective of this study was to isolate and screen the highly efficient copper-removing microorganisms from the petroleum hydrocarbon (PH)-contaminated sites in the Amazonian rain forest in Ecuador. Two bacterial strains (strain UEAB3 and UEAB6) have shown 100% microbial resistance on the nutrient medium containing 100 mM of MgCl₂, FeCl₃, and Al₂(SO₄)₃ separately. Though these two strains were less tolerant of ZnCl₂ and CuSO₄.5H₂O, they have proven 100% resistance at the lower concentrations of these two metals. According to atomic absorption spectroscopy (AAS) analysis, the filamentous fungi (strains UEAFr and UEAFg) were significantly (p<0.05) effective at bacteria in the biosorption (97–100%) of copper (5 mg L^?1) over 7 d. As per 16/18S rDNA sequences, UEAB3, UEAB6, UEAFr, and UEAFg were Bacillus thuringiensis, Bacillus cereus, Geomyces pannorum, and Geomyces sp., respectively. From these results, it can be comprehensively concluded that the isolated microbial cultures had a capacity to remove the copper metal from the liquid medium.     

Immunochemical and partial chemical characterization of fractions of membrane-bound smooth lipopolysaccharide-protein complex from Brucella abortus

Smooth lipopolysaccharide (sLPS) of Brucella abortus was prepared and fractionated by a modification of the procedures of Moreno et al. (J. Bac. 138:361–369, 1979). Washed B. abortus cells were disrupted by 21 freeze-quick thaw cycles with ultrasonication to separate the non-membrane-bound material. Ultrasonicated bacteria were used for preparation of membrane-bound sLPS ( f5, the main crude sLPS fraction described by Moreno et al.). Phenol extraction was repeated 3 times and then washed with H₂O 10 times to remove most of the chromogen, polysaccharides and nucleic acids, eliminating the need for enzyme treatment as described previously. The membrane-bound sLPS was fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation, these fractions required only 80 ng for positive ELISA, about 0.2 ng for positive Limulus lysate tests, and reacted well with precipitating antibodies in the serum from a strain 2308 infected cow. They had marked differences in precipitin curves and chemical composition. The protein content varied from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which implies that the proteins associated with LPS may also play important roles in the complex for the immunochemical interactions and the heterogeneity of B. abortus lipopolysaccharide protein complex. As compared with previous reports, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, was obtained. Group f5A, which had a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19 and 2308). The amount of other subfractions obtained varied with batches or strains of B. abortus. These results provide a new profile of the immunochemical reactivities and the heterogeneity on B. abortus smooth membrane-bound endotoxins.Abbreviations LPS lipopolysaccharides - sLPS smooth lipopolysaccharides - cLPS crude lipopolysaccharides - AH acid hapten - KDO 3-deoxyoctulosonic acid - ELISA enzyme-linked immunosorbent assay - f5 the main crude sLPS described by Mooreno et al. [2] - LAL limulus amoebocyte lysate test - HexN Hexosamine - PS phenol sulfuric method - O orcinol method     

Crystallization of Synthetic Escherichia coli-Type Lipid A

Synthetic Escherichia coli-type lipid A formed hexagonal plate crystals when it was precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl₂ and incubated in 70% ethanol containing 250 mM MgCl₂ at 4 C for 10 days. Analyses of crystals by electron diffraction and synchrotron X-ray diffraction showed that crystals consist of hexagonal lattices with the lattice constant (a side of the lozenge as a unit cell on the basal plane) of 4.62 Å and the longitudinal axis (perpendicular to the basal plane) of 49.3 ±1.3 Å. Results suggest that the previous finding that various kinds of R-form lipopolysaccharides crystallized but free lipid A isolated by acid hydrolysis from Re lipopolysaccharide did not crystallize under the same experimental conditions (Kato et al, J. Bacteriol., 172: 1516-1528, 1990) is due to structural changes of lipid A occurring during the procedure of isolation of free lipid A.     

Characterization of a dUTPase from the Hyperthermophilic Archaeon <Emphasis Type="Italic">Thermococcus onnurineus</Emphasis> NA1 and Its Application in Polymerase Chain Reaction Amplification

Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 (TNA1) revealed the presence of a 471-bp open reading frame with 93% similarity to the dUTPase from Pyrococcus furiosus. The dUTPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dUTP at about a 10-fold higher rate than dCTP. The protein behaved as a dimer in gel filtration chromatography, even though it contains five motifs that are conserved in all homotrimeric dUTPases. The dUTPase showed optimum activity at 80°C and pH 8.0, and it was highly thermostable with a half-life (t _1/2) of 170 min at 95°C. The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl₂, KCl, (NH₄)₂SO₄, and Triton X-100 concentrations, although it was reduced by bovine serum albumin. Addition of the dUTPase to polymerase chain reactions (PCRs) run with TNA1 DNA polymerase significantly increased product yield, overcoming the inhibitory effect of dUTP. Further, addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase. This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases, including Pfu and KOD.     

Response of fluoranthene-degrading bacteria to surfactants

A prerequisite for surfactant-enhanced biodegradation is that the microorganisms survive, take up substrate and degrade it in the presence of the surfactant. Two Mycobacterium and two Sphingomonas strains, degrading fluoranthene, were investigated for their sensitivity towards non-ionic chemical surfactants. The effect of Triton X-100 and Tween 80 above their critical micelle concentration on mineralization of [¹⁴C]-glucose and [¹⁴C]-fluoranthene was measured in shaker cultures. Tween 80 had no toxic effect on any of the tested strains. The surfactant inhibited fluoranthene mineralization by the hydrophobic Mycobacterium spp. slightly, but more than doubled that by the two less hydrophobic Sphingomonas strains. Triton X-100 inhibited fluoranthene mineralization by all strains, yet this was more pronounced for the Sphingomonas spp. Both surfactants caused cell wall permeabilization, as shown by transient colouring of surfactant-containing media. Inhibition of glucose mineralization, indicating non-specific toxic effects of Triton X-100, was observed only for the Sphingomonas strains and the toxicity was caused by micelle-to-cell interactions. These strains, however, appeared to recover from initial Triton X-100 toxicity within 50–500 h of exposure. The ratio of surfactant concentration to initial cell density was found to determine critically the bacterial response to surfactants. For both Sphingomonas and Mycobacterium strains, this work indicates that fluoranthene solubilized in surfactant micelles is only partially available for mineralization by the bacteria tested. However, our results suggest that optimal conditions for polycyclic aromatic hydrocarbon mineralization can be developed by selection of the proper surfactant, bacterial strains, cell density and incubation conditions. Received: 6 February 1998 / Received revision: 19 June 1998 / Accepted: 19 June 1998     

An improved method of transformation in Pseudomonads

Summary A protocol for producing competent Pseuclomonas aeruginosa, Pseudomonas putida, and Xanthomonas maltophilia was adapted and modified from existing methods. Cells were incubated on ice for 30 minutes in buffered 100 mM MgCl₂ followed by 30 minutes in buffered 100 mM CaCl₂ prior to addition of DNA. The MgCl₂-CaCl₂ incubation increased transformation efficiency two to three times, compared with protocols which use incubation in either Mg²⁺ or Ca²⁺, but not both.     

Chlorophyllase and Photsystem

• 1 Preliminary experiments showed that Triton X-100 at low concentrations (appr. 0.01%) induces reversible changes in some photosynthetic reactions in Phaeodactylum cells without influencing their absorption spectrum; presumably membrane permeability is effects. Although DC PIP can pass through the cell membrane in the presence of 0.01% Triton X-100, only a very low PS I activity, measured as MV photoreduction by DCPIP-ascorbate, is observed with whole cells.
• 2 MV photoreduction sensitized by membrane fragments isolated from Phaetdactylum, Euglena, Porphyridium and Synechococcus after French Press treatment is much higher with Phaeodactylum than with the other organisms. Spinach shroma membrane fragments show higher photosensitizing activity than grana fragments.
• 3 MV photoreduction in the above mentioned experiments is not influenced by DCMU (2 × 10^?6M) or sodium azide (0.01–0.05 M); KCN (4 × 10^?3M) has an inhibiting effect only with the blue-green alga Synechococcus. The reaction mechanism of chlorophyll-sensitized MV photoreduction is discussed.
• 4 Chlorophyll in an aqueous medium containing Triton X-100 (JsO.01%) sensitizes the photoreduction of MV by DCPIP ascorbate. A similar reaction is observed with chlorophyll combined with solubilized Phaeocdactylum chlorophyllase: in the latter case the presence of both MgCl₂ and DTT is required.
• 5 MV-photoreduction is concluded to be a very unreliable procedure for determining PS I activity in membranes if these membranes have been prepared in (the presence of detergents.
• 6 The results support the hypothesis that (prochlorophyllase is a PS I protein.
• 7 The capacity of PS I to reduce MV in vivo is discussed.

     

Isolation, Composition, and Structure of Membrane of Listeria monocytogenes

The plasma membrane of Listeria monocytogenes strain 42 was prepared by osmotic lysis of protoplasts with tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.2, containing MgCl₂ and glucose, followed by washing with NaCl and MgCl₂ in Tris buffer. Electron microscopy showed that the preparation was not contaminated with cytoplasmic material. The membrane preparation was composed of 55 to 60% protein, 1.5% ribonucleic acid, 0.1% deoxyribonucleic acid, 1.3 to 2.3% carbohydrate, 0.17 to 0.38% amino sugar, 0.2 to 0.4% rhamnose, 3.5 to 4.0% phosphorus, 10.5 to 12.0% nitrogen, and 30 to 35% lipid. Amino acid composition of the washed membrane showed some variation from that of the whole cells. Sulfur-containing amino acids were not present in the membrane hydrolysate. The membrane carbohydrate contained glucose, galactose, ribose, and arabinose. The membrane lipid was 80 to 85% phospholipid and 15 to 20% neutral lipid. The lipid contained 2.3 to 3.0% phosphorus, 2.5 to 3.0% carbohydrate, and a very small amount of nitrogen (0.2 to 0.3%). The phospholipid was of the phosphatidyl glycerol type. Electron micrographs of the washed membrane showed three layers. The outer and inner layers varied in thickness from 25 to 37 A and the middle layer from 20 to 25 A. The total thickness varied between 85 and 100 A. These preparations contained many vesicles which stained heavily with lead citrate. Some vesicles were also attached to the protoplast ghosts in the form of extrusions or intrusions, or both. Membrane preparations obtained by lysis of protoplasts in the absence of MgCl₂ were fragmented and contained less lipid (20 to 22%) and ribonucleic acid (0.3 to 0.5%) than preparations prepared with MgCl₂.     

Effects of detergent on the excited state structure and relaxation dynamics of the photosystem II reaction center: A high resolution hole burning study

Low temperature (4.2 K) absorption and hole burned spectra are reported for a stabilized preparation (no excess detergent) of the photosystem II reaction center complex. The complex was studied in glasses to which detergent had and had not been added. Triton X-100 (but not dodecyl maltoside) detergent was found to significantly affect the absorption and persistent hole spectra and to disrupt energy transfer from the accessory chlorophyll a to the active pheophytin a. However, Triton X-100 does not significantly affect the transient hole spectrum and lifetime (1.9 ps at 4.2 K) of the primary donor state, P680^*. Data are presented which indicate that the disruptive effects of Triton X-100 are not due to extraction of pigments from the reaction center, leaving structural perturbations as the most plausible explanation. In the absence of detergent the high resolution persistent hole spectra yield an energy transfer decay time for the accessory Chl a Q_Y-state at 1.6 K of 12 ps, which is about three orders of magnitude longer than the corresponding time for the bacterial RC. In the presence of Triton X-100 the Chl a Q_Y-state decay time is increased by at least a factor of 50.Abbreviations PS I photosystem I - PS II photosystem II - RC reaction center - P680, P870, P960 the primary electron donor absorption bands of photosystem II, Rhodobacter sphaeroides, Rhodopseudomonas viridis - NPHB nonphotochemical hole burning - TX Triton X-100 - DM Dodecyl Maltoside - Chl chlorophyll - Pheo pheophytin - ZPH ero phonon hole     

Thermotolerant phosphate solubilizing microorganisms and their interaction with mung bean

Several phosphate solubilizing microorganisms (PSM) were tested for their efficiency at 35°, 40° and 45°C. There was a marked variation in their ability to solubilise tricalcium phosphate and the effect was more pronounced at 45°C. Two bacterial and one fungal strain were found to be thermotolerant as they solubilised a large amount of tricalcium phosphate at the three tested temperatures. These thermotolerant strains were identified as Bacillus subtilis (TT₀), Bacillus circulans (TT₈) and Aspergillus niger (TT₁₀). Seed inoculation of mung bean showed a better establishment of temperature tolerant strains as revealed by the rhizosphere population. The inoculation improved nodulation, the available P₂O₅ content of the alluvial soil, root and shoot biomass, straw and grain yield and phosphorus and nitrogen uptake of the crop. Among the bacterial strains, the best effect on yield was obtained with B. subtilis. However, statistically it was equivalent to streptomycin resistant mutant (M-20) and Pseudomonas striata (27). A. niger was less effective than bacteria. Though superphosphate was found to be a better source of phosphate fertiliser, the use of rock phosphate (RP₄₀), coupled with phosphate solubilising bacteria (PSB), gave results comparable to superphosphate (SP₂₀) + PSB inoculants.     

Simultaneous phenanthrene and cadmium removal from contaminated soil by a ligand/biosurfactant solution

Surfactants and inorganic ligands are pointed as efficient to simultaneous removal of heavy metals and hydrophobic organic pollutants from soil. However, the biosurfactants are potentially less toxic to soil organisms than other chemical agents. Thus, in this study the efficiency of combinations of iodide (I⁻) ligand and surfactants produced by different bacterial species in the simultaneous removal of cadmium (Cd²⁺) and phenanthrene in a Haplustox soil sample was investigated. Four microbial surfactants and the synthetic surfactant Triton X-100 were tested with different concentrations of ligand. Soil samples contaminated with Cd²⁺ and phenanthrene underwent consecutive washings with a surfactant/ligand solution. The removal of Cd²⁺ increased with increased ligand concentration, particularly in solutions containing biosurfactants produced by the bacterial strains Bacillus subtilis LBBMA155 (lipopeptide) and Flavobacterium sp. LBBMA168 (mixture of flavolipids) and Triton X-100. Maximum Cd²⁺ removal efficiency was 99.2% for biosurfactant produced by Arthrobacter oxydans LBBMA 201 (lipopeptide) and 99.2% for biosurfactant produced by Bacillus sp. LBBMA111A (mixed lipopeptide) in the presence of 0.336 mol iodide l⁻¹, while the maximum efficiency of Triton X-100 removal was 65.0%. The biosurfactant solutions removed from 80 to 88.0% of phenanthrene in soil, and the removal was not influenced by the presence of the ligand. Triton X-100 removed from 73 to 88% of the phenanthrene and, differently from the biosurfactants, iodide influenced the removal efficiency. The results indicate that the use of a single washing agent, called surfactant-ligand, affords simultaneous removal of organic contaminants and heavy metals.     

Enhancement of transglutaminase production in <Emphasis Type="Italic">Streptomyces mobaraensis</Emphasis> as achieved by treatment with excessive MgCl<Subscript>2</Subscript>

In this study, we first tested the capacity for eight different salts as stress-mediated bioprocesses in the production of transglutaminase (TGase). A significant effect on the cell growth and TGase production was obtained with the highest yield of TGase being observed at 96 h of incubation (4.3 U/ml) when the basic medium was supplemented 0.10 M MgCl₂, as opposed to that observed with the basic medium control (2.1 U/ml at 120 h). Data from Western blot assays showed that transformation of pro-TGase to its mature enzyme occurred more rapidly in MgCl₂ medium. Furthermore, total protease, metalloprotease, and serine protease were also synthesized at a faster rate in the medium containing MgCl₂. The results demonstrate that MgCl₂ enhanced the production of key proteases involved in the activation of TGase biosynthesis. To explore the mechanism, viability assay was performed. The results show that MgCl₂ induced the mycelia differentiation, decreased cell growth rate, and stimulated cell death. We argue that TGase production was promoted by the stimulation of mycelium differentiation induced by MgCl₂ stress.     

Inactivation of thymidylate synthetase by a novel mechanism-based enzyme inhibitor: 1-(beta-D-2'-deoxyribofuranosyl) 8-azapurin-2-one 5'-monophosphate

The possible relationship of the soluble, “cytosolic” estradiol receptor with complex membranous and cytoskeletal structures of the cell matrix has been studied using a model erythrocyte system. Extraction of erythrocyte ghosts with a nonionic detergent (Triton X-100) under conditions that yield a cytoskeletal matrix reveals the presence of a limited number (less than 100) of specific sites having high affinity (k_d 10^?9 M) for the estradiol-receptor complex. The interation between the estradiol receptor and the cytoskeleton is critically dependent on temperature and it is improved by 25 mM KCl or NaCl and by 2.5 mM MgCl₂. The data suggest that the estradiol receptor, which has been generally considered to be freely “soluble” in the cytoplasm, may actually be physiologically associated in an integral manner with a complex cytoskeletal network in the cell cytoplasm.     

Purification of the membrane-bound proton-translocating inorganic pyrophosphatase from Rhodospirillum rubrum

The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl₂ and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers.