Differential detergent-solubilization of integral thylakoid membrane complexes in spinach chloroplasts. Localization of photosystem II, cytochrome b6-f complex and photosystem I

Progressive solubilization of spinach chloroplast thylakoids by Triton X-100 was employed to investigate the domain organization of the electron transport complexes in the thylakoid membrane. Triton/chlorophyll ratios of 1:1 were sufficient to disrupt fully the continuity of the thylakoid membrane network, but not sufficient to solubilize either photosystem I (PSI), photosystem II (PSII) or the cytochrome b6-f(Cyt b6-f) complex. Progressive with the Triton concentration increase (Triton/Chl greater than 1:1), a differential solubilization of the three electron transport complexes was observed. Solubilization of the Cyt b6-f complex from the thylakoid membrane preceded that of PSI and apparently occurred early in the solubilization of stroma-exposed segments of the chloroplast lamellae. The initial removal of chlorophyll (up to 40% of the total) occurred upon solubilization of PSI from the stroma-exposed lamella regions in which PSI is localized. The tightly appressed membrane of the grana partition regions was markedly resistant to solubilization by Triton X-100. Thus, solubilization of PSII from this membrane region was initiated only after all Cyt b6-f and PSI complexes were removed from the chloroplast lamellae. The results support the notion of extreme lateral heterogeneity in the organization of the electron transport complexes in higher plant chloroplasts and suggest a Cyt b6-f localization in the membrane of the narrow fret regions which serve as a continuum between the grana and stroma lamellae.     

Effect of the hydrophile-lipophile balance of non-ionic detergents (Triton X-series) on the solubilization of biological membranes and their integral b-type cytochromes.

The solubilization of four integral membrane proteins (i.e. cytochrome b-561 of the chromaffin granule membrane, cytochrome b5 of the endoplasmic reticulum and the mitochondrial b-type cytochrome(s) as well as cytochrome c oxidase) has been studied at 0 degrees C using the non-ionic detergents of the Triton X-series having the common hydrophobic 4(1,1,3,3-tetramethylbutyl)phenoxy (t-octyl-phenoxy) group and a variable average number (n) of polar ethylene oxide units added. Following a pre-extraction of peripheral membrane and matrix proteins with low and high salt concentration and a weak non-ionic detergent (Tween 20, average hydrophile-lipophile balance (HLB) = 16.7), the amount of heme proteins solubilized by subsequent Triton X-solutions was measured. With the detergents tested the degree of solubilization decreased in the sequence cytochrome b-561 greater than cytochrome b5 greater than mitochondrial cytochrome(s) b and parallelled the effect of the detergents on light scattering and the phospholipid to protein ratio of the three membranes. For all the b-cytochromes, the solubilizing power of the detergent increased with decreasing average length of the polar ethylene oxide chain and the hydrophile-lipophile balance as long as clouding did not occur (e.g. Triton X-114,n = 7.5 and HLB = 12.4). Thus, the greatest difference in the degree os solubilization of the three cytochromes was observed with Triton X-405 (n = 40 and HLB = 17.9). All the cytochromes were most efficiently solubilized (i.e. approx. 90%) by Triton X-100 (n = 9.5 and HLB = 13.5).     

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.     

Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).     

the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.     

Cytochrome b reducible by succinate in an isolated succinate dehydrogenase-cytochrome b complex from Bacillus subtilis membranes

In previous work with membranes of Bacillus subtilis, the succinate dehydrogenase complex was isolated by immunoprecipitation of Triton X-100-solubilized membranes. The complex included a polypeptide with an apparent molecular weight of 19,000, probably attributable to apocytochrome. This paper reports the further characterization of this cytochrome and its relation to the respiratory chain of B. subtilis. The cytochrome was identified as cytochrome b, and its difference absorption spectra showed maxima at 426, 529, and 558 nm at room temperature. The oxidized cytochrome had an absorption maximum at 413 nm. The cytochrome was reduced by succinate in the isolated succinate dehydrogenase complex and in Triton X-100-solubilized membranes. In whole membranes cytochromes b, c, and a were reduced by succinate. In membranes from a mutant containing normal cytochromes but lacking succinate dehydrogenase no reduction of cytochrome was seen with succinate. It was concluded that the isolated succinate dehydrogenase-cytochrome b complex is a functional unit in the intact B. subtilis membrane. An accompanying paper describes cytochrome b as a structural unit involved in the membrane binding of succinate dehydrogenase.     

Isolation and partial characterization of the cytochrome c oxidase of Micrococcus luteus (lysodeikticus)

The cell membrane of Micrococcus luteus (lysodeikticus) contains a respiratory chain composed of hemes a, b, and c, which contain 171, 457, and 407 pmol/mg protein, respectively. Cytochrome c oxidase, the heme a containing component, has been purified after solubilization in Triton X-100, by gel filtration on Sepharose 4B-CL ammonium sulfate precipitation and ion-exchange and affinity chromatographies on a yeast cytochrome c-Sepharose 4B column. The purified complex, which contains three polypeptides of apparent Mr 47,000, 31,000, and 19,000, has CN-sensitive ferrocytochrome c oxidase activity (Ki = 0.35 microM) and a characteristic absorption spectrum with maxima in the oxidized form at 595 and 426 nm and in the reduced form at 601 and 444 nm. The purified enzyme contains 17.4 nmol/mg protein and its copper content is 23.2 nmol/mg protein. The enzyme was purified about 100-fold with respect to its content in crude membranes. The total heme a yield, also with respect to crude membranes content, was 6.8%.     

Localization and Solubilization of the Respiratory Nitrate Reductase of Bacillus stearothermophilus

Membranes were isolated from Bacillus stearothermophilus 2184D by lysozyme digestion of the cell wall and subsequent differential centrifugation. Observations with the electron microscope indicate that such membranes are relatively intact and have a typical membrane appearance. Nitrate will preferentially oxidize the cytochrome b of such membranes. Approximately 80% of the total respiratory nitrate reductase activity of whole cells can be localized in the washed membrane fraction and the process of membrane isolation results in a sixfold purification of this enzyme. Of several detergents tested, sodium dodecyl sulfate, Triton 114, and Triton X-100 are most effective in converting reduced methyl viologen-nitrate reductase to a form which will not pellet at 130,000 x g. Density gradient analysis reveals that such detergent-mediated solubilization converts virtually all membrane protein to a form of lighter density.     

Incorporation of D-glucose-, L-alanine- and phosphate-transport systems from rat renal brush-border membranes into liposomes.

An extract of soluble proteins was prepared from a rat kidney brush-border membranes by Triton X-100 solubilization followed by centrifugation for 1 h at 100000g. Its protein composition was markedly different from that of the brush-border membranes. Proteoliposomes were formed by co-sonication of the Triton X-100-free extract with a naturally occurring mixture of phospholipids extracted from rat kidney. These proteoliposomes were shown to contain Na+-stimulated D-glucose-, L-alanine- and phosphate-transport systems.     

Characterization of membrane-bound electron transport enzymes from castor bean glyoxysomes and endoplasmic reticulum

Membranes purified from castor bean endosperm glyoxysomes by washing with sodium carbonate exhibited integral NADH:ferricyanide and NADH:cytochrome c reductase activities. The enzyme activities could not be attributed to contamination by other endomembranes. Purified endoplasmic reticulum membranes also contained the redox activities; and marker enzyme analysis indicated minimum cross contamination between glyoxysomal and endoplasmic reticulum fractions. The glyoxysomal redox activities were optimally solubilized at detergent to protein ratios (weight to weight) of 10 (Triton X-100), 50 (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and 100 (octylglucoside). Detergent in excess of the solubilization optimum was stimulatory to NADH:ferricyanide reductase and inhibitory to NADH:cytochrome c reductase. Endoplasmic reticulum redox activity solubilization profiles were similar to those obtained for glyoxysomal enzymes using Triton X-100. Purification of the glyoxysomal and endoplasmic reticulum NADH:ferricyanide reductases was accomplished using dye-ligand affinity chromatography on Cibacron blue 3GA agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of NADH:ferricyanide reductase preparations purified by rate-zonal density gradient centrifugation, affinity chromatography, and nondenaturing electrophoresis of detergent-solubilized glyoxysomal and endoplasmic reticulum membranes consistently displayed 32- and 33-kDa silver-stained polypeptide bands, respectively.     

Isolation of mitochondrial succinate: ubiquinone reductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using nonionic detergent.

The electron transfer complexes, succinate: ubiquinone reductase, ubiquinone: cytochrome c reductase, and cytochrome c: O2 oxidase were isolated from the mitochondrial membranes of Neurospora crassa by the following steps. Modification of the contents of the complexes in mitochondria by growing cells on chloramphenicol; solubilisation of the complexes by Triton X-100; affinity chromatography on immobilized cytochrome c and ion exchange and gel chromatography. Ubiquinone reductase was obtained in a monomeric form (Mr approximately 130 000) consisting of a flavin subunit (Mr 72 000) an iron-sulfur subunit (Mr 28 000) and a cytochrome b subunit (Mr probably 14 000). Cytochrome c reductase was obtained in a dimeric form (Mr approximately 550 000), the monomeric unit comprising the cytochromes b (Mr each 30 000), a cytochrome c1 (Mr 31 000), the iron-sulfur subunit (Mr 25 000), and six subunits without known prosthetic groups (Mr 9000, 11 000, 14 000, 45 000, 45 000, and 52 000). Cytochrome c oxidase was also isolated in a dimeric form (Mr approximately 320 000) comprising two copies each of seven subunits (Mr 9000, 12 000, 14 000, 18 000, 21 000, 29 000, and 40 000). The complexes were essentially free of phospholipid. Each bound one micelle of Triton X-100 (Mr approximately 90 000). After isolation, the bound Triton X-100 could be replaced by other nonionic detergents such as: alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers and acyl polyoxyethylene sorbitan esters.     

Exploring detergent insolubility in bovine hippocampal membranes: a critical assessment of the requirement for cholesterol

The phenomenon of detergent insolubility of bovine hippocampal membranes in Triton X-100 was monitored by estimating the presence of phospholipids in the insoluble pellet. This represents a convenient and unambiguous assay and reports the dependence of the extent of phospholipid solubilization on detergent concentration. The advantage of this approach is its ability to accurately determine the extent of detergent insolubility in natural membranes. Importantly, our results show that when suboptimal concentrations of Triton X-100 are used for solubilization, interpretations of the mechanism and extent of detergent insolubility should be made with adequate caution. At concentrations of Triton X-100 that leads to no further solubilization, ∼44% of phospholipids are left insoluble at 4 °C in bovine hippocampal membranes. Cholesterol depletion using methyl-β-cyclodextrin enhanced phospholipid solubilization at low detergent concentrations but produced no significant change in the amount of insoluble phospholipids at saturating detergent concentration. Progressive solubilization by the detergent resulted in insoluble membranes that contained lipids with higher fatty acyl chain order as reported by fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH). These results suggest that it is the presence of such lipids rather than their association with cholesterol that determines detergent insolubility in membranes.     

Exploring detergent insolubility in bovine hippocampal membranes: a critical assessment of the requirement for cholesterol

The phenomenon of detergent insolubility of bovine hippocampal membranes in Triton X-100 was monitored by estimating the presence of phospholipids in the insoluble pellet. This represents a convenient and unambiguous assay and reports the dependence of the extent of phospholipid solubilization on detergent concentration. The advantage of this approach is its ability to accurately determine the extent of detergent insolubility in natural membranes. Importantly, our results show that when suboptimal concentrations of Triton X-100 are used for solubilization, interpretations of the mechanism and extent of detergent insolubility should be made with adequate caution. At concentrations of Triton X-100 that leads to no further solubilization, approximately 44% of phospholipids are left insoluble at 4 degrees C in bovine hippocampal membranes. Cholesterol depletion using methyl-beta-cyclodextrin enhanced phospholipid solubilization at low detergent concentrations but produced no significant change in the amount of insoluble phospholipids at saturating detergent concentration. Progressive solubilization by the detergent resulted in insoluble membranes that contained lipids with higher fatty acyl chain order as reported by fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH). These results suggest that it is the presence of such lipids rather than their association with cholesterol that determines detergent insolubility in membranes.     

Effect of triton X-100 on the activity and solubilization of rat liver mitochondrial phosphatidylserine decarboxylase

It was shown that, among ionic and nonionic detergents tested, only Triton X-100 was able to stimulate the activity of rat liver phosphatidylserine decarboxylase, whereas other detergents were without effect or were inhibitory. The solubilization procedure of phosphatidylserine decarboxylase from mitochondrial membranes with Triton X-100 was elaborated. The dependence of the solubilized decarboxylase on the Triton X-100 to phosphatidylserine ratio and the inhibitory effect of Triton X-100 at its molar ratio to phospholipid higher than 5.6 was observed. No divalent cation requirement and no dependence of the ionic strength for the solubilized enzyme were observed. Kinetic parameters were determined.     

Application of phase partition method for the fractionation of hydrophobic membrane proteins]

Phase partition method in a two-phase polyethylene glycol-dextrane system has been applied to fractionation in Triton X-100 of hydrophobic membrane proteins from Micrococcus lysodeikticus. This method allowed to separate the cytochrome b556 from other cytochromes. Spectral and gel electrophoretic patterns of isolated cytochrome are given.     

Comparison of platelet-derived growth factor prepared from release products of fresh platelets and from outdated platelet concentrates

Treatment of synaptic membranes from rat brainstem and spinal cord with the nonionic detergent Triton X-100 at 1-10 microliters/mg protein caused a marked increase in glycine receptor (3H)strychnine binding expressed per mg of residual membrane protein. The effect was maximal (220 +/- 6% of control) at 5 microliters Triton/mg protein, while higher concentrations caused progressive loss of strychnine binding ability of membranes (27 +/- 6% at 25 microliters Triton/mg protein). The increase in strychnine binding caused by low Triton X-100 reflected an increase in membrane Bmax, the kD being unaffected by the treatment. The affinity of glycine analogues for receptor sites was not appreciably affected by the detergent either. The findings suggest an enrichment of the synaptic membrane preparation in glycine receptors, caused by the solubilization by Triton of membrane constituents not related to the receptor sites.     

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.     

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.     

Endocytosis of GPI-linked membrane folate receptor-alpha

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36- 38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100- resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.     

Differential Solubilization of γ-Aminobutyric Acid Receptive Sites from Membranes of Mammalian Brain

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized as to binding constants, pharmacologic specificities, and sodium dependence. The SPM fraction was then treated with various concentrations of Triton X-100 resulting in the loss of pharmacologic specificity, binding characteristics, and sodium dependence associated with +Na GABA receptive sites in SPM. The resulting junctional complex preparation (JC), i.e., a fraction enriched in junctional complexes, possessed only the pharmacologic specificity and binding constants associated with -Na receptive sites whether assayed in the presence or absence of 100 mM-NaCl. This is probably due to the detergent dispersal or solubilization of the +Na GABA receptive site. The binding constants, KD and Bmax, for -Na GABA binding in SPM were 170 nM and 4.4 pmol/mg protein, while in JC they were 186 nM and 3.7 pmol/mg protein. Under repeated washing the KD was reduced to 60 +/- 6.9 nM and the Bmax was reduced to 2.5 +/- 0.5 pmol/mg protein in JC, probably owing to the removal of endogenous ligand or inhibitor, and not to inhibition by residual Triton X-100. Multiple extraction with 0.1% or 0.5% Triton X-100 did not alter the KD or Bmax values for the binding of [3H]GABA to JC. Sodium-independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.