Localisation of the subunits of the photosynthetic reaction centers in the chromatophore membrane of Rhodospirillum rubrum.

Reaction centers were isolated with the detergent lauryl dimethyl amine oxide from chromatophore membranes of Rhodospirillum rubrum. The subunit composition of these reaction centers is similar to the one obtained from Rhodopseudomonas spheroides: three subunits with the molecular weights of 21 000, 24 000 and 29 000. Reaction centers prepared from chromatophores labeled with 131I were heavely labeled in their large subunit (H). The smaller subunits (L and M) contained only little label. Sonication during labeling yielded a slightly higher incorporation of 131I in subunit H compared to the smaller ones. It is concluded that the H protein is largely exposed at the cytoplasmic side of the membrane but might also be accessible for iodination on the inside of the membrane while the L and M proteins are almost completely embedded in the membrane. Iodination of spheroplasts results in only a slight binding of 131I to chromatophores and reaction centers.     

Hexane-solubilised reaction centre proteolipid complexes of Rhodopseudomonas sphaeroides

Reaction centres from Rhodopseudomonas sphaeroides, strain R-26, have been solubilised in hexane by the use of phospholipids and cations. Two procedures have been developed: (I) a two-step technique involving the formation of detergent-free proteoliposomes from detergent solubilised reaction centres and phospholipids and mixing these with hexane in the presence of cations; (II) directly sonicating detergent-solubilised reaction centres with phosopholipids before mixing with hexane and cations.The yield of the extracted complex varied with the ratios of protein, phospholipid and cations, species of phospholipid, and sonication time. The spectral characteristics of the complex in the organic phase were similar to those of detergent-solubilised reaction centres. The stability of the reaction centres appeared to be dependent on the presence of phospholipid and water in the hexane. The usefulness of the hexane solution as a model membrane system is discussed and its possible future applications are considered.     

Lateral organization of proteins in the chromatophore membrane ofRhodospirillum rubrum studied by chemical cross-linking

The organization of proteins in the chromatophore membrane, particularly of the reaction center and the light-harvesting polypeptide, was examined by the use of a hydrophobic and a hydrophilic cross-linking reagent, namely DSP (dithiobis-succinimidyl propionate) and glutaraldehyde. The linkage of proteins was studied by SDS polyacrylamide pore gradient electrophoresis. DSP was shown to link proteins within the core of the membrane. The subunit H of the reaction center is linked with DSP at a low concentration, either with itself or with other membrane proteins but not to the subunits M and L. In isolated reaction centers the subunits H are exclusively linked with each other. With increasing concentrations of DSP the bands of the subunits M, L, and the light-harvesting polypeptide disappear simultaneously from the gel, suggesting that these proteins are linked together. This hypothesis is supported by the finding that reaction centers isolated from chromatophores treated with DSP retain an appreciable amount of light-harvesting polypeptide. With increasing concentrations of the hydrophilic cross-linking reagent glutaraldehyde, the bands of all the three subunits of the reaction center, H, M, and L, progressively disappear from the gel, suggesting that they are linked together. The light-harvesting polypeptide remains free when this reagent is used.     

2-[8-14C]naphthyl 2-diazo-3,3,3-trifluoropropionate, a new carbene generating reagent for probing hydrophobic membrane domains

A new precursor of a lipophilic photolabel, 2-[8-14C]naphthyl 2-diazo-3,3,3-trifluoropropionate (NADIT) has been synthesized. The suitability of the reagent for labeling the hydrophobic core of membranes is demonstrated by studying its reactivity in chromatophores of Rhodospirillum rubrum G-9+. The label binds preferentially to the phospholipids and intrinsic membrane proteins. In isolated reaction centers treated with NADIT the hydrophobic subunits M and L are more labeled than the H subunit. The high reactivity, dark stability and ease of synthesis favors this very lipophilic reagent to identify the intrinsic hydrophobic sections of membrane proteins.     

Reconstitution and partial characterization of phospholipid flippase activity from detergent extracts of the Bacillus subtilis cell membrane

In bacteria, phospholipids are synthesized on the inner leaflet of the cytoplasmic membrane and must translocate to the outer leaflet to propagate a bilayer. Transbilayer movement of phospholipids has been shown to be fast and independent of metabolic energy, and it is predicted to be facilitated by membrane proteins (flippases) since transport across protein-free membranes is negligible. However, it remains unclear as to whether proteins are required at all and, if so, whether specific proteins are needed. To determine whether bacteria contain specific proteins capable of translocating phospholipids across the cytoplasmic membrane, we reconstituted a detergent extract of Bacillus subtilis into proteoliposomes and measured import of a water-soluble phospholipid analog. We found that the proteoliposomes were capable of transporting the analog and that transport was inhibited by protease treatment. Active proteoliposome populations were also able to translocate a long-chain phospholipid, as judged by a phospholipase A(2)-based assay. Protein-free liposomes were inactive. We show that manipulation of the reconstitution mixture by prior chromatographic fractionation of the detergent extract, or by varying the protein/phospholipid ratio, results in populations of vesicles with different specific activities. Glycerol gradient analysis showed that the majority of the transport activity sedimented at approximately 4S, correlating with the presence of specific proteins. Recovery of activity in other gradient fractions was low despite the presence of a complex mixture of proteins. We conclude that bacteria contain specific proteins capable of facilitating transbilayer translocation of phospholipids. The reconstitution methodology that we describe provides the basis for purifying a facilitator of transbilayer phospholipid translocation in bacteria.     

Phospholipid distribution in the microenvironment of the immunoglobulin E-receptor from rat basophilic leukemia cell membrane

It has been previously found that lipids were required to maintain intact the tetrameric structure of the receptor for immunoglobulin E (IgE) (Fc epsilon R) in detergent solutions [Rivnay, B., Rossi, G., Henkart, M., & Metzger, H. (1984) J. Biol. Chem. 259, 1212-1217, and references cited therein]. Failure of commercially obtained lipids to provide sufficient protection, however, underscored the necessity for development of additional analytical approaches. In order to identify the phospholipid distribution in the intimate natural environment of this receptor, both the plasma membrane vesicles and the ligand-receptor complex (IgE-Fc epsilon R) have been isolated by affinity chromatography. The phospholipids of both preparations were compared. After extensive washing with detergent lipid micelles, IgE-Fc epsilon R retained 0.1-1% of the total phospholipids in the purified plasma membrane. The receptor-bound lipids were shown to contain phosphatidylcholine and sphingomyelin; the content of the latter lipid was enriched 2-5-fold compared with that in the plasma membranes. This pattern was observed with several detergents employed for purification and under a variety of experimental conditions. In light of the general distribution of choline phospholipids in the outer leaflet of plasma membranes, this enrichment may not be a characteristic of this particular receptor exclusively. These observations should be particularly helpful in studies on aggregation-induced functions of the isolated Fc epsilon receptor. In general, the methods employed enable isolation of purified and lipid-protected integral proteins and also provide an appropriate reference source of intact membrane vesicles. These qualities render this approach useful in similar studies of other membrane proteins.     

Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits

The LolCDE complex of Escherichia coli belongs to the ATP-binding cassette transporter superfamily and mediates the detachment of lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane. The complex is composed of one copy each of membrane subunits LolC and LolE, and two copies of ATPase subunit LolD. To establish the conditions for reconstituting the LolCDE complex from separately isolated subunits, the ATPase activities of LolD and LolCDE were examined under various conditions. We found that both LolD and LolCDE were inactivated on incubation at 30 degrees C in a detergent solution. ATP and phospholipids protected LolCDE, but not LolD. Furthermore, phospholipids reactivated LolCDE even after its near complete inactivation. LolD was also protected from inactivation when membrane subunits and phospholipids were present together, suggesting the phospholipid-dependent reassembly of LolCDE subunits. Indeed, the functional lipoprotein-releasing machinery was reconstituted into proteoliposomes with E. coli phospholipids and separately purified LolC, LolD and LolE. Preincubation with phospholipids at 30 degrees C was essential for the reconstitution of the functional machinery from subunits. Strikingly, the lipoprotein-releasing activity was also reconstituted from LolE and LolD without LolC, suggesting the intriguing possibility that the minimum lipoprotein-releasing machinery can be formed from LolD and LolE. We report here the complete reconstitution of a functional ATP-binding cassette transporter from separately purified subunits.     

Kinetics and yields of bacteriochlorophyll fluorescence: redox and conformation changes in reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Induction of the bacteriochlorophyll fluorescence under rectangular shape of intense laser diode illumination (1 W cm(-2), 808 nm) was measured over wide time range from 10 mus to 4 s in whole cells, chromatophore and isolated reaction center protein of wild type and carotenoid-less mutant (R-26.1) of purple photosynthetic bacterium Rhodobacter sphaeroides. While the antenna-containing species showed large and positive variable fluorescence (F (v)) to initial fluorescence (F (0)) (F (v)/F (0) approximately 4.5 in whole cells), the isolated RC had negative change (F (v)/F (0) approximately -0.6) during photochemistry. In chromatophore from R-26.1, only seven times higher rate was measured than in isolated reaction center under identical experimental conditions. The enhancement effect of large antenna on the rate of photochemistry in chromatophore was partially compensated by the favorable pigment absorption properties in isolated RC. The transition from membrane bound to isolated form of the reaction center was probed by titration of zwitterionic detergent LDAO in chromatophore, and at 0.03% LDAO concentration, sharp change of the variable fluorescence was observed. The sudden drop was explained by the formation of LDAO micelles. After the photochemical phase, additional change of fluorescence yield could be observed in isolated RC considered as manifestation of long-living conformations of the trapped redox states of the protein characterized by non-exponential kinetics. Strong support was provided for use of the fluorescence induction to track structural and conformation changes at their earliest phases in chromatophores and isolated reaction centers.     

Probing the topology of proteins in the chromatophore membrane of Rhodospirillum rubrum G-9 with proteinase K

All the major membrane proteins of isolated chromatophore vesicles are eventually degraded upon incubation with the unspecific proteinase K. These proteins must therefore be exposed at least partially or temporarily on the cytosolic surface of the membrane which is exclusively accessible to the proteinase in intact chromatophore vesicles. That the vesicles are intact during the incubation with proteinase is demonstrated by the finding that cytochrome c₂, which is located in the interior of the vesicles, is protected from proteolytic attack. The degree of degradation of the various chromatophore proteins and the time taken for degradation differ characteristically. From the changes in intensity of the gel bands during the course of digestion it appears that reaction center subunit H is digested first, much faster than are subunits M and L. The near-infrared absorption spectrum of the chromatophores changes only after proteolytic degradation of these two pigment-carrying subunits. Fading of the band of the light-harvesting polypeptide is evident only after prolonged incubation. It seems that this is the most stable component of the chromatophore membrane. The light-harvesting polypeptide appears to be somewhat shortened eventually, leaving the protein conformation necessary for holding the pigments unchanged, as shown by the absorption spectrum. The possible topology of these major membrane components is discussed in the light of these findings.     

Phosphorus nuclear magnetic resonance studies of lipid-protein interactions: human erythrocyte glycophorin and phospholipids

Human erythrocyte glycophorin containing four molecules of phospholipid tightly bound to the protein was isolated from human red cell ghosts. This protein preparation was reconstituted into a digalactosyl diglyceride bilayer. The 31P NMR spectrum of this reconstituted membrane produced an axially symmetric powder pattern arising exclusively from the phospholipids bound to glycophorin. The width of the powder pattern, about 90 ppm, is about twice as broad as that normally exhibited by a phospholipid bilayer. The chemical shift tensor is perturbed relative to phospholipids in a bilayer. The spin-lattice relaxation rate of these protein-bound phospholipids is found to be nearly an order of magnitude faster than phospholipids in a bilayer. The results are consistent with phospholipids tightly bound to the membrane protein and undergoing rotational diffusion, perhaps as a complex of phospholipid and protein.     

Fusion of chromatophores derived from Rhodopseudomonas sphaeroides

Fusion of chromatophores, the photosynthetic membrane vesicles isolated from the intracytoplasmic membranes of Rhodopseudomonas sphaeroides, was achieved by the use of poly(ethylene glycol) 6000 as fusogen. Ultracentrifugation, electron microscopy, intrinsic density and isotope labeling were used to demonstrate chromatophore fusion. Although studies of the flash-induced shift in the carotenoid absorbance spectrum indicated that the membrane was rendered leaky to ions by either the fusion procedure or the increased size of the fused products, the orientation and integrity of fused chromatophores were otherwise demonstrated to be identical to control chromatophores by freeze-fracture electron microscopy, proteolytic enzyme digestion, enzymatic radioiodination, and transfer of chromatophore phospholipids mediated by phospholipid exchange protein extracted from Rps. sphaeroides.     

The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.

The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl-POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+-loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing delta psi with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex-OH treatment to an even higher rate than that found in the GTP-free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP-loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no 'valve' mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+-translocating site differs from the highly pH-dependent nucleotide-binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel-type H+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and subunit composition of the H+-ATPase complex from pea chloroplasts

The H+-ATPase complex from pea chloroplasts was isolated and partially purified. The complex incorporated into the phospholipid membrane can catalyze the 32Pi-ATP exchange reaction. The complex contains eight types of the subunits, five of which belong to the catalytic and three--to the hydrophobic moieties. The molecular weights of the subunits were determined.     

Agglutination of mouse erythrocytes by binding of non-choline phospholipids to a 70 000-Dalton protein

The structure of some phospholipids that cause agglutination of mouse erythrocytes has been studied. Haemagglutination is a property of non-choline-containing phospholipids; the phosphate group is essential and unsaturated fatty acids optimal. A protein of Mr 70 000 was isolated from mouse erythrocyte membranes which completely inhibited phospholipid-mediated erythrocyte agglutination. It is proposed that this protein is the phospholipid binding site on mouse erythrocytes and the ligand for the human B-lymphocyte receptor for mouse erythrocytes. Preliminary investigations suggest that a similar inhibitor of phospholipid-mediated agglutination is found in serum. Agglutination of mouse erythrocytes by phospholipid and specific inhibition by the 70 kDa membrane protein constitute a simple system for studying the interaction of phospholipid with protein.     

Brominated lipids identify lipid binding sites on the surface of the reaction center from Rhodobacter sphaeroides

This study describes the use of brominated phospholipids to distinguish between lipid and detergent binding sites on the surface of a typical alpha-helical membrane protein. Reaction centers isolated from Rhodobacter sphaeroides were cocrystallized with added brominated phospholipids. X-ray structural analysis of these crystals has revealed the presence of two lipid binding sites from the characteristic strong X-ray scattering from the bromine atoms. These results demonstrate the usefulness of this approach to mapping lipid binding sites at the surface of membrane proteins.     

The chemical carcinogen-induced enzyme, GDP-fucose: GM1 alpha 1----2 fucosyltransferase in rat liver and hepatoma: modulation by and association with phospholipids

The enzyme GDPFuc:GM1 alpha 1----2 fucosyltransferase, induced by chemical carcinogens in precancerous rat liver as well as rat hepatoma cells, was found previously to be membrane bound, and was inactivated by various detergents, while the activities of many other transferases are generally enhanced by detergents (Holmes, E.H. & Hakomori, S. (1983) J. Biol. Chem. 258, 3706-3717). The effects of phospholipids and detergents on rat hepatoma H35 cells, the conditions of solubilization and subsequent affinity chromatography of the enzyme, and a possible association of phospholipids with the enzyme have been studied with the following major results: The alpha 1----2 fucosyltransferase activity in Golgi membrane was diminished on treatment of membranes with phospholipase A1 or phospholipase C. The enzyme activity was stimulated 7-fold in the presence of cardiolipin or phosphatidylglycerol (and 3-fold by phosphatidylethanolamine) but not other phospholipids. The stimulatory effect of phosphatidylglycerol was eliminated when a variety of ionic or non-ionic detergents were added to the reaction mixture, with the exception of the cationic detergent G-3634-A, which provided a 10-fold total stimulation in the presence of phosphatidylglycerol. The kinetic analysis indicated that addition of phosphatidylglycerol has a negligible effect on apparent Km values but increases the Vmax of the enzyme 5- to 6-fold. The enzyme activity was solubilized by the dialyzable detergent CHAPSO without inhibition of the enzyme activity, and the solubilized enzyme in the presence of 0.4% CHAPSO is partially purified by chromatography on GDP-hexanolamine-Sepharose. Removal of CHAPSO from the affinity purified enzyme by dialysis resulted in a 66% loss of the original activity, which was restored by addition of phosphatidylglycerol. Chromatography of the affinity-purified enzyme with 3H-labeled phosphatidylglycerol on a Biogel A0.5 column indicated an association of the enzyme with the phospholipid that occurred only in the absence of detergent. These results suggest that phospholipid has a direct effect on the enzyme and that the inhibitory effect of detergents can be ascribable to disturbing interaction between phospholipids and the enzyme. A possible role of specific phospholipids on in vivo transferase activity for glycolipids is discussed.     

Activation of phospholipase hydrolysis in the process of oxidative cell damage]

Effect of tert-butyl hydroperoxide toxic action on phospholipase A2 activity and the changes in phospholipid composition from mastocytoma P815 cells were investigated. Oxidative damage of tumor cell membranes was accompanied by the release of arachidonic acid from membrane phospholipids and the accumulation of lysophosphatidylcholine, the product of phospholipase A2 reaction and a potent detergent. Tert-butyl hydroperoxide also increased relative contents of sphingomyelin, phosphatidylserine and phosphatidic acid in tumor cell membranes. It is possible that phospholipase A2 activation and the changes of phospholipid molecular species contents may cause the damage of cell membrane stability.     

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of a cross-linking complex of dinitrogenase reductase-activating glycohydrolase (DRAG) with membrane proteins from <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> chromatophores

Association of dinitrogenase reductase-activating glycohydrolase (DRAG) with membrane proteins of chromatophores has been investigated. The formation of a multicomponent complex between DRAG and membrane proteins was demonstrated in the presence of glutaraldehyde and EDC/NHS (N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride/hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid sodium salt). Complex formation was observed both in native chromatophore membrane and in chromatophores treated with 0.5 M NaCl in the presence of homogeneous DRAG and glutaraldehyde in cross-reaction. The molecular weight of the complex was around 200 kD, which is consistent with the association of DRAG with three or more chromatophore membrane proteins. A specific complex with molecular weight of about 75 kD was formed only in the presence of EDC/NHS in the cross-linking reaction. It was demonstrated that ammonium transport protein and P11 protein are possible candidates for association with DRAG in chromatophore membranes.