Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents.

The Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into sealed phospholipid vesicles using the method recently developed for bacteriorhodopsin (Rigaud, J.L., Paternostre, M.T. and Bluzat, A. (1988) Biochemistry 27, 2677-2688). Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, sodium cholate or dodecyl octa(oxyethylene) glycol ether (C12E8) and protein incorporation was studied at each step of the liposome solubilization process by each of these detergents. After detergent removal by SM-2 Bio-Beads the resulting vesicles were analyzed with respect to protein incorporation by freeze-fracture electron microscopy, sucrose density gradients and Ca2+ pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With octyl glucoside, direct incorporation of Ca(2+)-ATPase into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes homogeneous in regard to protein distribution. With the other detergents, optimal Ca(2+)-ATPase pumping activities were obtained when starting from Ca(2+)-ATPase/detergent/phospholipid micellar solutions. However, the homogeneity of the resulting recombinants was shown to be dependent upon the detergent used and in the presence of Triton X-100 or C12E8 different populations were clearly evidenced. It was further demonstrated that the rate of detergent removal drastically influenced the composition of resulting proteoliposomes: upon slow detergent removal from samples solubilized with Triton X-100 or C12E8, Ca(2+)-ATPase was found seggregated and/or aggregated in very few liposomes while upon rapid detergent removal compositionally homogeneous proteoliposomes were obtained with high Ca2+ pumping activities. The reconstitution process was further analyzed by centrifugation experiments and the results demonstrated that the different mechanisms of reconstitution were driven predominantly by the tendency for self-aggregation of the Ca(2+)-ATPase. A model for Ca(2+)-ATPase reconstitution was proposed which accounted for all our results. In summary, the advantage of the systematic studies reported in this paper was to allow a rapid and easy determination of the experimental conditions for optimal detergent-mediated reconstitution of Ca(2+)-ATPase. Proteoliposomes prepared by the present simple method exhibited the highest Ca2+ pumping activities reported to date in Ca(2+)-ATPase reconstitution experiments performed in the absence of Ca2+ precipitating agents.     

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by triton X-100, octyl glucoside, and sodium cholate

The mechanisms governing the solubilization by Triton X-100, octyl glucoside, and sodium cholate of large unilamellar liposomes prepared by reverse-phase evaporation were investigated. The solubilization process is described by the three-stage model previously proposed for these detergents [Lichtenberg, D., Robson, R.J., & Dennis, E.A.(1983) Biochim. Biophys. Acta 737, 285-304]. In stage I, detergent monomers are incorporated into the phospholipid bilayers until they saturate the liposomes. At that point, i.e., stage II, mixed phospholipid-detergent micelles begin to form. By stage III, the lamellar to micellar transition is complete and all the phospholipids are present as mixed micelles. The turbidity of liposome preparations was systematically measured as a function of the amount of detergent added for a wide range of phospholipid concentrations (from 0.25 to 20 mM phospholipid). The results allowed a quantitative determination of RSat, the effective detergent to lipid molar ratios in the saturated liposomes, which were 0.64, 1.3, and 0.30 for Triton X-100, octyl glucoside, and sodium cholate, respectively. The corresponding ratios in the mixed micelles, RSol, were 2.5, 3.8, and 0.9 mol of detergent/mol of phospholipid. The monomer concentrations of the three detergents in the aqueous phase were also determined at the lamellar to micellar transitions (0.18, 17, and 2.8 mM, respectively). These transitions were also investigated by 31P NMR spectroscopy, and complete agreement was found with turbidity measurements. Freeze-fracture electron microscopy and permeability studies in the sublytic range of detergent concentrations indicated that during stage I of solubilization detergent partitioning between the aqueous phase and the lipid bilayer greatly affects the basic permeability of the liposomes without significantly changing the morphology of the preparations. A rough approximation of the partition coefficients was derived from the turbidity and permeability data (K = 3.5, 0.09, and 0.11 mM-1 for Triton X-100, octyl glucoside, and sodium cholate, respectively). It is concluded that when performed systematically, turbidity measurements constitute a very convenient and powerful technique for the quantitative study of the liposome solubilization process by detergents.     

Structural characterization of the ATP-hydrolyzing portion of the coated vesicle proton pump

The ATP-hydrolyzing portion of the proton pump from clathrin-coated vesicles (isolated from calf brain) was solubilized with three nondenaturing detergents (cholate, octyl glucoside, and Triton X-100). The hydrodynamic properties of the solubilized (Mg2+)-ATPase were then determined by sedimentation analysis in H2O and D2O and gel filtration on Sepharose 4B. The coated vesicle (Mg2+)-ATPase migrated under all conditions as a single peak of activity. In cholate, the sedimentation coefficient (S20,w), Stokes radius (a), and partial specific volume (vc) were 8.25 (+/- 0.20) S, 68 (+/- 2) A, and 0.71 (+/- 0.03) cm3/g, respectively. In octyl glucoside and Triton X-100 these values were respectively 7.90 (+/- 0.20) and 7.45 (+/- 0.20) S, 68 (+/- 3) and 101 (+/- 5) A, and 0.74 (+/- 0.03) and 0.75 (+/- 0.03) cm3/g. Application of the Svedberg equation to these data gave a molecular weight for the protein-detergent complex of 217,000 +/- 21,000 (cholate), 234,000 +/- 26,000 (octyl glucoside), and 337,000 +/- 40,000 (Triton X-100). Assuming the protein binds one micelle of detergent, these values correspond to a protein molecular weight of 215,000 +/- 21,000 (cholate), 226,000 +/- 26,000 (octyl glucoside), and 247,000 +/- 40,000 (Triton X-100). The cholate-solubilized, gradient-purified (Mg2+)-ATPase, when combined with a 100,000 g pellet fraction, could be reconstituted by dialysis into phospholipid vesicles which displayed ATP-dependent proton uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional reconstitution of halorhodopsin. Properties of halorhodopsin-containing proteoliposomes

A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsin in cell envelope vesicles. Thus, the reconstitution yields halorhodopsin with both photochemical and transport activities restored. Freeze-fracture electron micrographs of the proteoliposomes showed unilammellar liposomes with numerous particles of 100-150 A diameter at the fracture faces. These should correspond to halorhodopsin aggregates, formed in the bilayer in an apparently concentration-dependent manner.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.     

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl2 and DIDS. The IC50 for HgCl2 was 0.16+/-0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9+/-0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.     

Reconstitution of CF0F1 into liposomes using a new reconstitution procedure

The H(+)-ATPase (ATP synthase) from chloroplasts was isolated, purified and reconstituted into phosphatidylcholine/phosphatidic-acid liposomes. Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100 and protein incorporation was studied at each step of the solubilization process. After detergent removal by SM2-Biobeads, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitution was obtained by insertion of the protein into preformed, detergent-saturated liposomes. The conditions for the reconstitution were optimized with regard to ATP synthesis driven by an artificially generated delta pH/delta psi. An important benefit of the new reconstituted CF0F1 liposomes is the finding that the rate of ATP synthesis remains constant up to 10 s, indicating a low basal membrane permeability.     

A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal

Equilibrium and kinetic aspects of Triton X-100 adsorption onto hydrophobic Bio-Beads SM2 were investigated in detail using the batch procedure originally described by Holloway, P.W. (1973) Anal. Biochem. 53, 304-308. The results demonstrated the importance of the initial detergent concentration, the amount of beads, the commercial source of the detergent, the temperature and the presence of phospholipids in determining the rates of Triton X-100 adsorption onto Bio-Beads. One of the main findings was that Bio-Beads allowed the almost complete removal of Triton X-100, whatever the initial experimental conditions. It was shown that monomeric as well as micellar detergent could be adsorbed and that a key factor in determining the rate of detergent removal was the availability of the free bead surface. Rates of detergent removal were found to be linearly related to the amount of beads even for bead concentrations above those sufficient to remove all the detergent initially present. Adsorptive capacity of phospholipids onto Bio-Beads SM2 was also analyzed and found to be much smaller (2 mg lipid per g of wet beads) than that of Triton X-100 (185 mg TX 100 per g of wet beads). A more general aspect of this work was that the use of Bio-Beads SM2 provided a convenient way for varying and controlling the time course of Triton X-100 removal. The method was further extended to the formation of liposomes from phospholipid-Triton X-100 micelles and the size of the liposomes was found to be critically dependent upon the rate of detergent removal. A general procedure was described to prepare homogeneous populations of vesicles. Freeze-fracture electron microscopy and permeability studies indicated that the liposomes thus obtained were unilamellar, relatively large and impermeable. Noteworthy, this new procedure was shown to be well suited for the reconstitution of different membrane transport proteins such as bacteriorhodopsin, Ca2(+)-ATPase and H(+)-ATPase.     

Optimization of detergents in solubilization and reconstitution of Aquaporin Z: A structural approach

BackgroundThe exceptional capacities of aquaporins in terms of water permeation and selectivity have made them an interesting system for membrane applications. Despite the multiple attempts for immobilizing the aquaporins over a porous substrate, there is a lack of studies related to the purification and reconstitution steps, principally associated with the use of detergents in solubilization and destabilization steps. This study analyzed the effect of detergents in Aquaporin Z solubilization, considering the purity and structural homogeneity of the protein.MethodsThe extraction process was optimized by the addition of detergent at the sonication step, which enabled the omission of the ultracentrifugation and resuspension steps. Two detergents, Triton X-100, and octyl-glucoside were also evaluated. Destabilization mediated by detergents was used as reconstitution method. Saturation and solubilization points were defined by detergent concentration and both, liposomes and proteoliposomes, were analyzed by size distribution and permeability assays. Detergent removal with Bio-beads was also analyzed.ResultsOctyl glucoside ensures structural stability and homogeneity of Aquaporin Z. However, high concentrations of detergents induce the presence of defects in proteoliposomes. While saturated liposomes create homogeneous and functional structures, solubilized liposomes get affected by a reassembly process, creating vesicle defects with anomalous permeability profiles.ConclusionsDetergent concentration affects the structural conformation of proteoliposomes in the reconstitution process.General significanceSince the destabilization process is dependent on vesicle, detergent, and buffer composition, optimization of this process should be mandatory for further studies. All these considerations will allow achieving the potential of Aquaporins and any other integral membrane protein in their applications for industrial purposes.     

The solubilization and morphological change of human platelets in various detergents

The solubilization of human gel-filtered platelets by octyl glucoside, Triton X-100, dodecylsulfate, and deoxycholate was compared from the analysis of (1) cell lysis, (2) marker leakiness, and (3) component solubility. These analyses all revealed that the effect of detergent concentration on the solubilization of platelets by these detergents was exerted in three stages, i.e., the prelytic, lytic, and complete platelet-lysis stages. These analyses also indicated several differences among platelets in these detergents. (i) The ratio of the platelet-saturation concentration (PSC) to critical micellar concentration (CMC) was about 1/2 for octyl glucoside. Triton X-100 and dodecylsulfate, while it was close to 1 for deoxycholate. (ii) Platelets in octyl glucoside. Triton X-100, and dodecylsulfate all showed parallel curves in cell lysis, protein solubilization and marker leakiness, while the platelet lysis in deoxycholate was identical to the phospholipid solubilization. (iii) The solubility curves of various components in Triton X-100 and deoxycholate were parallel. However, the solubility of cholesterol in octyl glucoside was lower than that of protein and phospholipid. In dodecylsulfate, the solubility of phospholipid and cholesterol was very low in comparison with that of protein. In addition, morphological studies using scanning electron microscopy (scanning EM) revealed that the solubilization by octyl glucoside or Triton X-100 might occur via membrane area expansion. On the other hand, the solubilization by dodecylsulfate or deoxycholate showed membrane vesiculation prior to cell lysis. Moreover, in the prelytic stage, the morphological change in platelets in octyl glucoside showed only concentration dependence by swelling to an ellipsoid and then to a sphere. However, the morphological change in platelets in the other three detergents was dependent not only on the detergent concentration but also on prolonged incubation. Specifically, in Triton X-100, the cells initially changed to spiculate discs and then reached their final shape as swollen discs with surface invagination. In dodecylsulfate and deoxycholate the morphological changes were almost the same. The cell initially deformed in shape to a spiculate disc and finally to a stretched-out flat form. The results are discussed according to the bilayer couple hypothesis. Also, in the prelytic stage, these detergents caused inhibition of the response of platelets to collagen and ADP-fibrinogen.     

Solubilization of Myelin Membranes by Detergents

The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS-polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.     

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl₂ and DIDS. The IC₅₀ for HgCl₂ was 0.16 ± 0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9 ± 0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.     

Structure and energy-linked activities in reconstituted bacteriorhodopsin--yeast ATPase proteoliposomes.

Bacteriorhodopsin-F₁·F₀ (mitochondrial oligomycin-sensitive ATPase complex) proteoliposomes have poor proton pumping and photophosphorylation activities when reconstituted by cholate dialysis. A considerable proportion of the bacteriorhodopsin is not incorporated by cholate dialysis, the particles being too large to be combined into liposomes. Much better reconstitution is achieved where the purple membranes are first fragmented by sonication. Optimal incorporation occurs where bacteriorhodopsin and the phospholipids are sonicated together, suggesting that some perturbation of the liposomes is necessary for successful integration. Since F₁·F₀ is denatured by sonication a two-step reconstitution procedure has been developed wherein bacteriorhodopsin is first incorporated by sonication, then F₁·F₀ by cholate dialysis. The vesicles have high phosphorylation rates and also catalyze postillumination [³²P]ATP formation where pyridine is present during first stage illumination.F₁·F₀ can also be incorporated into sonicated bacteriorhodopsin vesicles by “direct incorporation.” This depends on the presence of negatively charged amphiphiles such as cholate or phosphatidylserine in the membranes, and is stimulated by divalent metal cations. Optimum conditions for the various reconstitution procedures are described.     

The use of octyl beta-D-glucoside as detergent for hog kidney brush border membrane

Octyl beta-D-glucoside was synthetized from alpha-acetobromoglucose with an improved method yielding a very pure product with a sharp melting point (108-109 degrees C) and free of intermediate products as judged by IR and NMR spectra. The yield of the synthesis is 66% when referred to alpha-acetobromoglucose. The potency of this compound as a detergent on hog kidney brush border membranes was compared to the action of Triton X-100. Octyl glucoside preferentially extracts aminopeptidase M and gamma-glutamyltranspeptidase in a concentration-dependent manner. The more deeply imbedded membrane enzyme, alkaline phosphatase, was relatively resistent to the action of octyl glucoside. In contrast, Triton X-100 extracted all membrane proteins to about the same extent. Additionally it was found that octyl glucoside can be removed from membrane extracts by Biobead SM 2. The capacity of the beads is about 170 mg detergent/g of dry Biobead SM 2. Thus octyl glucoside seems to be a useful tool for solubilization and purification of brush border membranes proteins.     

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and antibodies

Exposure of rat liver mitochondrial membranes to octyl glucoside, Triton X-100, or Tween 20 solubilized an active and tetradecylglycidyl-CoA (TG-CoA)-insensitive carnitine palmitoyltransferase (presumed to be carnitine palmitoyltransferase II). The residual membranes after octyl glucoside or Triton X-100 treatment were devoid of all transferase activity. By contrast, Tween 20-extracted membranes were still rich in transferase; this was completely blocked by TG-CoA and thus was presumed to be carnitine palmitoyltransferase I. The residual carnitine palmitoyltransferase activity disappeared from the membranes upon subsequent addition of octyl glucoside or Triton X-100 and could not be recovered in the supernatant fraction. Antibody raised against purified rat liver transferase II (Mr 80,000) recognized only this protein in immunoblots from untreated liver mitochondrial membranes containing both transferases I and II. Tween 20-extracted membranes, which contained only transferase I, did not react with the antibody. Purified transferase II from skeletal muscle (also of Mr 80,000) was readily recognized by the antiserum, suggesting antigenic similarity with the liver enzyme. These and other studies on the effects of detergents on the mitochondrial [3H]TG-CoA binding protein provide further support for the model of carnitine palmitoyltransferase proposed in the preceding paper. They suggest that: 1) carnitine palmitoyltransferases I and II in rat liver are immunologically distinct proteins; 2) transferase I is more firmly anchored into its membrane environment than transferase II; 3) association of carnitine palmitoyltransferase I with a membrane component(s) is necessary for catalytic activity. While carnitine palmitoyltransferase I is a different protein in liver and muscle, it seems likely that both tissues share the same transferase II.     

Dynamic nature of the quaternary structure of the vesicular stomatitis virus envelope glycoprotein

The envelope glycoprotein (G protein) of vesicular stomatitis virus probably exists in the viral envelope as a trimer of identical subunits. Depending on the conditions of solubilization, G protein may dissociate into monomers. G protein solubilized with the detergent octyl glucoside was shown to exist as oligomeric forms by sedimentation velocity analysis and chemical cross-linking. G protein was modified with either fluorescein isothiocyanate or rhodamine isothiocyanate. Resonance energy transfer between fluorescein and rhodamine labels was observed upon mixing the two labeled G proteins in octyl glucoside. This result provided further evidence that G protein in octyl glucoside is oligomeric and indicated that the subunits are capable of exchange to form mixed oligomers. Resonance energy transfer was independent of G protein concentration in the range examined (10-80 nM) and was not observed when labeled G proteins were mixed with fluorescein or rhodamine that was not conjugated to protein. Resonance energy transfer decreased upon incorporation of G protein into Triton X-100, consistent with sedimentation velocity data that G protein in Triton X-100 is primarily monomeric. Kinetic analysis showed that the subunit exchange reaction had a half-time of about 3 min at 27 degrees C that was independent of G protein concentration. These data indicate that the exchange occurs through dissociation of G protein trimers into monomers and dimers followed by reassociation into timers. Thus, in octyl glucoside, G protein must exist as an equilibrium between monomers and oligomers. This implies that monomers are capable of self-assembly into trimers.     

Studies on the role of the oligomeric state and subunit III of cytochrome oxidase in proton translocation

Anion-exchange fast protein liquid chromatography in the presence of lauryldimethylamine N-oxide (LDAO) was introduced to separate cytochrome oxidase into different complexes that either did or did not contain subunit III. Both kinds of enzyme complex exhibited H+ translocation after reconstitution into phospholipid vesicles, but with a significantly (approx. 50-60%) reduced H+/e- ratio as compared with unchromatographed enzyme. The anion-exchange FPLC fractions of the enzyme (with or without subunit III) sedimented more slowly than the control enzyme upon sucrose gradient centrifugation in the presence of cholate and a high potassium phosphate concentration. When the control enzyme was subjected to the sucrose gradient centrifugation in the presence of LDAO or Triton X-100, instead of cholate, one band containing all subunits was observed, which sedimented slowly like the FPLC fractions. Transfer of this band to cholate medium, and reapplication on the sucrose gradient (with cholate), yielded both a slow- and a fast-migrating band after centrifugation. Enzyme complexes that sedimented slowly or rapidly in the sucrose gradients revealed longer and shorter elution times, respectively, in gel filtration FPLC. This suggests that these complexes corresponds to monomers and dimers of cytochrome oxidase. Solubilization of proteoliposomes and subsequent sucrose gradient centrifugation in cholate yielded one fast-migrating band for the untreated enzyme, but both a fast- and a slow-migrating band for the anion-exchange FPLC-treated enzyme, which was exclusively slow-migrating before reconstitution into liposomes. It is suggested that dimerisation of monomeric cytochrome oxidase may be favoured when the enzyme encounters a membranous milieu, and that the dimeric structure might be necessary for proton translocation.     

Interaction of tubulin with non-denaturing amphiphiles.

Soluble purified calf brain tubulin contains extensive and easily accessible regions capable of hydrophobic interactions. The binding of non-ionic and mild anionic detergents to this protein has been characterized by difference absorption spectroscopy and equilibrium gel chromatography with labelled ligands. Tubulin bound reversibly and co-operatively 0.42 +/- 0.05 g deoxycholate per g protein and bound octyl glucoside at a minimal stoichiometry of 0.26 g per g protein. Binding of deoxycholate and octyl glucoside perturbed the protein absorption, quenched the fluorescence, and produced a moderate change in the far u.v. circular dichroism of tubulin. These changes have been interpreted as the result of detergent binding near aromatic amino acids and the production of a structural change different from detergent-induced denaturation. Deoxycholate and octyl glucoside inhibited colchicine binding. Octyl glucoside and Triton X-100 inhibited the in vitro self-assembly of tubulin into microtubules, whereas small concentrations of deoxycholate were found to enhance microtubule formation.