Cell-free translation in reversed micelles.

Cell-free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin-2 by the wheat germ translation system solubilized in Brij 96 (oleyl-poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell-free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2+ ions necessary for the functioning of the translation apparatus.     

Morphology of lipid micelles containing lysolecithin.

Mixtures of lysolecithin with various phospholipids were studied by electron microscopy using negative staining. Mixtures of dipalmitoyllecithin and lysolecithin produced disc-shaped structures which were stacked in aggregates with a 6.0--6.4 nm repeat. The disc were 10--50 nm in diameter. The disc-shaped structures were best observed in equimolar mixtures of dipalmitoyllecithin and lysolecithin. When dipalmitoyllecithin was replaced by dimyristoyllecithin, the structures were rather different from those observed in the system containing dipalmitoyllecithin; a cylindrical micellar phase was predominant. Equimolar mixtures of egg lecithin and lysolecithin formed the more usual smectic, concentric lamellae (liposomes) and elongated rod-like micelles which might be bimolecular fragments of spherules. The radius of the rod-like micelles was about 6 nm. Structures of rod-like micelles were observed more frequently in the samples after incubation at room temperature and then further incubation at 0 degrees C. Equimolar mixtures of didecanoyllecithin and lysolecithin produced large amounts of elongated rod-like micelles. Beef brain sphingoymyelin showed disc-shaped structures when mixed with lysolecithin. Incorporation of cholesterol into the mixtures of dipalmitoyllecithin and lysolecithin changed the morphological structure; the size of the disc became larger and eventually liposomes were formed with an increase of cholesterol content. The structures observed in mixtures of dipalmitoyllecithin or sphingomyelin and lysolecithin closely resembled those observed in complexes of apolipoprotein and lipid.     

Solubilization of ribosomes in reverse micelles.

Pig liver ribosomes have been solubilized in reverse micelles constituted by bis (2-ethyl hexyl) sodium sulfosuccinate (AOT) in isooctane and 3.6% water, v:v. The micellar ribosomal solutions are transparent, show no significant scattering and permit direct spectroscopic observation of the ribosomes to be made. Ultraviolet absorption and circular dichroic spectra have been recorded and indicate that the ribosomes maintain in the micellar environment their structural integrity. Some possible applications of these micellar systems are discussed.     

PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar structures at high molar ratios of trition to phosphalipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A2 can utilize as substrate. Spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidycholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that of pure Triton X-100 micelles. The T1 values for the phsopholipid in the mixed micelles are found to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as membrane models, but the lines are somewhat sharper suggesting the possibility of less anisotropic motion in the mixed micelles than in the vesicles.     

Dimerization and reactivation of triosephosphate isomerase in reverse micelles.

The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.     

Studies on Triton X-100 detergent micelles.

Triton X-100 micelle formation at 25 degrees C was studied by use of sedimentation equilibrium and fluorescence spectroscopic techniques. The apparent molecular weight of the major Triton X-100 micelle was found to be 81250, indicating a micelle number of 125. A micelle number of 121 was obtained with fluorescence titration experiments, which showed one molecule of 1-anilino-8-naphthalene sulfonate binding per micelle with an apparent association constant of 0.9 x 10(5) M. The fluorescent titration experiments also indicated the presence of another TX-100 binding species of variable size.     

The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.

The kinetics of light-induced electron transfer in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides were studied in the presence of the detergent lauryldimethylamine-N-oxide (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P+ reflects recombination from the reduced acceptor quinones, QA- or QB-. The secondary quinone, QB, which is loosely bound to the RC, determines the rate of this process. Electron transfer to QB slows down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau P approximately 1 s, whereas charge recombination in RCs lacking QB generates a fast phase with time tau AP approximately 0.1 s. The amount of quinone bound to RC micelles can be reduced by increasing the detergent concentration. The characteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (at high detergent concentration), is about 1.2-1.5 s, in sharp contrast to expectations from previous models, according to which the time of the slow component should approach the time of the fast component (about 0.1 s) when the quinone concentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics of electron transfer in isolated RCs with the following key features: 1) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from QB- to P+; 2) The exchange of quinone between the detergent "phase" and the QB binding site within the same RC micelle is much faster than electron transfer between QA- and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n) > or = 1, the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of QA). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau P, and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between QA and QB (LAB), and the association equilibrium constant of quinone binding at the QB site (KQ+). We found that LAB = 22 +/- 3 and KQ = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the QB binding site in detergent-solubilized RCs is difficult to achieve with hydrophobic quinones. This has important implications for the interpretation of apparent dependencies of QB function on environmental parameters (e.g. pH) and on mutational alterations. The model accounts for the effects of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significance for the study of quinone-binding membrane proteins in detergent solutions.     

Superoxide dismutase inhibits lipid peroxidation in micelles.

Superoxide dismutase (SOD) taken in minor concentrations (a few U/ml) displays a pronounced inhibiting effect on the chain oxidation of methyl linoleate and methyl linolenate (but not methyl oleate) induced by 2,2'-azobis(2-amidinopropan) dihydrochloride (AAPH) in micellar solutions of sodium dodecyl sulfate and Triton X-100 in phosphate buffer, pH 7.40, at 37.0 degrees C. The inhibition is evidently caused by purging the system from O(2)*(-). The latter suggests the formation of O(2)*(-) (HO(2)* in the course of peroxidation, most likely, via beta-decay of lipid peroxy radical (LO(2)*. Thermodynamic estimations verify a rather high probability of beta-decay of LO(2)* produced from polyunsaturated fatty acids by contrast to that produced from saturated and monoenic fatty acids. It is speculated that O(2)*(-) (HO(2)*, being an amphiphilic, reactive and highly mobile species, participates in intermicellar (interliposomal) transfer of free valence during lipid peroxidation in microheterogeneous systems.     

Energy transfer by chlorophyll beta in detergent micelles.

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyll beta-containing detergent micelles with Triton X-100 were studied in a concentration range of c equal to 0.4 muM-0.6mM chlorophyll beta and cd equal to 0.4-7.0 mM Triton X-100. The concentration-dependent depolarization obeys F?rster's theory of depolarization of fluorescence with a transfer distance parameter R0 equal to 43 plus or minus 2 A. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yield n/n0 equal to 1/[1+(c/c1/2)-2] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149-161). With increasing chlorophyll beta concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyll beta molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyll beta yields chlorophyll beta homotransfer may be about 3-26% of the homotransfer chlorophyll alpha yields chlorophyll-alpha depending on the ratio of their concentrations.     

Association of myelin basic protein with detergent micelles.

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being 3.58 +/- 0.12 and 2.30 +/- 0.15 for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively 1.34 +/- 0.10 for deoxycholate (at 0.12 ionic strength) and 4.0 +/- 0.5 for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strenght in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and deterent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single poly-peptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

Studies on adrenaline-induced lipolysis in artificial lipid micelles.

Lipid micelles were prepared by incubating a mixture of glycerides (triolein, diolein, and monoolein), and lecithin in Krebs-Ringer phosphate buffer at 37 degrees C for 30 min. It was found that adrenaline stimulated the release of free fatty acids in a lipolytic system consisting of the lipid micelles and adipose tissue lipase. Adrenaline did not increase the cyclic AMP content of the reaction mixture. Dibutyryl cyclic AMP, theophylline, and phospholipase C increased the rate of lipolysis in the system but cyclic AMP and phospholipase D did not.     

Targeted worm micelles

Giant and stable worm micelles formed from poly(ethylene glycol) (PEG)-based diblock copolymer amphiphiles have the potential advantage compared to smaller assemblies for delivery of a large quantity of hydrophobic drugs or dyes per carrier. Here we show that worm micelles can be targeted to cells with internalization and delivery of nontoxic dyes as well as cytotoxic drugs. Constituent copolymers are end-biotinylated to mediate high affinity binding of worm micelles to both avidin-bearing surfaces and biotin-specific receptors on smooth muscle cells. Pristine worm micelles, that lack biotin, show much less frequent and nonspecific point attachments to the same surfaces. Biotinylated worm micelles prove stable in aqueous solution for at least a month and also prove capable of loading, retaining, and delivering hydrophobic dyes and drugs. The results thus demonstrate the feasibility of targeted delivery by polymeric worm micelles.     

Interactions of colipase with bile salt micelles. 1. Ultracentrifugation studies.

A detailed investigation by ultracentrifugation of the colipase-taurodeoxycholate system showed the formation of well-defined mixed associations with a sedimentation coefficient of about 2.2S. The fact that these associations were only detectable above the critical micelle concentration of the salt indicated that micelles rather than monomers were bound to the cofactor. Two technical difficulties must be overcome before the weight of the associations could be measured with a reasonable accuracy. Firstly, the partial specific volume of the associations was determined using a digital microdensimeter and the interferometric system of the ultracentrifuge for concentration determinations. Secondly, due to the fact that micelle concentrations could not be equilibrated by dialysis, even after an extended period of time, an appropriate dilution of the ligand in the buffer compartment was necessary in order to compensate for its fixation by colipase in the solution. Then, the ionic strength dependence of the weight of the associations was found to vary in parallel with that of the micelles and to be in each case equal to the sum of the weights of one colipase molecule and one micelle. Therefore, colipase can be expected to contain a single high affinity site for bile salt micelle binding.     

Phospholipid-based reverse micelles

Physicochemical investigations on the aggregation of phospholipids (mainly phosphatidylcholines) in organic solvents are reviewed and compared with the aggregation behaviour of phospholipids in aqueous medium. In particular we review the data showing that phosphatidylcholines (lecithins) form reverse micellar structures in certain apolar solvents. In these systems not only low molecular weight compounds but also catalytically active enzymes and entire cells can be solubilized. In addition, highly viscous phosphatidylcholine gels can be obtained in organic solvents upon solubilizing a critical amount of water. Generally, phospholipid-based reverse micelles can be regarded as thermodynamically stable models for inverted micellar lipid structures possibly occurring in biological membranes.     

Interactions of colipase with bile salt micelles. 2. Study by dialysis and spectrophotometry.

The finding reported in the preceding paper that colipase is able to bind one sodium taurodeoxycholate micelle per molecule was confirmed by dialysis and spectrophotometry. Dialysis in the presence of labelled sodium taurodeoxycholate provided a direct qualitative proof of taurodeoxycholate binding to colipase. This binding was found to occur only above the critical micelle concentration. But, dialysis did not give any information about the composition of the associations, because equilibrium was not attained at the end of the assays. Addition of sodium taurodeoxycholate above the critical micelle concentration was also observed to induce a strong perturbation of the ultraviolet spectrum of one or several of the three tyrosines of colipase. The variation of the perturbation as a function of sodium taurodeoxycholate concentration was consistent with the binding of a single micelle to colipase. The dissociation constant calculated in "micelle molarity" was approximately 1 X 10(-4) M. The colipase-bile salt micelle association can fix one molecule of lipase to form a ternary complex which represents an interesting model of a protein-protein interaction mediated by an organized lipid structure. The ternary complex is probably also a model for lipase-substrate interactions in the presence of an amphipath.