On the size-dependent disintegration of small unilamellar phosphatidylcholine vesicles in rat plasma. Evidence of complete loss of vesicle structure.

The destruction of small unilamellar egg phosphatidylcholine vesicles in rat plasma was monitored by measuring release of encapsulated 125I-poly(vinylpyrrolidone) or carboxyfluorescein and by determining transfer of radiolabelled phosphatidylcholine to plasma lipoproteins by means of gel filtration. The susceptibility of the vesicles to the destructive action of plasma increased with decreasing vesicle size, as observed by incubating plasma with individual fractions constituting the small-vesicle peak on Sepharose CL-2B. This results in selective destruction of small vesicles when heterogeneous vesicle populations are incubated with plasma. Samples of homogeneous vesicle populations were incubated with a wide range of plasma concentrations, which resulted in extents of solute and phospholipid release ranging from 10 to 90%. When the extents of solute release were plotted against the extents of lipid release a linear, virtually 1:1, relationship was found, for both carboxyfluorescein and poly(vinylpyrrolidone) as the solute. This suggests that the release of solutes from small unilamellar phosphatidylcholine vesicles as a result of their interaction with plasma (lipo)proteins involves the total destruction of a fraction of the vesicles, the magnitude of which is determined by the vesicle: plasma ratio. Our results argue against a previously presented view suggesting that the interaction between such vesicles and plasma results in the formation of pores through which encapsulated solutes diffuse at Mr-dependent rates [Kirby & Gregoriadis (1981) Biochem. J. 199, 251-254]. The discrepancies between the two studies in observations as well as in interpretation are discussed.     

A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection

A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.     

Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.     

Enzymatic synthesis of polymers containing nicotinamide mononucleotide.

Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.     

Entrapment of proteins in phosphatidylcholine vesicles.

The trapping efficiency of globular proteins in four different types of phosphatidylcholine vesicles was systematically studied. Vesicles were generated in a mixture of 125I-labeled proteins of various molecular weights. The trapped proteins were separated from untrapped proteins by gel filtration and ultrafiltration and subsequently analyzed by gel electrophoresis and autoradiography. Entrapment of proteins was demonstrated by their resistance to trypsin digestion. The relative amount of each entrapped protein species was then compared to that of the original protein solution. In multilamellar vesicles and large unilamellar vesicles, proteins of molecular weight up to 97 000 had the same trapping efficiency as sucrose. In small unilamellar vesicles generated by either sonication or ethanol injection, however, the relative trapping efficiency of protein decreased progressively as the molecular weight of the protein became greater. For example, the trapping efficiency of alpha-amylase (Mr 97 000) was only half of that for sucrose. The apparent decrease in trapping efficiency with the protein's molecular weight in small unilamellar vesicles canbe accounted for by the combination of the bound water layer at the vesicle's internal surface and the steric hindrance when protein is captured during vesicle formation.     

Phase transitions of phospholipid single-wall vesicles and multilayers. Measurement by vibrational raman spectroscopic frequency differences

Raman spectroscopic frequency differences between selected carbon-carbon stretching modes of lipid hydrocarbon chains were determined as a function of temperature for use in monitoring lipid phase transition behavior and acyl chain disorder in both multilamellar and single-wall vesicles. Transition temperatues detected by this procedure for pure dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine multilayers were observed at $\text{39±1 °}\text{C}$ and $\text{23±1 °}\text{C}$, respectively. Although the phase transition for unilamellar vesicles of dipalmitoyl phosphatidylcholine occurred at nearly the same temperature as the multilayers, the crystal-liquid crystalline transition for the single-shell vesicles appeared to span a slightly broader temperature range, a characteristic consistent with irregularities in the packing arrangement of the hydrocarbon chains. Within the precision of the Raman spectroscopic method, however, the temperature behavior of both the multilamellar and the unilamellar dimyristoyl phosphatidylcholine assemblies appeared nearly identical. The temperature profile for the Raman frequency differences of an excess water sonicate of 25 mol percent cholesterol in dipalmitoyl phosphatidylcholine served as an example of the effect upon lipid phase transition characteristics of a bilayer component intercalated between the acyl chains. For this particular cholesterol-lipid system the phase transition was broadened over a 30 °C temperature range, in contrast to the narrow 5?4 °C range observed for pure multilayer and single-shell vesicle particles.     

Separation of oligo(adenosine diphosphate ribose) fractions with various chain lengths and terminal structures.

Oligo(adenosine diphosphate ribose) preparations with chain lengths of 3 to 10 adenosine diphosphate ribose units were fractionated according to their chain lengths and their terminal structures by hydroxyapatite column chromatography and then polyacrylamide gel electrophoresis. The peak fractions from the hydroxyapatite column were each separated into two distinct subfractions by gel electrophoresis. The two subfractions were found to differ in chain length and terminal structure. A linear correlation was observed between the mobility and the logarithm of the chain length of oligo(adenosine diphosphate ribose) on gel electrophoresis, irrespective of the terminal structure.     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.     

A simple method for elimination of RNAase contamination from DNAase preparations

RNAase which usually contaminates commercial pancreatic DNAase preparations can be removed by affinity chromatography on agarose-coupled anti-RNAase antibodies. RNA treated with purified DNAase can be re-isolated intact, as determined by polyacrylamide gel electrophoresis under denaturing conditions. This method might be applicable to purification of other preparations which are used in RNA research, such as PNPase (polynucleotide phosphorylase) and specific antibodies for polysome immunoprecipitation. The non-specific binding of DNAase in our system is less than 5% and the loss of specific activity of DNAase I is less than 1%.     

Fusogenic activity of δ-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin δ-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of δ-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Fusogenic activity of delta-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin delta-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of delta-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.     

The turnover number for band 3-mediated sulfate transport in phosphatidylcholine bilayers

The anion transport system of the human erythrocyte membrane was reconstituted in unilamellar phosphatidylcholine vesicles, and a vesicle subpopulation of a narrow size distribution was isolated from the sample by gel filtration. In this subpopulation, the turnover number of the transport protein (the band 3 protein) for sulfate transport was determined. It was found that, in the reconstituted system, the protein transports sulfate 5-10 times faster than in the human erythrocyte membrane.     

The interaction of saccharides with lipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying

The fusion of small unilamellar vesicles of phosphatidylcholines during freeze-thawing and freeze-drying/rehydration, and the suppression of fusion under these conditions by various saccharides, was investigated by gel filtration on Sepharose 4B, quasielastic light scattering, high-resolution 1H-NMR, ESR spin labeling, and differential scanning calorimetry. Freeze-thawing and freeze-drying of aqueous small unilamellar vesicle suspensions in the presence of sufficient sucrose had no significant effect on the average size and size distribution of small unilamellar vesicles. In the presence of sucrose the structural integrity and the permeability properties of the phosphatidylcholine bilayers were retained during freeze-thawing and freeze-drying. A comparison of the stabilizing effect of sucrose with that of trehalose and glucose showed that the stabilization is not sugar-specific but is a general property of saccharides. The fraction of small unilamellar vesicles recovered after freeze-thawing depended on the saccharide/phosphatidylcholine molar ratio. The mechanism of the cryoprotective effect involves binding of the sugar to the phospholipid polar group, probably through hydrogen bonding.     

Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions.     

Curvature elasticity and refolding of OmpA in large unilamellar vesicles

The stability of OmpA in large unilamellar vesicles of dilauroyl phosphatidylcholine was studied using different concentrations of urea. The effective energy of unfolding, as determined from refolding experiments, is greater than that for small sonicated unilamellar vesicles by an amount that is compatible with estimates of the elastic energy of highly curved vesicles. The on-rate for refolding and insertion is slower for large unilamellar vesicles than for small unilamellar vesicles, which indicates a contribution of vesicle strain also to the free energy of the transition state.     

Interaction of cytochrome c with liposomes: covalent labeling of externally bound protein by the fluorescent probe, azidonaphthalenedisulfonic acid, enclosed in the inner aqueous compartment of unilamellar vesicles

The photoreactive fluorescent probe, 3-azidonaphthalene-2,7-disulfonic acid (ANDS) was encapsulated in the inner aqueous compartment of small unilamellar liposomes, prepared from egg phosphatidylcholine (PC) +/- 20 mol% dihexadecylphosphate (DHP). After adding cytochrome c externally to a suspension of these vesicles, the probe was activated by ultraviolet irradiation, and the protein was separated from the lipids. When negatively charged (egg PC/DHP) vesicles at low ionic strength were used, which form an electrostatic complex with cytochrome c, the protein was labeled by ANDS. This process depended on irradiation time, and was inhibited by increasing the ionic strength of the medium. Labeling was not observed with isoelectric (egg PC) vesicles. These observations suggest that electrostatic binding of cytochrome c to the bilayer is accompanied by intramembrane penetration to such a depth that the protein can communicate with the inner membrane-water interface.     

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (T_c) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [¹⁴C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T_c of the lipid and increases with temperature above the T_c. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.