A membrane-bound fluorescent probe to detect phospholipid vesicle-cell fusion

Summary Pyrenesulfonylphosphatidylethanolamine has been incorporated into sonicated phospholipid vesicles to provide a fluorescent signal from a membrane-bound probe whose spectrum is sensitive to the local concentration of dye molecules. When vesicle material was taken up by viable mouse splenocytes, the disappearance of the pyrene excimer fluorescence emission peak that accompanied dilution of the vesicle membrane lipid could be quantitated. One can thus measure, by a simple and rapid procedure, a new parameter which is related to the extent of vesicle-cell fusion and which is independent of the transfer of aqueous vesicle contents to the cell cytoplasm.Abbreviations used 6-CF 6-carboxyfluorescein - HBSS Hanks Balanced Salt Solution - DMPC dimyristoylphosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - EYL egg yolk lecithin - PE phosphatidylethanolamine - PEG polyethylene glycol - PLV phospholipid vesicle - PSPE pyrenesulfonylphosphatidylethanolamine     

Measurement of rotational motion in membranes using fluorescence recovery after photobleaching.

A method has been developed for the measurement of the rotational motion of membrane components. In this method fluorescent molecules whose transition dipole moments lie in a given direction are preferentially destroyed with a short intense burst of polarized laser radiation. The fluorescence intensity, excited with a low intensity observation beam of polarized laser radiation, changes with time as the remaining fluorescent molecules rotate. The feasibility of the method has been demonstrated in a study of the rotation of the fluorescent lipid probe, dil ([bis,-2-(N-octadecyl-3,3-dimethyl-1-benzo[b]pyrrole]-trimethincyanine iodide) incorporated into membranes composed of distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC) and 0.20 mol% cholesterol, below the main chain-melting transition temperatures of the phosphatidylcholines. Rotation times in the 0.6-800 s range were observed. The fluorescence recovery (or decay) curves are in satisfactory agreement with theoretical calculations.     

Affinity purification of lipid vesicles

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.     

Use of the fluorescent weak acid dansylglycine to measure transmembrane proton concentration gradients

The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient.     

Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.     

Kinetics of lipid rearrangements during poly(ethylene glycol)-mediated fusion of highly curved unilamellar vesicles.

In an effort to increase our understanding of the molecular rearrangements that occur during lipid bilayer fusion, we have used different fluorescent probes to characterize the lipid rearrangements associated with poly(ethylene glycol) (PEG)-mediated fusion of DOPC:DL(18:3)PC (85:15) small, unilamellar vesicles (SUVs). Unlike in our previous studies of fusion kinetics [Lee, J., and Lentz, B. R., Biochemistry 36, 6251-6259], these vesicles have mean diameters of 20 nm compared to 45 nm. Surprisingly, we found significant inter-vesicle lipid mixing at 5 wt % PEG, well below the PEG concentration required (17.5 wt %) for vesicles fusion. Lipid movement rate between bilayers (or inter-leaflet movement) increased abruptly at 10 wt % PEG, and the rate of lipid mixing increased thereafter with increasing amounts of PEG. The characteristic time of lipid mixing between outer leaflets (tau approximately equal to 24 s) was comparable to that observed at and above PEG concentrations needed to induce fusion (17.5 wt %) of either 20 or 45 nm vesicles. We also found that slower lipid mixing (tau approximately equal to 267 s) between fusing vesicles occurred on the same time scale or slightly faster than vesicle contents mixing (tau approximately equal to 351 s). In addition, our measurements showed that lipids redistributed across the bilayer on a time scale just slightly faster than pore formation (tau approximately equal to 217 s). This is the first demonstration of trans-bilayer movement of lipids during fusion. We also found that water was excluded from the bilayer (tau approximately equal to 475 s) during product maturation. These observations suggest that fusion in smaller vesicles (approximately 20 nm) proceeds via a multistep mechanism similar to that we reported for somewhat larger vesicles, except that two intermediates are no longer clearly resolved.     

Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes

Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.     

Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.     

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope.

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.     

Interaction of mucin with cholesterol enriched vesicles: role of mucin structural domains

We utilized fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to examine the role of gallbladder mucin (GBM) in promoting the aggregation and/or fusion of cholesterol enriched vesicles. By fluorescent labeling either the vesicle or the mucin, we could examine the change in vesicle size as well as changes in mucin's diffusion constant. Both FRAP and FCS show that GBM has a profound effect in inducing vesicles to aggregate/fuse, particularly after overnight incubation. GBM mucin domains (either protease digested or reduced GBM) are not as effective as native GBM. Intact GBM alone was able to shorten crystal appearance time and increase the number of crystals nucleated by polarized optical microscopy. In summary, our findings would suggest that both glycosylated and nonglycosylated domains of GBM are involved in early aggregation of cholesterol enriched vesicles but that this effect is reversible in the absence of nonglycosylated domains.     

A Monte Carlo study of giant vesicle morphologies in nonequilibrium environments

It is known that giant vesicles undergo dynamic morphological changes when exposed to a detergent. The solubilization process may take multiple pathways. In this work, we identify lipid vesicle shape dynamics before the solubilization of 1,2-dioleoyl-sn-glycero-3-phosphocholine giant vesicles with Triton X-100 (TR) detergent. The violent lipid vesicle dynamics was observed with laser confocal scanning microscopy and was qualitatively explained via a numerical simulation. A three-dimensional Monte Carlo scheme was constructed that emulated the nonequilibrium conditions at the beginning stages of solubilization, accounting for a gradual addition of TR detergent molecules into the lipid bilayers. We suggest that the main driving factor for morphology change in lipid vesicles is the associative tendency of the TR molecules, which induces spontaneous curvature of the detergent inclusions, an intrinsic consequence of their molecular shape. The majority of the observed lipid vesicle shapes in the experiments were found to correspond very well to the numerically calculated shapes in the phase space of possible solutions. The results give an insight into the early stages of lipid vesicle solubilization by amphiphilic molecules, which is nonequilibrium in nature and very difficult to study.     

The binding of a neutral aromatic molecule to a negatively-charged lipid membrane. II. Kinetics and mechanism

The kinetics of the binding of the fluorescence indicator N-phenyl naphthylamine to bilayer vesicles of C12-methyl-phosphatidic acid have been investigated by means of the temperature-jump relaxation technique utilizing fluorescence light detection. Single-exponential relaxation curves were observed, with time constants in the range 0.2-3 ms. The concentration dependence of the relaxation time yielded an apparent association rate constant (expressed in terms of monomeric phospholipid) of k(on) = 5 x 10(6) M(-1) s(-1) in aqueous solution at 25 degrees . The activation energy and viscosity dependence associated with the binding rate show that this process is actually diffusion-controlled. The theory of diffusion-controlled reactions then allows a determination of the average size of the bilayer vesicles and of the true rate constant for the association of the indicator molecules with the vesicles. Assuming spherical geometry for the vesicles, the values are: r(ves) = 190 A, which corresponds to 20000 lipid molecules per vesicle and k'(on) = 1 x 10(11) M(-1) s(-1) (25 degrees). The correctness of this size-determination was confirmed semi-quantitatively by electron microscopy. Since in fact a distribution of vesicle sizes must be present, a discussion is included of the relaxation function which the system is expected to take in the general case. Biological implications of diffusion control for the transport of non-polar substances and for lipid mixing are indicated.     

An efficient method for introducing defined lipids into the plasma membrane of mammalian cells

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).     

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.     

Phospholipid packing asymmetry in curved membranes detected by fluorescence spectroscopy

There are distinct differences in the molecular packing of phospholipid molecules in the inner and outer membrane monolayers of small lipid vesicles; a small radius of curvature imparts an asymmetry to the interface between these two monolayers. I have used an amphiphilic fluorescent probe, N-[5-(dimethylamino)naphthalenyl-1-sulfonyl]glycine (dansylglycine), to determine if this asymmetry in molecular packing leads to the existence of different environments for fluorescent probes resident in the membrane. Dansylglycine is highly sensitive to the dielectric constant of its environment, and the fluorescence signal from membrane-bound dye is distinct from that in the aqueous medium. When dansylglycine is first mixed with vesicles, it rapidly partitions into the outer monolayer; the subsequent movement of dye into the inner monolayer is much slower. Because of the time lag between the initial partitioning and the subsequent translocation, it is possible to measure the emission spectrum from membrane-bound dye before and after translocation, thus distinguishing the two potential environments for dansylglycine molecules. In the outer membrane monolayer of small dipalmitoylphosphatidylcholine vesicles, dye fluorescence emission is maximal at 530 nm, corresponding to a dielectric constant of 7 for the medium surrounding the fluorophore. For dye in the inner monolayer, emission is maximal at 519 nm, corresponding to a dielectric constant of 4.7. The results suggest that water molecules are excluded more efficiently from the dye binding sites of the inner membrane monolayer than they are from those of the outer monolayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral mobility in membranes as detected by fluorescence recovery after photobleaching.

The evaluation of lateral diffusion coefficients of membrane components by the technique of fluorescence recovery after photobleaching (FRAP) is often complicated by uncertainties in the values of the intensities F(O), immediately after bleaching, and F(infinity), after full recovery. These uncertainties arise from instrumental settling time immediately after bleaching and from cell, tissue, microscope, or laser beam movements at the long times required to measure F(infinity). We have developed a method for precise analysis of FRAP data that minimizes these problems. The method is based on the observation that a plot of the reciprocal function R(tau) = F(infinity)/[F(infinity)-F(tau)] is linear over a large time range when (a) the laser beam has a Gaussian profile, (b) recovery involves a single diffusion coefficient, and (c) there is no membrane flow. Moreover, the ratio of intercept to slope of the linear plot is equal to tau 1/2, the time required for the bleached fluorescence to rise to 50% of the full recovery value, F(infinity). The lateral diffusion coefficient D is related to tau 1/2 by tau 1/2 = beta w2/4D where beta is a defined parameter and w is the effective radius of the focused laser beam. These results are shown to indicate that the recovery of fluorescence F(tau) can be represented over a large range of percent bleach, and recovery time tau by the relatively simple expression F(tau) = [ F(o) + F(infinity) (tau/tau 1/2)]/[1 + tau/tau 1/2)]. FRAP data can therefore be easily evaluated by a nonlinear regression analysis with this equation or by a linear fit to the reciprocal function R(tau). It is shown that any error in F(infinity) can be easily detected in a plot of R(tau) vs. tau which deviates significantly from a straight line when F(infinity) is in error by as little as 5%. A scheme for evaluating D by linear analysis is presented. It is also shown that the linear reciprocal plot provides a simple method for detecting flow or multiple diffusion coefficients and for establishing conditions (data precision, differences in multiple diffusion coefficients, magnitude of flow rate compared to lateral diffusion) under which flow or multiple diffusion coefficients can be detected. These aspects are discussed in some detail.     

Asymmetric pore distribution and loss of membrane lipid in electroporated DOPC vesicles.

An externally applied electric field across vesicles leads to transient perforation of the membrane. The distribution and lifetime of these pores was examined using 1,2-di-oleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid vesicles using a standard fluorescent microscope. The vesicle membrane was stained with a fluorescent membrane dye, and upon field application, a single membrane pore as large as approximately 7 microm in diameter was observed at the vesicle membrane facing the negative electrode. At the anode-facing hemisphere, large and visible pores are seldom found, but formation of many small pores is implicated by the data. Analysis of pre- and post-field fluorescent vesicle images, as well as images from negatively stained electron micrographs, indicate that pore formation is associated with a partial loss of the phospholipid bilayer from the vesicle membrane. Up to approximately 14% of the membrane surface could be lost due to pore formation. Interestingly, despite a clear difference in the size distribution of the pores observed, the effective porous areas at both hemispheres was approximately equal. Ca(2+) influx measurements into perforated vesicles further showed that pores are essentially resealed within approximately 165 ms after the pulse. The pore distribution found in this study is in line with an earlier hypothesis (E. Tekle, R. D. Astumian, and P. B. Chock, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:11512--11516) of asymmetric pore distribution based on selective transport of various fluorescent markers across electroporated membranes.     

Insertion kinetics of a denatured alpha helical membrane protein into phospholipid bilayer vesicles

Membrane protein folding has suffered from a lack of detailed kinetic studies, particularly with regard to the insertion of denatured protein into lipid bilayers. We present a detailed in vitro kinetic study of the association of a denatured, transmembrane alpha helical protein with lipid vesicles. The mechanism of folding of Escherichia coli diacylglycerol kinase from a partially denatured state in urea has been investigated. The protein associates with lipid vesicles to give a protein, vesicle complex with an apparent association constant of 2 x 10(6) M(-1) s(-1). This association rate approaches the diffusion limit of the protein, vesicle reaction. The association of the protein with lipid vesicles is followed by a slower process occurring at observed rate of 0.031 s(-1), involving insertion into the bilayer and generation of a functional oligomer of diacylglycerol kinase. Protein aggregation competes with vesicle insertion. The urea-denatured protein monomers begin to aggregate as soon as the urea is diluted. This aggregation is faster than the association of the protein with vesicles so that most protein aggregates before it inserts into a vesicle. Increasing the vesicle concentration favours insertion of protein monomers, but at high vesicle concentrations monomers are primarily in separate vesicles and do not associate to form functional oligomers. Irreversible aggregation limits the yield of functional protein, while the data also suggest that lipid vesicles can reverse another aggregation reaction, leading to the recovery of correctly folded protein.     

Dopamine β-hydroxylase distribution in density gradients: Physiological and artefactual implications

Knowledge of the vesicular origin of circulating dopamine β-hydroxylase (DβH) is indispensable for any attempts to explain the parallelism or lack of it between circulating enzyme and catecholamines as they may relate to physiological stress, forms of hypertension, neurological disorders, and the response to pharmacological agents. The present study represents an effort to evaluate and to place in proper perspective data based on the DβH activity found in the region of the light vesicle peak of noradrenaline (NA), which is used as a quantitative measure of a population of small terminal vesicles. Distributions of vesicles and subvesicular components are compared with DβH and NA in sucrose-D₂O density gradients used to prepare relatively pure fractions of large dense cored vesicles (LDV) from bovine splenic nerve. Although NA in sedimentable particles of the light vesicle peak is likely to be a valid measure of a small vesicle population, the following is demonstrated: (1) A substantial fraction (25%–37%) of the total sedimentable DβH acitivity can be proven to distribute in the region of the light vesicle peak from a tissue with an insignificant small vesicle population. Based on studies of vesicles from sequential nerve segments, this enzyme activity probably corresponds to a population of “immature” LDV which are undergoing axoplasmic transport and have not synthesized their full complement of transmitter. (2) Physical lysis which depletes the matrix of LDV causes redistribution of DβH activity from the heavy vesicle peak into the region of the light vesicle peak. Analogously, DβH associated with exocytosed LDV and retrograde transport particles is also likely to contaminate the region of the light vesicle peak. (3) Based on available data, it can be calculated that each small dense cored vesicle could contain only 0.1–0.5 molecules of DβH and that a contamination of only 0.016% LDV can account for all of the DβH reported to occur in the light vesicle peak of normal rat vas deferens preparations.     

Hydrodynamic radii and lipid transfer in prostasome self-fusion.

The characteristics of human prostasomal vesicles have been investigated by three methods, namely, dynamic light scattering, transfer of a lipophylic fluorescent dye (R18), and electron microscope appearance. The vesicle preparations were stable for a long time and their diameters were in the range of 200 nm. The exposure to acidic pH values (about 5) increased both particle radii and the transfer of R18. The microscopic appearance was also affected by the pH value. We infer that these changes are due to a self-fusion of prostasome vesicles; this fusion is protein-dependent since various methods used by us and able to affect the protein component of membranes (boiling, extraction of lipid and liposome preparation, treatment with pronase) all abolished the effect seen at pH 5 on intact particles.