Nonmonotonic trends in electrolyte-induced injury to rapidly cooled erythrocytes.

Rapid freeze-thaw injury to erythrocytes and erythrocyte ghosts has been shown to be strongly cation dependent. For the Group I ions this dependence is nonmonotonic in nature with injury increasing in the order Li+ less than Na+ less than Cs+ less than K+. Injury can be reduced by the inclusion in the freezing media of saccharide cryoprotectants or by the substitution with less injurious cations, e.g., Mg2+ or (CH3)4N+. In contrast to the situation observed with cations injury with anions follows Hofmeister lyotropic power series with injury increasing with decreasing hydrated ionic radius. Careful choice of electrolyte species allows injury to be reduced to levels comparable to that afforded by saccharide cryoprotectants. A possible mechanism for the nonmonotonic trends in injury observed with cations is considered.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

Acetylcholine receptor-controlled ion flux in electroplax membrane vesicles: Identification and characterization of membrane properties that affect ion flux measurements

Summary Several intrinsic properties of acetylcholine receptor-rich membrane vesicles prepared fromElectrophorus electricus, which need to be considered in measurements of receptor-mediated ion flux, have been identified. One of these properties is a slow exchange of inorganic ions in the vesicles. The slow exchange of ions is not related to the receptor-mediated flux of ions and accounts for 30–35% of the efflux observed. A method to separate this process from the receptor-controlled flux has been developed. It has also been shown, using a light-scattering method, that aggregation-disaggregation of the vesicles can interfere with the efflux measurements, and a method to overcome this problem has been developed. The difference in the amplitude of effluxes induced by saturating amounts of carbamylcholine and gramicidin has been investigated and has been shown not to be due to a receptor-controlled process; therefore, the amplitude difference does not need to be considered in understanding the receptor-controlled process.     

Vesicles of variable sizes produced by a rapid extrusion procedure

Previous studies from this laboratory have shown that large unilamellar vesicles can be efficiently produced by extrusion of multilamellar vesicles through polycarbonate filters with a pore size of 100 nm (Hope, M.J., Bally, M.B., Webb, G. and Cullis, P.R. (1985) Biochim. Biophys. Acta 812, 55-65). In this work it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar or plurilamellar vesicles by utilizing filters with pore sizes ranging from 30 to 400 nm. The unilamellarity and trapping efficiencies of these vesicles can be significantly enhanced by freezing and thawing the multilamellar vesicles prior to extrusion. This procedure is particularly applicable when very high lipid concentrations (400 mg/ml) are used, where extrusion of the frozen and thawed multilamellar vesicles through 100 and 400 nm filters results in trapping efficiencies of 56 and 80%, respectively. Freeze-fracture electron microscopy revealed that vesicles produced at these lipid concentrations exhibit size distributions and extent of multilamellar character comparable to systems produced at lower lipid levels. These results indicate that the freeze-thaw and extrusion process is the technique of choice for the production of vesicles of variable sizes and high trapping efficiency.     

Insufficiency of Copper Ion Homeostasis Causes Freeze-Thaw Injury of Yeast Cells as Revealed by Indirect Gene Expression Analysis

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.Yeast (Saccharomyces cerevisiae) cells are exposed to various environmental stresses such as freeze-thaw, high-temperature, osmotic, and air-drying stresses during commercial processes. Freeze-thaw stress is important in the bread-making process, because frozen dough baking has become a major technology (3). Frozen dough baking improves labor conditions for bakers and enables them to provide fresh baked goods for consumers (3). Because frozen dough baking involves freeze-thaw treatment, it exposes yeast cells to freeze-thaw stress, which leads to a significant decrease in the fermentation ability and viability of yeast cells (called “freeze-thaw injury”) (8). The freeze-thaw injury of yeast cells depends on many factors, including freezing periods, freezing temperature, and the physiology of yeast cells (2, 5, 16, 17). Clarification of the changes in the cell physiology of yeast cells caused by freeze-thaw stress is important, because bakers are eager to extend the shelf life of frozen dough. In this study, we attempted to determine the changes in yeast cell physiology due to freeze-thaw injury by indirect gene expression analysis, which is described below.Freezing subjects yeast cells to low temperature, ice crystal formation in the cells, and dehydration from the cells. This causes both physical damage to cellular components, such as the cell wall, membrane, and proteins, and formation of reactive oxygen species (ROS) (13, 15). Superoxide anions and free radicals are generated in yeast cells during the freeze-thaw process (18), and ROS generation during the thawing process is increased, depending on the freezing period (5, 18). Oxidative stress generated by freeze-thaw treatment enhances the damage to cellular components (9, 25). Because modulation of intracellular levels of ROS after freeze-thaw treatment is required to protect against toxicity, ROS scavenging systems such as glutathione, catalase, and superoxide dismutase (SOD) are believed to be important for freeze-thaw tolerance of yeast cells (1, 2, 17). In particular, copper/zinc SOD (Cu/Zn SOD), which plays a role in oxygen radical detoxification, is necessary to confer full tolerance to freeze-thaw injury (18). Heavy metal ions, such as iron ions and copper ions, are important transition metals for the detoxification of oxygen radicals in yeast cells (6).Although there have been several studies on the mechanisms of freeze-thaw injury (5, 17, 18, 24), the mechanisms are complex and have not yet been fully elucidated. We therefore examined freeze-thaw injury by indirect gene expression analysis, which was conducted during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. Indirect gene expression analysis may be advantageous for such an examination, because changes of gene expression may reflect the physiology of the freezing-state cell. We hypothesized that the genes involved in freeze-thaw injury may upregulate during postthaw incubation. To elucidate the physiological changes during freeze-thaw injury, we carried out indirect gene expression analysis using yeast cells after they had been frozen for different periods. The upregulated genes in the indirect gene expression analysis were extracted by clustering methods. We found that the genes involved in metal ion homeostasis were specifically upregulated. The importance of the genes extracted by the clustering was confirmed by phenotypic analysis using the deletion strains of the extracted genes. We also showed, by physiological analysis, that insufficiency of copper ion homeostasis causes freeze-thaw injury.     

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219- 221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.     

A loss in the plasma membrane ATPase activity and its recovery coincides with incipient freeze-thaw injury and postthaw recovery in onion bulb scale tissue

Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase.     

Autonomic innervation of the arteriovenous anastomoses in the dog tongue

Summary The innervation of the arteriovenous anastomoses in the dog tongue has been investigated. At the lightmicroscopic level, the vessels were found to be densely supplied with adrenergic and AChE-positive nerve plexuses and less densely with the quinacrine-binding nerve plexus. At the electron-microscopic level, at least two apparently different types of axon profiles were identified: 1) Small vesicle-containing axons, characterized by many small granular vesicles, variable numbers of small clear vesicles and large granular vesicles. Storage of endogenous amines and uptake of exogenous amines into most small granular vesicles and many large granular vesicles was demonstrated. These axons stained only lightly with reaction products for AChE activity and thus seemed to be adrenergic in nature. Some axons contained numerous large granular vesicles, whose cores occasionally stained with uranyl ions; this suggests a co-localization of ATP or peptides as neurotransmitters. 2) Small granular vesicle-free axons, containing small clear vesicles and large granular vesicles in variable ratio. Most cores of these large granular vesicles were heavily stained with uranyl ions. No storage or uptake of amine into the synaptic vesicles was detected. Some axons appeared to be typically cholinergic, some, typically non-adrenergic, noncholinergic, and the rest, intermediate between the two. All axons stained heavily with reaction products for AChE activity, suggesting their cholinergic nature.     

Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline

The relative effectiveness of two natural cryoprotectants, proline and trehalose, in preserving membrane structure and function during freezing was studied. Isolated vesicles of sarcoplasmic reticulum (SR) from lobster muscle (Homarus americanus) were employed to study changes in structure and function during rapid freeze-thaw conditions. Both proline and trehalose were shown to effectively preserve the structure (assessed with freeze fracture) and function (assessed by the ability of the membranes to transport calcium) in the frozen vesicles. As a first step toward determining the mechanism of cryoprotection by these compounds, we have investigated their effectiveness in inhibiting freezing induced fusion between phospholipid vesicles. Pamiltoyloleoyl-phosphatidylcholine: phosphatidylserine (85:15 mole ratio) small unilamellar vesicles (SUVs) were made incorporating one of the following fluorescent probes, and energy donor, cholesteryl anthracene-9-carboxylate, or an energy acceptor, nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine to investigate the amount of membrane mixing during rapid freeze-thaw cycles, and storage at -20 degrees C. Membrane mixing was measured as an energy transfer from donor to acceptor when donor vesicles and acceptor vesicles were mixed before a particular freezing treatment. Membrane mixing was correlated with structural changes in these membranes by freeze-fracture analysis. Both trehalose and proline were found to be more effective in preventing membrane mixing between SUVs than the standard protectants, glycerol and dimethylsulfoxide.     

Isolation and nitrogenase activity of vesicles from Frankia sp. strain EAN1pec.

Vesicles, specialized cell structures thought to be the site of nitrogen fixation in the actinorhizal bacteria, were isolated from Frankia sp. strain EAN1pec by using French pressure disruption of mycelia followed by differential and isopycnic gradient centrifugation. The isolated vesicles reduced acetylene when incubated anaerobically with Mg2+ ions, ATP, and dithionite. No nitrogenase activity was detected in the disrupted mycelial fractions. Vesicles permeabilized by freeze-thaw or detergents showed increased rates of acetylene reduction due to increased permeability of dithionite. The effect on nitrogenase activity of different ATP concentrations was the same in normal and permeabilized vesicles. The endogenous respiratory rate of vesicles was significantly lower than that of mycelia, and the respiration rate of vesicles did not increase following the addition of succinate. The low respiratory activity of vesicles and their apparent dependence on externally supplied ATP for acetylene reduction suggest that the energy and reducing power for nitrogen fixation may be supplied from the mycelia to which they are attached.     

Control of Pro-Oxidant Activity of Cupric Ions by Entrapment in Unilamellar Lipid Vesicles

As a demonstration of a potential means of delivering and controlling the biochemical and biological activity of metal ions, cupric ions have been trapped in unilamellar phospholipid vesicles. The activity of these cupric ion-containing vesicles as catalysts of the autoxidation of ascorbate and epinephrine has been investigated. A marked increase in autoxidation rate was observed on release of the cupric ion on addition of detergent. When an azobenzene-containing photochromic lipid was incorporated in the bilayer membrane of the vesicles, the release of cupric ions could be initiated by irradiation with ultraviolet light. In the dark, these vesicles remained stable for at least several weeks. Photo-controlled release of liposomally-entrapped species might find application in areas similar to those where 'caged' reagents are presently used.     

Electrostatic forces control the penetration of membranes by charged solutes

Using fluorescent, anionic dyes such as carboxyfluorescein as model solutes, it is shown that the forces allowing such solutes to be retained within sealed lipid vesicles, against a large concentration gradient, can be primarily electrostatic in nature. At temperatures distant from that of the ordered-fluid lipid phase transition a small number of the anionic dye molecules trapped within lipid vesicles are capable of traversing the lipid bilayer and establishing an electrical diffusion potential across the membrane. Further solute movement can then only occur with the concomitant permeation of ions which restore electrical balance. A significant flux of dye can be triggered by (a) increasing the permeability of the membrane to ions (for example by the addition of ionophores such as gramicidin, or by allowing the lipid to approach a phase transition) or by (b) adding lipophilic counterions such as tetraphenylborate or dinitrophenol to the system.     

Electrostatic forces control the penetration of membranes by charged solutes

Using fluorescent, anionic dyes such as carboxyfluorescein as model solutes, it is shown that the forces allowing such solutes to be retained within sealed lipid vesicles, against a large concentration gradient, can be primarily electrostatic in nature. At temperatures distant from that of the ordered-fluid lipid phase transition a small number of the anionic dye molecules trapped within lipid vesicles are capable of traversing the lipid bilayer and establishing an electrical diffusion potential across the membrane. Further solute movement can then only occur with the concomitant permeation of ions which restore electrical balance. A significant flux of dye can be triggered by (a) increasing the permeability of the membrane to ions (for example by the addition of ionophores such as gramicidin, or by allowing the lipid to approach a phase transition) or by (b) adding lipophilic counterions such as tetraphenylborate or dinitrophenol to the system.     

Preparation and some properties of giant liposomes and proteoliposomes

Optimal conditions for formation of giant liposomes and proteoliposomes were investigated. A suspension of small unilamellar vesicles made of various phospholipids in a buffer of 0-3 M KCl, 0.1 mM EDTA, and 20 mM MOPS (pH 7.0) was subjected to a freeze-thaw treatment. Giant multilamellar liposomes of diameter ranging from 10 to 60 microns were found to form from phospholipid mixtures containing phosphatidylethanolamine as a major component and phosphatidylserine as a minor component. The concentration of KCl optimal for the giant vesicle formation was 30-500 mM. By applying a patch-pipette to a giant liposome, suitable conditions for obtaining a high-resistance (giga-ohm) seal were sought. It was found that use of a patch-pipette of relatively small tip diameter (less than 1 micron), the presence of divalent metal cations in the suspension medium and inflation of vesicles in a hypotonic solution facilitated giga-seal formation. In a suspension of asolectin (soybean phospholipid) vesicles which had been subjected to the freeze-thaw treatment, giant unilamellar vesicles were found. They could be held on the tip of a suction pipette and impaled with a microelectrode filled with an EGTA solution. Small unilamellar proteoliposomes were prepared by the cholate-dialysis method from asolectin and sarcoplasmic reticulum vesicles, and were subjected to a freeze-thaw cycle. When the ratio of exogenous phospholipid to protein was larger than 10, giant multilamellar vesicles were formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance

 It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band ³¹P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters ≤30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From ³¹P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles. Received: 2 September 1999 / Revised version: 27 February 2000 / Accepted: 27 February 2000     

Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures

Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to −3.9 C resulted in 80% lysis of the protoplasts. At −3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl ≥ Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.     

Effects of solute concentration on the entrapment of solutes in phospholipid vesicles prepared by freeze-thaw extrusion.

Phospholipid vesicles prepared by the freeze-thaw extrusion method contain internal solute concentrations which are much higher than the external values (entrapment ratios much greater than 1). This concentrating effect is a complex function of the total impermeant solute concentration in the medium used to prepare vesicles, the presence or absence of permeant solutes in the medium and the apparent competitive binding interactions between solutes and phospholipid. Increases in water phase solute concentration during freezing are thought to underlie the concentrating phenomenon, while osmotic pressure driven lysis of vesicles during thawing appears to limit its magnitude. By judicious selection of solute concentration and physical properties, further increases in the entrapment ratio should be obtainable, improving the usefulness of these vesicles as drug delivery vesicles and experimental systems.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes.

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca2+-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca2+-influx. With purified lumisomes it has been shown that Ca2+-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with 22Na and 42D indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Ultracytochemistry of the synaptic ribbons in the rat pineal organ

Summary The synaptic complexes of the rat pinealocytes are neither cholinergic nor adrenergic. In the synaptic vesicles, a neurotransmitter carrier substance of lipid nature reacting with OsO₄-Zn I₂ mixture (similar to that present in both cholinergic and adrenergic vesicles) was not found.In addition, there were no indications of glucose-6-phosphatase or thiamine-pyrophosphatase activity in the synaptic vesicles. Thus, it appears that the synaptic vesicles do not originate from the rough or smooth endoplasmic reticulum.The synaptic ribbons do not contain carbohydrates, are of protein nature and possess some chemical resemblance to microtubules and microtubular bouquets.Appropriate ultracytochemical reactions have not shown detectable quantities of sodium and calcium ions in pinealocyte synaptic complexes.Grateful acknowledgment is made to Mr. P.-A. Milliquet for technical assistance and to Dr. T. Jalanti (C.M.E., Lausanne) for his help in the use of the X-ray microanalyser.Dedicated to Professor Dr. med. G. Töndury on the occasion of his 70th birthday.     

Reconstitution of ciliary membranes containing tubulin

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.