Reconstitution Medium Research Articles

Assessment of automated off-line solid-phase extraction LC-MS/MS to monitor EPA priority endocrine disruptors in tap water, surface water, and wastewater.

EPA method 539.1 recently introduced an expanded list of priority endocrine-disrupting compounds (EDCs), some of which were also included in the Unregulated Contaminant Monitoring Rule 3 (UCMR3). Though standardized methods are available for drinking water, analysis of steroid hormones and bisphenol A (BPA) at the ultra-trace level remains challenging. This study set out to evaluate the suitability of automated off-line solid-phase extraction (SPE) liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the determination of EPA-priority EDCs in environmental water matrixes (tap water, surface water, and wastewater influents and effluents). The target molecules included 14 steroid hormones (altrenogest, androstenedione, equilenin, equilin, α-estradiol, β-estradiol, estriol, estrone, ethinylestradiol, levonorgestrel, medroxyprogesterone, norethindrone, progesterone, testosterone) and BPA. Factors that may influence the analytical performance were assessed. This involved, for instance, testing combinations of SPE materials from different brands and protocol variations. Several materials presented absolute extraction efficiencies in acceptable ranges. Initial sample pH, nature of reconstitution medium, and mobile phase salt concentration were among the potential factors affecting analyte signal. Storage conditions (different preservative agents) possibly exerted the strongest influence, in agreement with the literature. Limits of detection were in the range of 0.03-0.5ng/L in drinking water, 0.1-0.5ng/L in surface water, and 0.16-1ng/L in wastewater. Method validation also involved testing linearity, accuracy, and precision in reagent water and matrix-matched extracted calibrants. The method was applied to field-collected water samples in Eastern Canada. Summed EDC concentrations remained low in tap water (<LOQ-0.92ng/L), while higher detection frequencies and contamination levels were reported in riverine surface waters (2.6-37ng/L) and municipal wastewaters (10-424ng/L).

Effects of waterborne luteinizing hormone and follicle-stimulating hormone on reproduction of the rotifer Brachionus calyciflorus (Monogononta: Brachionidae)

This study used freshwater rotifers to evaluate the effects of two endocrine disrupting compounds (EDCs), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which can be considered emergent contaminants in urban and rural wastewater and are of ecotoxicological importance. LH stimulates the synthesis of testosterone, whereas FSH promotes the maturation of follicles and sperm in vertebrates and invertebrates. However, in rotifers, there are no reports of the effects of chronic exposure to these hormones when added to reconstituted culture medium, as a way to study potential adverse effects that might occur in the environment. Therefore, we studied the reproductive effects of the rotifer Brachionus calyciflorus Pallas 1766 using a 4-day reproductive assay. Our results indicate that LH has a significant effect in increasing the production of females, males, and cysts, while FSH had no significant effect compared to control treatment. Additionally, our results indicate that LH exposure resulted in 0.33% of organisms being deformed, whereas FSH exposure resulted in 1.09% of organisms being deformed. Deformations included: (a) abnormal growth of lorica, (b) joined foot-head, (c) deformed anterior spine, and (d) deformed parthenogenetic eggs. The organisms with LH-induced deformations did not reproduce and only lived 48 h after 4 days of exposure, while those with FSH-induced deformities survived 15 days and produced 105 cysts with a hatching percentage of 58.10%. Our goal was to contribute to the knowledge of endocrine systems and endocrine hormones of rotifers, to explain the potential mechanism of endocrine disruption that results in adverse effects in freshwater rotifers.

Inappropriateness of medication use and associated health risks: A cross-sectional study from Pakistan

Purpose : To evaluate the patterns of medication use and medication appropriateness among people in different regions of Pakistan. Methods : A cross-sectional study involving 410 individuals in four provinces of Pakistan (Punjab, Sindh, Baluchistan and Khyber Pakhtunkhwa) as well as the capital city, Islamabad, via a convenient sampling method. The questionnaire used comprised three major sections: (a) medication use patterns of the population, (b) medication storage and wastage practices, and (c) development of secondary disease after medication use. The questionnaires were completed under the supervision of pharmacists trained for the purpose. Results : The majority of the study population preferred bottle caps for taking oral liquid medicines (83.9 %), and the proportion using bottle caps was highest in Punjab (55.2 %) followed by Sindh (21.8 %). A significant proportion of the population consider the use of very hot water as a reconstitution medium for suspensions (36.3 %). In many cases, suspensions were constituted by mere estimation of the vehicle used (56.1 % from Punjab, 18.9 % from Sindh, 16.1 % from KPK, 7.7 % from Baluchistan, and 1.1 % from Islamabad). Significant differences were observed in the handling of leftover drugs among respondent populations, as well as the overall diverse modes of disposal recorded (43.9 %). Doctors (46.6 %) and relatives were the main sources of instructions on medicine usage (37.6 %). A disinclination to purchase a full course of medicine was observed. Knowledge of medicine storage and measuring tools for oral liquids showed striking peculiarities among the various study populations. Conclusion : Inappropriate medication use among the general population of Pakistan requires educational interventions and awareness programs to promote rational drug use in the society. Keywords : Inappropriate, medication, population, healthcare, use

An Impedance-Based Method to Determine Reconstitution Time for Freeze-Dried Pharmaceuticals

The reconstitution of freeze-dried products is usually determined by visual inspection with the naked eye. This can inevitably lead to significant variability in the ability to detect complete reconstitution of the dried solid. It was thus the goal of our study to assess an automated method to monitor reconstitution of a freeze-dried protein drug product in its primary packaging. A newly developed measuring device was used to measure impedance. This was achieved by detecting minor changes in impedance of the reconstitution medium, which occurred because of solid material dissolving during the dissolution process. This measurement system was capable of consistently detecting the dissolution of the last visible residues of freeze-dried lyophilisates. The endpoint of reconstitution was defined at an impedance change of less than 1 Ω for at least 7 s. Finally, we compared reconstitution times determined by the automated impedance method with results obtained by a visual method. In contrast to human operators, the new method delivered both accurate and precise results. Besides detection of the reconstitution endpoint, the impedance method and apparatus can monitor reconstitution endpoints as well as reconstitution kinetics. This standardized method can therefore advantageously be used for the determination of the reconstitution endpoint.

Modulation of Plasma Membrane Ca2+-ATPase by Neutral Phospholipids: EFFECT OF THE MICELLE-VESICLE TRANSITION AND THE BILAYER THICKNESS

The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca(2+) pump (PMCA). We found that Ca(2+)-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca(2+)-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA.

Characterization of the Physical Stability of a Lyophilized IgG1 mAb after Accelerated Shipping-Like Stress

Upon exposure to shaking stress, an IgG1 mAb formulation in both the liquid and lyophilized state formed subvisible particles. Because freeze-drying was expected to minimize protein physical instability under these conditions, the extent and nature of aggregate formation in the lyophilized preparation were examined using a variety of particle characterization techniques. The effects of formulation variables such as residual moisture content, reconstitution rate, and reconstitution medium were also examined. Upon reconstitution of shake-stressed lyophilized mAb, differences in protein particle size and number were observed by microflow digital imaging, with the reconstitution medium having the largest impact. Shake stress had minor effects on the structure of protein within the particles as shown by SDS-PAGE and FTIR analysis. The lyophilized mAb was shake stressed to different extents and stored for 3 months at different temperatures. Both extent of cake collapse and storage temperature affected the physical stability of the shake-stressed lyophilized mAb upon subsequent storage. These findings demonstrate that physical degradation upon shaking of a lyophilized IgG1 mAb formulation includes not only cake breakage, but also results in an increase in subvisible particles and turbidity upon reconstitution. The shake-induced cake breakage of the lyophilized IgG1 mAb formulation also resulted in decreased physical stability upon storage.

Paclitaxel/β-cyclodextrin complexes for hyperthermic peritoneal perfusion – Formulation and stability

Due to its low aqueous solubility paclitaxel is currently formulated in a Cremophor EL ®/ethanol mixture. However, the vehicle of this formulation causes several side-effects. Our objective was to formulate a tensioactive-free and solvent-free paclitaxel solution, which can be used for a hyperthermic intraperitoneal chemoperfusion procedure (HIPEC). The potential of chemically modified β-cyclodextrins to form complexes with paclitaxel was investigated as a means to increase the aqueous solubility of paclitaxel. Methylated β-CDs (randomly methylated and 2,6-dimethylated) showed the best ability to solubilise paclitaxel compared to sulfobutyl-ether- and hydroxypropyl-β-CD. The minimal ratio of paclitaxel versus randomly methylated-β-cyclodextrin (RAME-β-CD) yielding 100% inclusion efficiency was 1/20 (mol/mol). Paclitaxel/RAME-β-CD inclusion complexes prepared via freeze drying were stable for at least 6 months when stored at 4 °C. A 5 mg/ml paclitaxel solution was formulated using paclitaxel/RAME-β-CD-complexes. Upon dilution of these solutions, no precipitation was seen. After 24 h storage at room temperature or 2 h at HIPEC conditions (41.5 °C) the 1/40 (mol/mol) ratio showed the highest stability at paclitaxel concentrations of 0.1 and 0.5 mg/ml. When hydroxypropyl methylcellulose (HPMC) was added to the reconstitution medium, the stability significantly increased, offering the opportunity to reduce the amount of RAME-β-CDs in the formulation.

Freeze-drying of proteins from a sucrose-glycine excipient system: Effect of formulation composition on the initial recovery of protein activity

The purpose of this study was to investigate the effect of sucrose-glycine excipient systems on the stability of selected model proteins during lyophilization. Recovery of protein activity after freeze-drying was examined for the model proteins lactate dehydrogenase and glucose 6-phosphate dehydrogenase in a sucrose-glycine-based excipient system in which the formulation composition was systematically varied. In a sucrose-only excipient system, activity recovery of both model proteins is about 80% and is independent of sucrose concentration over a range from 1 to 40 mg/mL. When both sucrose and glycine are used and the ratio of the 2 excipients is varied, however, activity recovery decreases in a pattern that is consistent with the inhibition of activity recovery by glycine crystals, despite the presence of an adequate amount of sucrose to afford protection. Annealing of sucrose-glycine formulations causes a small but significant decrease in activity recovery relative to unannealed controls, whereas no annealing effect is observed with sucrose-only formulations. Addition of 0.01% polysorbate 80 to the formulation resulted in complete recovery of activity, irrespective of the sucrose-glycine ratio or annealing. Addition of the same concentration of polysorbate 80 to the reconstitution medium caused an increase in activity recovery for each formulation, but the overall pattern remained unchanged. The data are consistent with an interfacial model for lyophilization-associated loss of protein activity involving denaturation at a solid/freeze-concentrate interface.

The improved dissolution and prevention of ampoule breakage attained by the introduction of pretreatment into the production process of the lyophilized formulation of recombinant human Interleukin-11 (rhIL-11)

Lyophilized protein formulations sometimes pose problems such as the formation of a cloudy solution upon reconstitution. Ampoule or vial breakage can also occur during the production processes of lyophilized pharmaceutical products. Various efforts have been made to overcome those difficult problems. In this study, we introduce a particular temperature program into the production process of a recombinant human Interleukin-11 (rhIL-11) lyophilized formulation containing sodium phosphates (Na 2HPO 4/NaH 2PO 4, pH 7.0) and glycine in an attempt to improve its dissolution properties and to prevent ampoule breakage from occurring. The formulation was pretreated by nucleating ice and maintaining the solution overnight at a temperature of −6 °C. The solution was then completely frozen at a lower temperature. This pretreatment proved successful in not only producing a lyophilized cake which readily disintegrated and dissolved in the reconstitution media, but also prevented ampoule breakage from occurring during the production processes. In contrast, a lyophilized cake produced without the pretreatment created a cloudy solution particularly when reconstituted using water for injection contaminated with aluminum (Al 3+), although the solution became transparent within 20–30 min. The pretreatment induced the crystallization of sodium dibasic phosphate (Na 2HPO 4) in the freeze-concentrate whereas direct freezing without the pretreatment did not crystallize the salt. Thermal analyses (DSC and TMA) showed that amorphous sodium dibasic phosphate in the freeze-concentrate became crystallized upon heating, accompanied by an increase in volume, which probably caused the ampoule breakage that occurred without the pretreatment. Although power X-ray diffraction (PXRD) experiments suggested that, with or without the pretreatment, glycine assumed the β-form and sodium phosphate stayed amorphous in the final products, an electrostatic interaction between dibasic phosphate anions and rhIL-11, a highly cationic protein, would only exist in the lyophilized cake produced without the pretreatment. This interaction is highly likely because aluminum facilitates the formation of a cloudy solution upon reconstitution possibly by using the divalent anions which effectively reduce electrostatic repulsions between aluminum and the protein to form an aggregate structure that is not readily soluble. The pretreatment would circumvent the interaction by crystallizing the sodium salt before freezing creating a relatively soluble lyophilized cake that is much less sensitive to aluminum.

Effect of excipients on the stability and aerosol performance of nebulized aviscumine.

Pulmonary delivery is an attractive alternative route to deliver protein drugs that are currently delivered by injection. Inhalation therapy via nebulizers is a well accepted way for pulmonary application of proteins considering the formulation difficulties of MDIs or DPIs. This research presents the effect of variable excipients on the stability and aerosol performance of freeze-dried aviscumine after reconstitution and nebulization. Aviscumine formulations containing 100 mmol/L Tris buffer, 0.1% (w/v) Polysorbate 80, 0.01% (w/v) Na(2)-EDTA and 8% (w/v) Hydroxyethyl starch have been lyophilized and reconstituted with a buffered isotonic solution pH 8. The aviscumine activity was determined by a binding assay directly after reconstitution and after nebulization with a PariBoy air-jet nebulizer, a Multisonic and a Systam ultrasonic nebulizer. The stabilization of aviscumine by the addition of variable buffer salts to the reconstitution medium, such as 50, 100, and 200 mmol/L Tris buffer, 20 and 100 mmol/L phosphate buffer, and 20 and 100 mmol/L Tricine buffer, was studied. About 50% of aviscumine activity was lost after 20 min nebulization time without any additives. Nevertheless, higher buffer concentrations confer greater stability. About 70% of the aviscumine activity could be retained by the addition 0.03% octanoyl-N-methylglucamide and 100 mmol/L Tricine to the reconstitution medium.

Effect of cryoprotectants on the stability and aerosol performance of nebulized aviscumine, a 57-kDa protein

Nebulization of aqueous drug solutions is a suitable delivery system for pulmonary application of proteins because it can easily produce droplets small enough to reach the alveolar region. However, proteins are sensitive to nebulization. Therefore, stabilizers need to be added which on the other hand influence the aerosol performance, such as average droplet size or mass output. This research presents the effect of various cryoprotectants such as Na-polyphosphate, CaCl 2×6H 2O and MgSO 4×7H 2O on the stability and aerosol performance of freeze-dried aviscumine after reconstitution and nebulization using three different nebulizers. Formulations containing Tris-buffer, polysorbate 80, Na 2-EDTA and HES450 were lyophilized and reconstituted with a buffered isotonic solution containing 100 mmol/l Tricine-buffer pH 8, 0.03% (w/v) octanoyl- N-methylglucamide, 150 mmol/l NaCl and a cryoprotectant. The aviscumine activity was determined by a binding assay. The addition of 0.2% Na-polyphosphate to the reconstitution medium led to retention of approx. 73% of the aviscumine activity after 20 min nebulization with the Systam ® ultrasonic nebulizer. It has been observed that 84 and 72% of the activity were retained by the addition of 10 mmol/l CaCl 2×6H 2O using PariBoy ® air-jet and Multisonic ® ultrasonic nebulizer, respectively. In addition, a decrease in the mean droplet size with increasing the cryoprotectant concentration has been observed. A relationship between the average droplet size, surface tension and viscosity depending on the used cryoprotectant type and concentration could be established.

H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1-liposomes.

The H(+)/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H(+)-translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pH(in), could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid-base transition at different [ATP]/([ADP][P(i)]) ratios as a function of deltapH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the deltapH at equilibrium, deltapH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][P(i)]) ratio as a function of deltapH (eq) and of the transmembrane electrochemical potential difference, delta micro approximately (H)(+) (eq), resulted in H(+)/ATP ratios of 3.9 +/- 0.2 at pH 8.45 and 4.0 +/- 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 +/- 2 kJ/mol at pH 8.45 and 36 +/- 3 kJ/mol at pH 8.05.

The Effects of Tween 20 and Sucrose on the Stability of Anti‐L‐Selectin during Lyophilization and Reconstitution

We have chosen an anti-L-selectin antibody as a model protein to investigate the effects of sucrose and/or Tween 20 on protein stability during lyophilization and reconstitution. Native anti-L-selectin secondary structure is substantially retained during lyophilization in the presence of sucrose (1 or 0.125%). However, aggregation of the protein during reconstitution of lyophilized protein powders prepared without sucrose is not reduced by the presence of sucrose in the reconstitution medium. Aggregate formation upon reconstitution is completely inhibited by freeze drying the protein with sucrose and reconstituting with a 0.1% Tween 20 solution. Tween 20 (0.1%) also partially inhibits loss of native anti-L-selectin secondary structure during lyophilization. However, upon reconstitution the formulations lyophilized with Tween 20 contain the highest levels of aggregates. The presence of Tween in only the reconstitution solution appears to inhibit the transition from dimers to higher order oligomers. Potential mechanism(s) for the Tween 20 effects were investigated. However, no evidence of thermodynamic stabilization of anti-L-selectin conformation (e.g., by Tween 20 binding) could be detected.

Vero cells, but not oviductal cells, increase the hatching frequency and total cell count of mouse blastocysts partly by changing energy substrate concentrations in culture medium.

To investigate the embryotrophic mechanisms of Vero and oviductal cells coculture. Mouse embryos were cultured in Chatot, Ziomek, and Bavister medium (CZB), in modified CZB media (MM) with nutrient concentrations adjusted to that found in conditioned media after different periods of Vero cells or oviductal cells culture, in reconstituted medium (RM) containing the purified > 100-kDa components of Vero cell conditioned medium that had been reconstituted with CZB medium, and cocultured with Vero cells with an interposing membrane. The blastulation rate was not different among embryos cultured in different Vero-cell-derived MMs. Nine-hour Vero-cell-derived MM significantly increased the total cell number and hatching frequency of the embryos. There was no difference in these parameters with oviductal-cell-derived MMs. The RM of Vero cells did not possess embryotrophic activity. The presence of a porous membrane between Vero cells and embryos did not affect the embryotrophic activity of coculture. Vero cells, but not oviductal cells, improved mouse embryo development partly by modifying the energy substrate concentration in culture medium.

Physicochemical Characteristics of Dehydrated Apple Cubes Reconstituted in Sugar Solutions

ABSTRACTWater solutions with different sugar compositions and concentrations were used to reconstitute air‐dried and freeze‐dried apple pieces. The mass transfer was determined between dehydrated fruit pieces and solutions during rehydration as well as some physicochemical properties of the reconstituted apple pieces. Varying the concentration of sugar solutions used for reconstitution medium, made it possible to achieve different levels of water absorption and solids loss or uptake. In addition, water activity (aw), freezing point, amount of freezable water and firmness correlated linearly with soluble solids content.

The effect of the reconstitution medium on aggregation of lyophilized recombinant interleukin-2 and ribonuclease A.

The effect of the reconstitution medium on aggregation of lyophilized recombinant interleukin-2 and ribonuclease A.

A new strategy for enhancing the stability of lyophilized protein: the effect of the reconstitution medium on keratinocyte growth factor.

Protein stabilization during lyophilization has previously focused on optimization of the formulation as well as the freezing and dehydration process parameters. However, the effect of the reconstitution medium has been largely neglected. We have investigated its effect on aggregate formation using recombinant keratinocyte growth factor (KGF). The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer. Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein. These results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.

Functional reconstitution of the primary quinone acceptor, Q A, in the Photosystem II core complexes

PS II reaction center core complexes consisting of CP47, D1, D2 and I proteins and α and β subunits of cytochrome b-559 were isolated from spinach PS II particles using a detergent solution containing 10 mM n-octyl β- d-thioglucopyranoside (OTG) and 17.5 mM n-octyl β- d-glucopyranoside (OG). Retention of plastoquinone-9 (PQ-9) in the particles was controlled at different levels from 0.1 to 0.7 per reaction center on average by changing the conditions of the detergent treatments, but primary quinone acceptor (Q A) activity was not detected in every preparation. PQ-9, lipids and proteins extracted from the original PS II particles into the detergent solution were separated by several steps of column chromatography to be reconstituted into the isolated complexes. The purified PQ-9 was reinserted into the vacant Q A site of the complex, but the Q A activity was not restored by itself. However, when the reinsertion of PQ-9 was carried out together with the protein fraction involving the H, L and nuclear encoding 4.1 kDa proteins and the extracted lipids, Q A activity was successfully restored in the resulting complexes. The addition of PQ-9 in the reconstitution medium in a molar ratio of 2:1 with the reaction center is sufficient to restore Q A function in the PQ-9-depleted complex, suggesting that the added PQ-9 is reinserted in the native Q A site of the complex and functions as the primary quinone acceptor under the influence of the H, L, and/or 4.1 kDa proteins and the lipids.

Co-reconstitution of the H +-ATP synthase and cytochrome [formula omitted] complex from a thermophilic cyanobacterium : High ATP yield and mutual effects on the enzymatic activities

The H +-ATP synthase and the cytochrome b-563 c-554 (Q bc) complex were selectively solubilized and isolated from the thylakoid membranes of the thermophilic cyanobacterium Synechococcus 6716. The enzymes were co-reconstituted into proteoliposomes with the native cyanobacterial lipids by detergent dialysis. After (co-)reconstitution of the protein complexes in variable stoichiometries, the different enzymatic activities were analyzed. Optimization of the functionally coupled enzymes led to a compromise for the weight ratios of 20 mg lipid and 0.5 mg of both complexes per ml of reconstitution medium. The average diameter (around 300 nm) and zeta potential of these proteoliposomes were determined. The oxidoreductase activity of reconstituted Q bc complex was found to be stimulated by the presence of co-reconstituted (inactive) ATP synthase. Conversely, ATP hydrolysis was inhibited or stimulated by the protonmotive force generated by the Q bc complex, co-reconstituted in the right-side out or inside-out orientation, respectively. In proteoliposomes containing trapped horse-heart cytochrome c the proton movement induced by the Q bc complex resulted in an ATP production of about 1 μmol ATP per mg of ATP synthase within 1 min. The mutual stimulation and high ATP synthesis activity may indicate improved incorporation and/or some kind of functional association of these proteins in the membrane, although as yet aspecific protein-stabilizing effects cannot be excluded.

Reconstitution of the human placental 5-hydroxytryptamine transporter in a catalytically active form after detergent solubilization.

The 5-hydroxytryptamine (5-HT; serotonin) transporter was solubilized from purified human placental brush border membranes by cholate in the presence of urea, and the solubilized transporter was reconstituted into proteoliposomes in a functionally active form. Solubilization of the membranes with cholate in the absence of urea inactivated the transporter. The reconstitution procedure involved precipitation of the solubilized proteins and simultaneous removal of cholate and urea by poly(ethylene glycol), and incorporation of the precipitated proteins into proteoliposomes in the presence of asolectin by a freeze-thaw/sonication technique. Optimal conditions included the use of 6% poly(ethylene glycol) during the precipitation step and an asolectin/protein ratio of 10:1 during the reconstitution step. K+ was present in the reconstitution medium. The reconstituted proteoliposomes showed the ability to transport 5-HT against a concentration gradient when an inwardly directed NaCl gradient was imposed. The 5-HT transport system in the proteoliposomes had an absolute requirement for Na+ and Cl-. The system was specific for 5-HT and was inhibited by imipramine, paroxetine and fluoxetine. The Na+/Cl-/5-HT stoichiometry was found to be 1:1:1. The transport process was electrically silent, indicating that one K+ ion was countertransported for each 5-HT molecule. The reconstituted 5-HT transporter showed high affinity for 5-HT, with an apparent Michaelis-Menten constant of 0.34 +/- 0.01 microM. It is concluded that the human placental 5-HT transporter can be solubilized and reconstituted into proteoliposomes in a transport-competent form and that the characteristics of the reconstituted transporter are similar to those of the native transporter.