Water- and solute-accessible spaces of purified peroxisomes. Evidence that peroxisomes are permeable to NAD+.

Peroxisomes were purified from liver homogenates from rats, treated with the peroxisome proliferator clofibrate, by a combination of differential centrifugation and isopycnic centrifugation in iso-osmotic self-generating Percoll gradients. Structural integrity of the peroxisomes appeared to be preserved as evidenced by a high degree of catalase latency, the absence of catalase release during purification and the exclusion of inulin (mol.wt. +/- 5000). Spaces for water and solutes were measured after incubation of the peroxisomes in iso-osmotic sucrose with radioactive water or solutes and separation of the organelles from their media by centrifugation through an organic layer. Extraperoxisomal water was corrected for by the use of radioactive dextran or inulin. The sucrose, glucose, urea, methanol and acetate-accessible spaces were identical, suggesting that these spaces represent the volume in which molecules that can cross the membrane distribute. This volume equalled 50-65% of the water space. Urate and NAD+, a cofactor of peroxisomal beta-oxidation of fatty acids, also distributed in this volume, but were also partly bound. Urate and NAD+ binding was not abolished by sonication, which released the bulk of matrix catalase activity, but NAD+ binding was seriously diminished. The peroxisomal water and sucrose spaces were estimated to be 107 microliters and 55 microliters per g of liver tissue from a clofibrate-treated rat. From quantitative morphometric data [Anthony, Schmucker, Mooney & Jones (1978) J. Lipid Res. 19, 154-165] and our marker enzyme analyses, as well as from our experimentally determined water spaces of mitochondrial and microsomal fractions, it could be calculated that the volume contamination by lysosomes, mitochondria and microsomes did not exceed 1, 8 and 6% respectively. Our data indicate that apparently intact peroxisomes are permeable to a number of small molecules, including NAD+. Whether the NAD+-binding sites in sonicated peroxisomes mirror the likely existence of a membrane carrier requires further investigation.     

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na+, with a positive cooperativity. Na+ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Peroxisomal membrane permeability and solute transfer

The review is dedicated to recent progress in the study of peroxisomal membrane permeability to solutes which has been a matter of debate for more than 40 years. Apparently, the mammalian peroxisomal membrane is freely permeable to small solute molecules owing to the presence of pore-forming channels. However, the membrane forms a permeability barrier for 'bulky' solutes including cofactors (NAD/H, NADP/H, CoA, and acetyl/acyl-CoA esters) and ATP. Therefore, peroxisomes need specific protein transporters to transfer these compounds across the membrane. Recent electrophysiological studies have revealed channel-forming activities in the mammalian peroxisomal membrane. The possible involvement of the channels in the transfer of small metabolites and in the formation of peroxisomal shuttle systems is described.     

Evidence for nucleotide-dependent passive H+ transport protein in the plasma membrane of barley roots

Plasma membranes were isolated from barley roots by two-phase partitioning, and octylglucoside-soluble and -insoluble fractions were obtained. The insoluble fractions were reconstituted into liposomes, and the plasma membrane H(+)-ATPase was shown to participate in MgATP-dependent H(+) transport activity. The H(+) transport was decreased when the octylglucoside-soluble fraction was reconstituted together with the insoluble fraction. The decrease was not due to inhibition of the H(+)-ATPase, but rather was likely due to the increased H(+) leakage from the proteoliposome. The octylglucoside-soluble fraction was, therefore, reconstituted in the liposomes and the passive H(+) transport was determined using the pH jump method. A pH gradient across the membranes was generated by the pH jump, and the gradient was found to be dissipated by passive H(+) transport. The H(+) transport required ATP, K(+), and valinomycin. The H(+)-transport also occurred when ADP, AMP, GTP, or ATP-gamma-S was present instead of ATP, and did not occur when the octylglucoside-soluble fraction was boiled before the reconstitution. These findings suggest that nucleotide-dependent H(+ )transport protein is present in the plasma membrane of root cells.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na⁺, with a positive cooperativity. Na⁺ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Dissociation of the sodium-ion-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and reconstitution of the active complex from the isolated subunits

Oxaloacetate decarboxylase was reconstituted from the purified alpha subunit and a Triton X-100 extract of bacterial membranes devoid of this protein. Upon freezing of oxaloacetate decarboxylase in salt solutions, the enzyme was split into subunits and the catalytic activity was abolished. The catalytically active decarboxylase complex was reconstituted by decreasing the salt concentration of the dissociated sample. The conditions for the inactivation were critical for an optimum recovery of catalytically active enzyme during reconstitution, and modest dissociating conditions generally improved the yield of the reconstitutively active decarboxylase. The dissociated enzyme has been separated by chromatography on avidin-Sepharose into two fractions: fraction I, that was not retained on the column, consisted of the beta + gamma subunits, and fraction II consisted of the biotin-containing alpha subunit. Oxaloacetate decarboxylase was reconstituted from a mixture of the isolated alpha and beta + gamma subunits. The Na+ transport activity was recovered, if a mixture of subunits alpha and beta + gamma was incorporated into liposomes, or by a sequential reconstitution, starting with the formation of proteoliposomes with the integral membrane proteins beta + gamma and completed by an attachment of the peripheral subunit alpha.     

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl2 and DIDS. The IC50 for HgCl2 was 0.16+/-0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9+/-0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.     

The phosphate translocator of the chloroplast envelope. Isolation of the carrier protein and reconstitution of transport

This report describes the solubilization and purification of the phosphate translocator of spinach chloroplasts and the reconstitution of its activity by incorporation into liposomes. (1) Prior to the isolation, the carrier is specifically labelled by treatment with 2,4,6-trinitrobenzenesulfonic acid and NaB[³H]H₄. (2) After preextraction of purified envelope membranes with Brij 58 for removing other loosely bound membrane proteins, the phosphate translocator is extracted with Triton X-100. After passing the resulting extract over a DEAE-Sepharose column followed by sucrose density gradient ultracentrifugation, the translocator protein is purified to apparent homogeneity. The 5–6-fold purification thus obtained concurs with earlier findings that the phosphate translocator protein represents 15–20% of the envelope membrane protein. This highly purified protein is suitable for studies of the hydrodynamic parameters of the translocator. (3) Since the exposure to detergents affects the activity of the translocator protein, alternatively, a rapid batch procedure for the purification of the translocator protein employing hydroxyapatite is used, yielding within 15 min the phosphate translocator protein of about 70% purity. (4) After incorporation of this protein fraction into liposomes, a specific transport of phosphate into these liposomes is observed, which van be terminated by inhibitor stop with pyridoxal 5′-phosphate. This uptake is only observed when the liposomes have been preloaded with phosphate or 3-phosphoglycerate, but not with 2-phosphoglycerate. Thus, like in intact chloroplasts, also the reconstituted transport facilitates an obligatory and specific counter exchange of anions. The apparent K_m for the transport of phosphate by this reconstituted system is about 0.8 mM, which is comparable to the corresponding value in intact chloroplasts. The calculated turnover of 150–300 min⁻¹ (20°C) accounts for 3–6% of the original activity.     

Carnitine acyltransferases in rat liver peroxisomes

Carnitine acyltransferase activities, as well as acetyl-CoA, octanyl-CoA, and palmityl-CoA hydrolase activities, were assayed in mitochondrial, peroxisomal, and endoplasmic reticulum fractions after isopycnic density sucrose gradient fractionation of rat liver homogenates. Both the forward and reverse assays show that carnitine acetyltransferase and carnitine octanyltransferase are associated with peroxisomes, mitochondria, and endoplasmic reticulum, while carnitine palmityltransferase was detected in mitochondria. Palmityl-CoA and octanyl-CoA hydrolase activities were found in all but the leading edge of the peroxisome peak of the gradient. The palmityl-CoA hydrolase in peroxisomal fractions was due to lysosomal contamination since the activity coincided with the lysosomal marker, acid phosphatase. The substrate specificity for carnitine octanyltransferase activity was maximum with medium-chain-length derivatives (about 20 nmol/ min/mg protein) and decreased as the acyl length increased until very low activity (<1 nmol/min/mg protein) was obtained with palmityl-CoA. When acyltransferases in peroxisomes were assayed by measuring acylcarnitine formation, nearly theoretical amounts of acetylcarnitine and octanylcarnitine were formed, but lesser quantities of 12 and 14 carbon acylcarnitines and very low amounts of palmitylcarnitine were detected. The presence of a broad spectrum of medium-chain and short-chain carnitine acyltransferases in peroxisomes is consistent with a role for carnitine for shuttling short-chain and medium-chain acyl residues out of peroxisomes. Carnitine acyltransferase activity was not detected in peroxisomes from spinach leaves.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

钙库调控的钙通道Orai1体外研究方法的建立

目的:建立钙通道Orai1的体外研究方法。方法:利用脂质体重组技术,将体外纯化的Orai1蛋白重组到脂质体膜上,利用蔗糖密度梯度离心来检测其重组效率及Orai1蛋白在脂质体膜上的结构,并利用钙染料Fura-2检测脂质体内钙离子的释放。结果:成功制备了脂质体及体外纯化了GST-Orai1融合蛋白,蔗糖密度梯度离心结果证明GST-Orai1蛋白成功重组到脂质体上,以及Orai1蛋白以多聚体的形式定位在脂质体膜上。钙离子释放实验证明脂质体内钙离子包装完好,可用于后续Orai1钙通道的功能研究。结论:利用脂质体重组技术建立了一种新的Orai1的研究方法,能够更直接有效地研究其功能及其活化机制。     

Isolation and characterization of transposon Tn5-induced mutants of Pseudomonas perfectomarina defective in nitrous oxide respiration.

Outer membranes of Haemophilus influenzae type b were fractionated to yield Triton X-100-insoluble material and lipopolysaccharide and phospholipids. Liposomes reconstituted from lipopolysaccharide and phospholipids were impermeable to sucrose (Mr, 342) and to a high-molecular-weight dextran (average Mr, 6,600). When the Triton X-100-insoluble material was introduced into the reconstituted liposomes, the vesicles became permeable to sucrose, raffinose (Mr, 504), and stachyose (Mr, 666) and fully retained dextrans of Mr greater than 1,500. Inulin (average Mr, 1,400) was tested for its efflux from the reconstituted outer membrane vesicles; 62% of the added inulin was trapped. The molecular weight exclusion limit for the outer membrane of H. influenzae type b was therefore estimated at approximately 1,400. A protein responsible for the transmembrane diffusion of solutes was purified from H. influenzae type b by extraction of whole cells with cetyl trimethyl ammonium bromide. When this extract was passed over DEAE-Sepharose, three protein-containing peaks (I, II, and III) were eluted. Peaks I and II contained mixtures of proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; when tested for their pore-forming properties, these proteins were unable to render liposomes of lipopolysaccharide and phospholipid permeable to sucrose. Peak III contained only one molecular species of protein of molecular weight 40,000; this protein acted as a porin in reconstituted vesicles. The molecular weight exclusion limit for 40,000-molecular-weight protein matched the estimate of approximately 1,400 which was determined for outer membranes. A series of homologous saccharides of increasing degree of polymerization was prepared from agarose by hydrolysis with beta-agarase and fractionation on gel filtration chromatography. These oligosaccharides of Mr, 936, 1,242, 1,548, and 1,854 were assayed for retention by the complete vesicles containing 40-kilodalton protein and lipopolysaccharide and phospholipids. All of these oligosaccharides were lost by efflux through the porin. Since the molecular conformation of the largest oligosaccharide is an elongated semirigid helix, it is suggested that the pore formed by the 40-kilodalton protein does not act as a barrier to the diffusion of this compound.     

Purification, reconstitution, and steady-state kinetics of the trans-membrane 17 beta-hydroxysteroid dehydrogenase 2

Human membrane 17 beta-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17beta-hydroxysteroids into their inactive keto forms in a variety of tissues. 17 beta-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-beta-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-beta-D-maltoside-destabilized liposomes prepared from l-alpha-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 micromol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 micromol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 +/- 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17 beta-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K(m) of NAD being 110 +/- 10 microM and that of NADP 9600 +/- 100 microM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20 alpha-dihydroprogesterone as substrates were also determined.     

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl₂ and DIDS. The IC₅₀ for HgCl₂ was 0.16 ± 0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9 ± 0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.     

The sucrose carrier of the plant plasmalemma. III. Partial purification and reconstitution of active sucrose transport in liposomes.

The proteins from plasma membranes from sugar beet leaves were solubilized by 1% CHAPS and separated by size exclusion chromatography and by ion-exchange chromatography. The fractions enriched in sucrose transporter were monitored in three ways: differential labeling, ELISA, and reconstitution in proteoliposomes. When the plasma membranes were differentially labeled by N-ethylamaleimide in the presence of sucrose, a major peak of differential labeling was found at 120 kDa upon gel filtration. When this peak was recovered, denaturated by sodium dodecyl sulfate and reinjected on the gel filtration column, it yielded a peak of differential labeling at 42 kDa. When unlabeled membranes were used, the fractions eluted from the column were monitored by ELISA for their ability to recognize a serum directed against a 42 kDa previously identified as a putative sucrose carrier. The results paralleled those obtained by differential labeling, i.e. a major ELISA-reactive peak was found at 120 kDa upon gel filtration, and this peak yielded a peak most reactive at 40 kDa after denaturation. The 120 kDa peak prepared from unlabeled membranes was further separated on a Mono-Q column. The fractions were monitored by ELISA as described above, and reconstituted into proteoliposomes using asolectin. Active transport of sucrose, but not of valine could be observed with the reconstituted 120 kDa fraction. When the eluates from the Mono-Q column were reconstituted, the fractions exhibiting highest transport activity were enriched with a 42 kDa band. The data provide the first report concerning reconstitution of sucrose transport activity and confirm the involvement of a 42 kDa polypeptide in sucrose transport.     

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase.Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O-(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes.Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K_m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.     

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.     

Purification and reconstitution of the high affinity choline transporter

The high-affinity choline transporter has been solubilized from synaptosomal membranes by various detergents. The solubilized carrier protein has been incorporated into liposomes after removal of the detergent by dialysis. Using the reconstitution of choline transport activity as an assay, the components catalyzing choline translocation were purified from the detergent extract by ion-exchange chromatography on a Mono-Q column followed by immunoaffinity chromatography. Monitoring the active fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis and isoelectrofocussing gave one major protein with an apparent molecular weight of about 90,000 and an isoelectric point of pH 4.7. The isolated protein appeared to be heavily glycosylated as shown by lectin binding; upon treatment with endoglycosidase F the polypeptide was degraded to an apparent molecular weight of about 65,000. Accumulation of choline into liposomes reconstituted with the purified protein was driven by artificially imposed sodium gradients and inhibited by hemicholinium-3.