Reconstitution and partial purification of the sodium and chloride-coupled glycine transporter from rat spinal cord

The (Na+ + Cl-)-coupled glycine transporter has been solubilized from rat spinal cord with 2% cholate and purified 6-7-fold using Wheat Germ Agglutinin-Sepharose 4B. Transport activity - as determined upon reconstitution of the fraction into liposomes - was retained on the column and eluted by N-acetylglucosamine. When the glycoprotein fraction was depleted of the N-acetylglucosamine and applied to a second round of lectin-chromatography, the glycine transport activity was retained and again could be eluted by the sugar. The transporter activity reconstituted from the glycoprotein fraction retains the same features displayed in the synaptic plasma membrane vesicles, namely an absolute dependence on sodium and chloride, electrogenicity and efflux and exchange properties. These observations indicate that the (Na+ + Cl-)-coupled glycine transporter is a glycoprotein.     

Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain.

The sodium- and potassium-coupled L-glutamate transporter from rat brain has been purified to near homogeneity by reconstitution of transport as an assay, assuming that inactivated and active transporters cochromatograph. The purification steps involve lectin chromatography of the membrane proteins solubilized with 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), fractionation on hydroxylapatite, and ion-exchange chromatography. The specific activity is increased 30-fold. The actual purification is higher since 3-5-fold inactivation occurs during the purification. The efficiency of reconstitution was about 20%. The properties of the pure transporter are fully preserved. They include ion dependence, electrogenicity, affinity, substrate specificity, and stereospecificity. Sodium dodecyl sulfate-polyacrylamide electrophoresis revealed one main band with an apparent molecular mass of around 80 kDa and a few minor bands. Comparison of polypeptide composition with L-glutamate transport activity throughout the fractionation procedure reveals that only the 80-kDa band can be correlated with activity. The GABA transporter, which has the same apparent molecular mass (Radian et al., 1986), is separated from it during the last two purification steps. Immunoblot experiments reveal that the antibodies against the GABA transporter only reacted with fractions exhibiting GABA transport activity and not with those containing the glutamate transporter. We conclude that the 80-kDa band represents the functional sodium- and potassium-coupled L-glutamate transporter.     

Structural and functional studies on the sodium- and chloride-coupled gamma-aminobutyric acid transporter: deglycosylation and limited proteolysis

The sodium- and chloride-coupled gamma-aminobutyric transporter, an 80-kDa glycoprotein, has been subjected to deglycosylation and limited proteolysis. The treatment of the 80-kDa band with endoglycosidase F results in its disappearance and reveals the presence of a polypeptide with an apparent molecular mass of about 60 kDa, which is devoid of 125I-labeled wheat germ agglutinin binding activity but is nevertheless recognized by the antibodies against the 80-kDa band. Upon limited proteolysis with papain or Pronase, the 80-kDa band was degraded to one with an apparent molecular mass of about 60 kDa. This polypeptide still contains the 125I-labeled wheat germ agglutinin binding activity but is not recognized by the antibody. The effect of proteolysis on function was examined. The transporter was purified by use of all steps except that for the lectin chromatography [Radian, R., Bendahan, A., & Kanner, B.I. (1986) J. Biol. Chem. 261, 15437-15441]. After papain treatment and lectin chromatography, gamma-aminobutyric transport activity was eluted with N-acetylglucosamine. The characteristics of transport were the same as those of the pure transporter, but the preparation contained instead of the 80-kDa polypeptide two fragments of about 66 and 60 kDa. The ability of the anti-80-kDa antibody to recognize these fragments was relatively low. The observations indicate that the transporter contains exposed domains which are not important for function.     

Lectin binding of solubilized opiate receptors: evidence for their glycoprotein nature

Lectin affinity chromatography was used to demonstrate that digitonin-solubilized opiate receptors contain a carbohydrate moiety. Receptors solubilized from toad, rat, chicken, bovine and human brains were retained on columns of wheat germ agglutinin (WGA)-agarose and eluted specifically with N-acetylglucosamine. The fraction retained and subsequently eluted ranged from 40–60% of the applied receptors. The eluted receptor was enriched approx. 30-fold. Evidence is presented which shows that the site of lectin interaction is functionally independent of the opiate binding site.     

Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain

Using the reconstitution conditions developed recently (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865) we have now purified the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain to apparent homogeneity. A partially purified transporter preparation was passed over wheat germ agglutinin-Sepharose 6MB and non-bound proteins were washed away. The transport activity, as expressed upon reconstitution of the protein into liposomes, was eluted by a solution containing Triton X-100 and N-acetylglucosamine. The specific transport activity was increased almost 400-fold over that of the crude extract. Taking into account an approximately 2.5-fold inactivation during the lectin column chromatography, the actual purification is about 1000-fold. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the active fractions revealed one band of 80 kDa and small amounts of a band which ran at an apparent molecular mass of 160 kDa. The ratio between the two could be experimentally changed such as, for instance, by lyophilization. Polyclonal antibodies were prepared against the 80-kDa band which also cross-reacted with the 160-kDa band, indicating that the latter apparently represents a dimer form of the first. Using Protein A-Sepharose Cl-4B and the antibody against the 80-kDa band, we were able to quantitatively immunoprecipitate the potential gamma-aminobutyric acid transport activity from a crude transporter preparation. The pure transporter preparation exhibited the same features of the transporter in synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogeneity, affinity, and efflux and exchange properties. We conclude that the 80-kDa band represents the gamma-aminobutyric acid transporter.     

Reconstitution and purification of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain

The sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain has been highly purified. Synaptic plasma membranes from rat brain were extracted with cholate in the presence of 10% ammonium sulfate. The soluble extract was incorporated into liposomes consisting of asolectin and crude brain lipids. Brain lipids markedly enhanced the transport activity. The resulting proteoliposomes catalyzed sodium- and chloride-coupled gamma-aminobutyric acid transport which, in the presence of internal potassium, was greatly (up to 20-fold) stimulated by valinomycin. Using this transport of the reconstituted system as an assay, the transporter was purified by the following steps. The cholate extract was fractionated by ammonium sulfate. The activity was not precipitated by 50% but could be precipitated by 70% ammonium sulfate. The cholate and ammonium sulfate were removed on a Sephadex G-50 column. Subsequently, the transporter was partially purified on DEAE-cellulose in a mixture of Triton X-100 and octyl glucoside. The active fractions were chromatographed on a hydroxylapatite column in the presence of Triton X-100. Although the increase in specific activity was only up to 100-fold, this was due to partial inactivation. The actual purification was at least 1000-fold. The purified transporter exhibited the same features of the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity, and a similar affinity. The sodium dodecyl sulfate gel pattern indicated that a major protein ran as a 24-kDa band. This band may represent the gamma-aminobutyric acid transporter.     

Purification of the sodium- and chloride-coupled glycine transporter from central nervous system.

We have recently developed a reconstitution assay which allows the rapid determination of sodium- and chloride-dependent glycine transport activity of many fractions (López-Corcuera, B., and Aragón, C. (1989) Eur. J. Biochem. 181, 519-524). In this paper we report the purification of the sodium- and chloride-coupled glycine transporter from pig brain stem. Transporter is solubilized from plasma membrane vesicles with 2% cholate and purified by sequential chromatography on phenyl-Sepharose, wheat germ agglutinin-Sepharose, and hydroxylapatite columns, followed by a 5-20% sucrose density gradient fractionation. Taking into account the inactivation suffered by the transporter, a final increase in specific activity of about 450-fold is achieved. Although two polypeptides with apparent molecular masses of 100 and 37 kDa are progressively enriched during the chromatographic steps, only the 100-kDa band comigrates with transport activity along the density gradient. This band is finally isolated to apparent homogeneity in the active fractions. We conclude that the 100-kDa band represents the glycine transporter. Finally, the pure transporter can be reconstituted into liposomes, retaining the absolute dependence on sodium and chloride gradients, the electrogenicity, the glycine affinity, the substrate specificity, and the sensitivity to group-selective modifiers characteristic of the native transporter.     

The accessibility of a novel reentrant loop of the glutamate transporter GLT-1 is restricted by its substrate

The excitatory neurotransmitter glutamate is removed from the synaptic cleft by several related sodium- and potassium-coupled transporters. They thereby restrict the neurotoxicity of this transmitter. Based on the accessibility of single cysteines to the large sulfhydryl reagent 3-N-maleimidyl(propionyl)biocytin, we have proposed a topological model for the astroglial glutamate transporter GLT-1 (Grunewald, M., Bendahan, A. and Kanner, B. I. (1998) Neuron 21, 623-632). Because of several unexpected observations, we have investigated the topological disposition of 19 cysteine residues engineered into a loop proposed to be intracellular. We have probed the accessibility of these cysteines to small and large sulfhydryl reagents. The impermeant hydrophilic sulfhydryl reagent [(2-trimethylammonium)ethyl] methanethiosulfonate inhibits transport activity only at two of these positions, weakly at G365C and potently at A364C. Glutamate and its nontransportable analogue dihydrokainate markedly protect A364C transporters against this impermeant reagent. Using a biotinylated maleimide, we found that, among the 14 mutants tested with it, only A364C is accessible to it from the extracellular side. This, together with our previous observations, indicates that the loop-including amino acid residues 354, 359, 373, and 379-is largely intracellular, but a short region of it forms a reentrant pore-loop-like structure, the accessibility of which is dependent on the conformation of the transporter.     

Counterflow of L-glutamate in plasma membrane vesicles and reconstituted preparations from rat brain

Membrane vesicles from rat brain exhibit sodium-dependent uptake of L-[3H]glutamate in the absence of any transmembrane ion gradients. The substrate specificity of the process is identical with (Na+ + K+)-coupled L-glutamate accumulation. Although these vesicles are prepared after osmotic shock and are washed repeatedly, they contain about 1.5 nmol/mg of protein endogenous L-glutamate, apparently located inside the vesicles. The affinity of the process (Km approximately 1 microM) is similar to that of (Na+ + K+)-dependent accumulation by the L-glutamate transporter. Membrane vesicles have been disrupted by the detergent cholate, and the solubilized proteins have been subsequently reconstituted into liposomes. The reconstituted proteoliposomes also exhibit the above uptake--with the same characteristics--provided they contain entrapped cold L-glutamate. Counterflow is optimal when sodium is present on both sides of the membrane, but partial activity is still observed when sodium is present either on the inside or on the outside. Increasing the L-glutamate concentration above the Km results in counterflow completely independent of cis sodium. The initial rate of counterflow is 100-200-fold lower than that of net trans potassium dependent flux. The rate of net flux in the presence of trans sodium or lithium is about 10-fold lower than when choline or Tris are used instead. However, the rate of counterflow (no internal potassium present) was not stimulated by replacing internal sodium or lithium by internal choline. Therefore, optimal functioning of the transporter requires internal potassium while internal sodium and lithium are inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific elution from hydroxylapatite of the mitochondrial phosphate carrier by cardiolipin

The role of cardiolipin in the purification of the mitochondrial phosphate carrier by hydroxylapatite has been investigated. Without added cardiolipin, the reconstituted phosphate-transport activity in the hydroxylapatite eluate is small and only confined to the first fraction. With cardiolipin added to the extract, the eluted activity is much higher and present until fraction 6. The activity retained by hydroxylapatite in the absence of cardiolipin is eluted after addition of this phospholipid to the column. The requirement of added cardiolipin diminishes on increasing the concentration of solubilized mitochondria. The hydroxylapatite eluate contains five protein bands in the Mr-region of 30 000-35 000, which are differently distributed in the various fractions. Among these, only the presence and the relative amount of band 3 of Mr 33 000 corresponds to the phosphate transport activity. Cardiolipin is the only phospholipid tested which causes elution of band 3 from hydroxylapatite; on the other hand, it prevents the elution of band 2 and retards that of band 5 (the ADP/ATP carrier). Band 1 starts to appear in the second fraction even without cardiolipin. On increasing the concentration of cardiolipin, in the first fraction of the hydroxylapatite eluate band 3 increases and the contamination of band 4 decreases. Under optimal conditions a preparation of band 3 about 90% pure and with high reconstituted phosphate transport activity is obtained. It is concluded that the elution of the phosphate carrier from hydroxylapatite requires cardiolipin and that the phosphate carrier is identical with (or with part of) band 3 of the hydroxylapatite eluate.     

Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the Vmax. In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.     

Partial purification and characterization of taurodeoxycholate 7α-monooxygenase

Taurodeoxycholate 7α-monooxygenase was partially purified from rat liver microsomes. The enzyme was solubilized with cholate, fractionated with polyethylene glycol and chromatographed on a Sepharose 4B column with cholate as ligand. The enzyme activity was eluted from the column into the fraction eluted with 50 mM phosphate buffer containing cholate and KCl, whereas the benzphetamine demethylase activity was eluted in the non-bound fraction. Thus it was established that both enzymes are different entities. The taurodeoxycholate 7α-monooxygenase activity was reconstituted from the partially purified cytochrome P-450, highly purified NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine and NADPH.     

Solubilization in good yield of active opiate binding sites from mammalian brain

Active opiate binding sites have been solubilized from mammalian brain cell membranes. The presence of 0.5-0.1 M NaCl during treatment of membranes from rat brain, human frontal cortex, and bovine corpus striatum with glycodeoxycholate or digitonin resulted in the extraction of active opiate binding sites in yields ranging up to 43%. The criteria for solubility of the sites were their inability to sediment at 10(5) x g after 2 hr and their apparent molecular weight of 3- 4 x 10(5) as determined by gel filtration. The receptors in solution resemble the membrane-bound sites with respect to saturability, stereo-specificity, sensitivity to heat and reagents, and high affinity for opioid ligands. The interaction of solubilized sites with immobilized lectins was used to demonstrate the glycoprotein nature of the opiate receptor. Soluble receptors from all species studied were retained by wheat germ agglutinin(WGA)-agarose and could be specifically eluted with N-acetylglucosamine. No retention of solubilized material was observed with eight other lectins examined, including horseshoe crab lectin, a sialic acid specific agglutinin. The receptor protein eluted from WGA columns was enriched 25-50-fold over the crude soluble fraction.     

Isolation and functional reconstitution of a 38-kDa chloride channel protein from bovine tracheal membranes.

Secretion of chloride ions via apically located anion-selective channels in epithelia regulates fluid formation and cytosolic Cl- homeostasis. In order to understand the biochemical basis of Cl- channel function, we attempted to isolate this transporter from bovine tracheal apical membranes. Initially, peripheral polypeptides were removed from apically enriched vesicles by washing with alkaline buffer (pH 10.8) containing 2 mM CHAPS. The resulting pellet contained 50-60% of the original protein and displayed 2-fold enhanced Cl- channel activity compared to untreated vesicles. The pellet was treated with Triton X-100, and the solubilized proteins were separated on the cationic exchanger CM-cellufine. Washing the resin with a pH 8.0-8.3 buffer eluted a fraction with enriched Cl- channel activity. This fraction contained less than 5% of the total solubilized protein. A subsequent separation was performed using the anionic exchanger AM-cellufine. The highest activity was found in the fractions eluted by 80-120 mM KCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a major 38,000-Da protein band. This band was electroeluted from the gel under nondenaturing and nonreducing conditions and reconstituted into phosphatidylcholine liposomes. KCl-loaded vesicles containing the purified 38-kDa protein transported up to 5 nmol of 125I-/mg of protein/5 min. This value was 15-fold higher than the uptake measured in vesicles reconstituted with total solubilized membrane proteins and 4-fold higher compared to the CM-cellufine-enriched fraction. The observed 125I- uptake was 90% inhibited by 100 microM 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate or 10 microM valinomycin. In summary, we have developed a biochemical protocol for the isolation of a 38 kDa protein mediating potential-dependent and 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate-sensitive Cl- channel activity.     

Ferrous-iron-dependent uptake of L-glutamate by a mesophilic,mixotrophic iron-oxidizing bacterium strain OKM-9

Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.     

The role of N-glycosylation in transport to the plasma membrane and sorting of the neuronal glycine transporter GLYT2

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.     

Lectin-binding domains on laminin

Chicken erythrocytes have been found to have at least two kinds of phospholipase A2. The first is a soluble enzyme from the cytosole fraction and has no calcium sensitivity. The second can be extracted from the plasma membrane fraction with the nonionic detergent Triton X-100. In this study the membrane-bound enzyme was partially purified by affinity chromatography on phosphatidylcholine-Sepharose, and its specific activity was increased 1100-fold compared with that of the cell homogenate without nuclei. It has an optimum pH of 8.5 and required calcium for maximum activity. It showed the specificity for both phosphatidylcholine and phosphatidylethanolamine, but reacted preferentially on the former substrate. Analysis by concanavalin A-Sepharose affinity chromatography revealed that the membrane-bound phospholipase A2 was retained on the resin and could be eluted specifically with a haptenic sugar, methyl alpha-D-mannopyranoside. The enzyme seems to be either a concanavalin A-binding glycoprotein or a part of a complex with certain concanavalin A-binding glycoproteins.     

Cholesterol is required for the reconstruction of the sodium- and chloride-coupled, gamma-aminobutyric acid transporter from rat brain

The reconstruction of the purified sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain into asolectin liposomes requires the addition of brain lipids (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865). The reconstitution assay was used to identify the component(s) from brain lipids responsible for the stimulation during the fractionation of brain lipids. The distribution of the active component was found to be similar to that of cholesterol. Furthermore, cholesterol was found to mimic the effect of brain lipids and it stimulated the transport activity up to 20-fold. Optimal reconstituted transport activity was achieved with mixtures of cholesterol and any one of several phospholipids, such as phosphatidylcholine, phosphatidylserine or phosphatidylglycerol. gamma-Aminobutyric acid transport in these liposomes of defined composition exhibited all the properties of the native transporter, such as the absolute dependence on sodium and chloride and electrogenicity. Cholesterol could not be replaced by cholest-4-en-3one and other steroids, and thus its effect is probably not due to effects on membrane fluidity. The requirement was also not due to effects on intactness of the liposomes or incorporation of proteins into them. Furthermore it was found that the reconstitution of the sodium and potassium coupled L-glutamic acid transporter from rat brain also required cholesterol. However, in this case the optimal activity was reached by 4-5-fold lower levels of cholesterol than those necessary for gamma-aminobutyric acid transport. When cholesterol depletion from the transporters was incomplete, addition of exogenous brain lipids was not required. Thus, if the cholesterol was still associated with the transporter proteins, its final concentration, as a fraction of the total lipids present in the reconstitution mixture, was only about 0.01 mol%. Thus, it is likely that the effects of cholesterol are due to direct interactions with the cotransporters and not to an average effect on membrane properties.     

Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction.

The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.     

The interaction of Sendai virus glycoprotein-bearing recombinant vesicles with cell surfaces

Sendai virus glycoproteins HN and F were purified by immunoaffinity chromatography from virions disrupted by beta-D-octylglucoside. The purified glycoproteins were reconstituted in recombinant vesicles with phosphatidylcholine or phosphatidylethanolamine and phosphatidylserine. P815 or EL-4 cells treated with glycoprotein HN/F-phosphatidylcholine recombinant vesicles acquired the glycoproteins and retained them in the plasma membrane for 4 h as demonstrated by surface immunofluorescence specific for each protein. Cells treated with glycoprotein HN-phosphatidylcholine recombinant vesicles initially bore glycoprotein HN on the surface but the protein eluted within 2 h. Surfaces of cells treated with glycoprotein F-phosphatidylcholine recombinant vesicles did not acquire the glycoprotein. Cells treated with glycoprotein HN-phosphatidylethanolamine: phosphatidylserine recombinant vesicles or glycoprotein F-phosphatidylethanolamine: phosphatidylserine recombinant vesicles in the presence of 5 mM Ca2+ acquired each protein for at least 2 h. Experiments showed that the acquired glycoproteins capped with antibody and that when glycoproteins HN and F were together on the surface they co-capped. Acquired viral glycoproteins did not co-cap with intrinsic H-2 glycoproteins.