Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 64,000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n-octyl-glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an Mr of 43,000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the Mr 64,000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent Kd 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent Kd value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.     

Chromatographic characterization of nitrobenzylthioinosine binding proteins in band 4.5 of human erythrocytes: purification of a 40 kDa truncated nucleoside transporter

DEAE-column-purified band 4.5 polypeptides of human erythrocyte membranes are mostly glucose transporters with nucleoside transporters as a minor component. The purpose of the present work was to differentially identify and isolate the nucleoside transporters in band 4.5 free from glucose transporters. Equilibrium binding studies demonstrated that the band 4.5 preparation binds nibrobenzylthioinosine (NBTI), a potent nucleoside transport inhibitor, at two distinct sites, one with a high affinity (dissociation constant, KD of 1 nM) with a small capacity, BT (0.4 nmol/mg protein), and the other with a low affinity (KD of 15 microM) with a large BT (14-16 nmol/mg protein). The BT of the low-affinity site was equal to that of the cytochalasin B binding site in the preparation. A gel-filtration chromatography of band 4.5 photolabeled with [3H]NBTI and [3H]cytochalasin B identified three polypeptides of apparent Mr 55,000, 50,000 and 40,000. Of these, the 55 kDa polypeptide was specifically labeled by cytochalasin B (p55GT), indicating that it is a glucose transporter. Both the 50 and 40 kDa polypeptides were labeled with NBTI at low ligand concentrations (less than 0.1 microM), which was abolished by an excess (20 microM) of nitrobenzylthioguanosine, indicating that they are two forms (p50NT and p40NT, respectively) of the high affinity NBTI binding protein or nucleoside transporter. At higher (not less than 10 microM) NBTI concentrations, however, p55GT was also labeled with NBTI, indicating that the low-affinity NBTI binding is due to a glucose transporter. Treatment of band 4.5 with trypsin reduced the p50NT labeling with a concomitant and stoichiometric increase in the p40NT NBTI labeling without affecting the high-affinity NBTI binding of the preparation. These findings indicate that the nucleoside transporter is slightly smaller in mass than the glucose transporter and that trypsin digestion produces a truncated nucleoside transporter of apparent Mr 40,000 which retains the high-affinity NBTI binding activity of intact nucleoside transporter. Both p55GT and p50 NT were coeluted in a major protein fraction, P1 in the chromatography, while p40NT was eluted separately as a minor protein fraction, P1a. All three polypeptides formed mixed dimers, which were eluted in a fraction PO. We have purified and partially characterized the truncated nucleoside transporter, p40NT. The purified p40NT may be useful for biochemical characterization of the nucleoside transporter.     

Reconstitution studies of the human erythrocyte nucleoside transporter

The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.     

Monoclonal antibody to the human glucose transporter that differentiates between the glucose and nucleoside transporters

A monoclonal antibody to the glucose transporter has been prepared with band 4.5 (Mr 45,000-65,000) from human erythrocyte ghosts as antigen. This antibody, designated 7F7.5, is of the IgG2b type. The antibody bound exclusively to proteins in the band 4.5 region of immunoblots of human erythrocyte ghosts separated on sodium dodecyl sulfate-polyacrylamide gels. Immobilized 7F7.5 antibody removed glucose transport activity from solubilized alkaline-treated ghosts. The material that was eluted from the immobilized antibody matrix migrated primarily in the band 4.5 region of electrophoretic gels and bound the antibody in immunoblots. To test the specificity of the antibody, glucose and nucleoside transporters in alkaline-treated human erythrocyte ghosts were affinity labeled with [3H]cytochalasin B and [3H]-S-(nitrobenzyl)thioinosine (NBMPR), respectively. Both of these transporters are band 4.5 proteins and "copurify" by DEAE-cellulose chromatography. A filter paper assay was developed to assess the presence of the labeled transporters. Immobilized 7F7.5 antibody bound 99% of the labeled glucose transporter. In contrast, only 3% of the specifically labeled nucleoside transporter bound to the immobilized antibody. Furthermore, the antibody did not remove nucleoside transport or NBMPR binding activities from detergent solution. The antibody recognized two tryptic fragments, Mr 23,000 and 18,000, which contain the cytochalasin B binding site of the glucose transporter. By immunoblot, the monoclonal antibody recognized the glucose transporter in cultured human IM9 lymphocytes, synovial cells, and HBL 100 mammary cells but not cells of murine or rat origin. These results indicate that the glucose and nucleoside transporters are distinct proteins which can be distinguished by monoclonal antibody 7F7.5. The method developed to quantitate covalently labeled glucose and nucleoside transporters should have broad applicability as a rapid and easy method for determining the recovery of affinity-labeled membrane proteins in detergent solution during purification. Because of the location of the epitope, the antibody itself should prove to be a valuable tool in establishing the molecular basis for the function and regulation of the glucose transporter.     

Proteolytic cleavage of [3H]nitrobenzylthioinosine-labelled nucleoside transporter in human erythrocytes.

The transmembrane topology of the nucleoside transporter of human erythrocytes, which had been covalently photolabelled with [3H]nitrobenzylthioinosine, was investigated by monitoring the effect of proteinases applied to intact erythrocytes and unsealed membrane preparations. Treatment of unsealed membranes with low concentrations of trypsin and chymotrypsin at 1 degree C cleaved the nucleoside transporter, a band 4.5 polypeptide, apparent Mr 66 000-45 000, to yield two radioactive fragments with apparent Mr 38 000 and 23 000. The fragment of Mr 38 000, in contrast to the Mr 23 000 fragment, migrated as a broad peak (apparent Mr 45 000-31 000) suggesting that carbohydrate was probably attached to this fragment. Similar treatment of intact cells under iso-osmotic saline conditions at 1 degree C had no effect on the apparent Mr of the [3H]nitrobenzylthioinosine-labelled band 4.5, suggesting that at least one of the trypsin cleavage sites resulting in the apparent Mr fragments of 38 000 and 23 000 is located at the cytoplasmic surface. However, at low ionic strengths the extracellular region of the nucleoside transporter is susceptible to trypsin proteolysis, indicating that the transporter is a transmembrane protein. In contrast, the extracellular region of the [3H]cytochalasin B-labelled glucose carrier, another band 4.5 polypeptide, was resistant to trypsin digestion. Proteolysis of the glucose transporter at the cytoplasmic surface generated a radiolabelled fragment of Mr 19 000 which was distinct from the Mr 23 000 fragment radiolabelled with [3H]nitrobenzylthioinosine. The affinity for the reversible binding of [3H]cytochalasin B and [3H]nitrobenzylthioinosine to the glucose and nucleoside transporters, respectively, was lowered 2-3-fold following trypsin treatment of unsealed membranes, but the maximum number of inhibitor binding sites was unaffected despite the cleavage of band 4.5 to lower-Mr fragments.     

Mammalian nitrobenzylthioinosine-sensitive nucleoside transport proteins. Immunological evidence that transporters differing in size and inhibitor specificity share sequence homology.

Polyclonal antibodies were raised against the nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter of human erythrocyte membranes. On Western blots of these membranes they labeled the broad "band 4.5" region (average apparent M(r) 55,000), which contains both the nucleoside and glucose transport proteins. However, they did not recognize the glucose transporter when this was prepared free of nucleoside transporter by expression from a cDNA clone. Their specificity for the nucleoside transporter was confirmed by the ability to immunoadsorb NBMPR- but not cytochalasin B-binding sites from a detergent-solubilized mixture of band 4.5 proteins. Although a large proportion of the antibodies recognized extracellular epitopes, these appeared to be located primarily on the polypeptide moiety of the glycoprotein, as demonstrated by the ability of the antibodies strongly to label the deglycosylated transporter (apparent M(r) 45,000) on Western blots. The antibodies were species-cross-reactive, recognizing nucleoside transporters from pig and rabbit erythrocytes and from rat liver. The pig protein is similar to the human transporter in its inhibitor sensitivity but is considerably larger (apparent M(r) 57,000 after deglycosylation). In contrast, the rat protein is similar in size to the human transporter (apparent M(r) 45,000 after deglycosylation) but much less sensitive to the inhibitors dilazep and dipyridamole. These findings indicate that despite their differences in size and inhibitor specificity, the NBMPR-sensitive nucleoside transporters of these mammalian species are related in amino acid sequence.     

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.     

An improved purification procedure for rat liver lysosomal phospholipase A1

A purification procedure is presented for the isolation of lysosomal acid phospholipase A1 (PLA1) from livers of non-pretreated rats, in a high yield and purity. The purification starts from a crude mitochondrial-lysosomal fraction. PLA1 is solubilised and subsequently purified by chromatography on concanavalin A-Sepharose, by chromatofocusing, and by gel filtration. After chromatofocusing, the enzyme is already purified 50200-fold with a yield of 50%, and after gel filtration 56600-fold with a yield of 7%. Purified PLA1 exhibits a specific activity of approx. 8.2 mumol phosphatidylethanolamine (preferred substrate) hydrolysed per min per mg protein, and upon chromatofocusing an apparent isoelectric point of 5.3 Gel filtration of purified PLA1 suggests a molecular mass of about 29 kDa, whereas in SDS-PAGE two proteins of 27 kDa and 55 kDa (mass ratio about 1/2) were visualised.     

Purification and characterisation of bovine spleen ADPase.

ADPase has been purified from bovine spleen. Electrophoresis revealed a minor contaminent in the preparation that may represent ADPase that has been decreased in size by limited proteolysis during extraction and purification. The native enzyme behaves on SDS/PAGE as a 100-kDa protein but ADPase is a glycoprotein and so its electrophoretic behaviour may be anomalous. The apparent molecular mass is decreased to 70 kDa after removal of carbohydrate by treatment with a glycosidase. The use of a cross-linking reagent followed by electrophoresis suggests that the enzyme is composed of a single subunit. The specific activity of the purified material was 115 U/mg protein. The enzyme catalyses the hydrolysis of nucleoside di- and tri-phosphates but nucleoside monophosphates are not acted upon. The Km for ADP (approx. 10-15 microM) is unaffected by the state of purification of the enzyme, but catalytic activity of the purified material is stimulated by inclusion of detergent in the assay system and by calcium ions. Maximum activity is seen at pH 8.0-8.5 with ADP as substrate but the optimum shifts to 7.5-8.0 for ATP hydrolysis.     

Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody.

Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross-reactivity by inducing nonspecific protein aggregation.     

Identification, purification, and characterization of the rat liver golgi membrane ATP transporter

Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.     

Purification of a soluble monoisozyme of nucleoside diphosphate kinase from chick brain: Exploitation of ionic characteristics

A procedure for the rapid purification of nucleoside diphosphate kinase, 24 h with a single operator, from the chick brain soluble fraction is described. The influence of the ionic conditions on the association-disassociation properties of the enzyme are exploited to obtain yields of 30% from the crude homogenate. The enzyme has been purified 500-fold with a maximal specific activity of 1500 μmol/min/mg at 25°C (using thymidine diphosphate as the phosphate acceptor and ATP as the donor) and is demonstrated to be monoisozymic.     

Purification and characterization of wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase

The 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic phosphates (N greater than p) to nucleoside 2'-phosphates has been purified 16,000-fold to near homogeneity from wheat germ. The purified enzyme is a single polypeptide with a molecular weight of 23,000-24,000. It has a pH optimum of 7.0. The apparent Km values for A greater than p, G greater than p, C greater than p, and U greater than p are 13.1, 9.2, 25.2, and 25.3 mM, respectively. Vmax values for A greater than p, G greater than p, C greater than p, and U greater than p are 2090, 280, 2140, and 600 mumol/min/mg of purified protein, respectively. Wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase does not hydrolyze 2',3'-cyclic esters in cyclic phosphate-terminated oligoribonucleotides or in nucleoside 5'-phosphate, 2',3'-cyclic phosphate (pN greater than p). This is in contrast to the 3'-phosphodiesterase activity associated with a wheat germ RNA ligase which hydrolyzes cyclic phosphate-terminated oligonucleotides and pN greater than p substrates much more efficiently than nucleoside 2',3'-cyclic phosphates. The enzyme characterized in this work appears to be the only known 2',3'-cyclic nucleotide 3'-phosphodiesterase specific for 2',3'-cyclic mononucleotides.     

Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose transporter

Human red cell membranes were isolated and partially stripped of peripheral proteins by gel filtration of hemolysates on a Sepharose CL-4B column at pH 8 connected in tandem to a Sepharose CL-6B column at pH 10.5. The eluted material was washed by centrifugations, once at pH 10.5 and twice at pH 12. In this way, water-soluble proteins and peripheral membrane proteins were thoroughly removed, and 0.2 g of integral membrane proteins could be prepared within 10 h from 0.2 litre of red cells. The exposure to high pH did not lower the D-glucose transport activity, and electrophoretically pure glucose transport protein could be isolated from this preparation. Gel filtration in sodium dodecyl sulfate separated the integral membrane components into four fractions, one of them containing 4.5-material; gel electrophoresis showed about 14 zones and two-dimensional electrophoresis resolved up to 100 mostly minor components, among which the glucose transporter focused around pH 7. However, purified glucose transporter focused around pH 8. Glucose and nucleoside transport proteins were co-purified in active form on DEAE-cellulose and a fraction isolated by adsorption to Mono Q was used for immunization of mice and production of monoclonal antibodies. One hybridoma produced antibodies that reacted with material in the 4.5-region, possibly the glucose transport protein, and not with band 3-material. Upon two-dimensional electrophoresis of integral membrane components that had been solubilized with octyl glucoside the immunoreactive and the silver-stained 4.5-material focused in a broad range from pH 6 to pH 9. A possible explanation for this heterogeneity might be interaction between the glucose and nucleoside transport proteins and negatively charged lipids.     

Photoaffinity labelling of nucleoside-transport proteins in plasma membranes isolated from rat and guinea-pig liver.

Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.     

Biomembrane affinity chromatographic analysis of nitrobenzylthioinosine binding to the reconstituted human red cell nucleoside transporter

Solute interactions with membrane proteins can be analyzed by biomembrane affinity chromatography (BAC), previously applied to the human red cell glucose transporter. As a novel example, frontal BAC analysis of interactions between the nucleoside transport inhibitor nitrobenzylthioinosine (NBTI) and immobilized reconstituted nucleoside and glucose transporters from human red cells revealed two binding sites, presumably corresponding to the two transporters. The affinities and amounts of sites were determined by use of a double rectangular hyperbolic equation. The Kd value for NBTI binding to the nucleoside transporter in egg phospholipid proteoliposomes was 0.38 +/- 0.08 nM (22 degrees C, I = 0.16, pH 7.4), lower than previously reported for reconstituted systems. The molar ratio between the amounts of nucleoside transporter sites for NBTI and glucose transporter sites for cytochalasin B was 4.5 +/- 0.6%.     

Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site.

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.     

PURIFICATION OF RAT BRAIN CHOLINE ACETYLTRANSFERASE1

Abstract— The purification of choline acetyltransferase (ChAc) has been hampered by the increasing instability of the enzyme in the course of purification. By working with a high concentration of protein and by adding glycerol to the enzyme, the stability was increased. The purification was performed by centrifuging twice, at low and high salt concentrations, precipitation by ammonium sulphate and chromatography on carboxymethyl–Sephadex, hydroxylapatite and Sephadex G 100. The final steps were performed by using chromatography on an immunoabsorbent; this consists of agarose-coupled gammaglobulins of antisera devoid of any activity against ChAc itself and directed against other proteins still present in the purest ChAc preparation achieved by conventional biochemical techniques. The purest rat brain ChAc preparation had a specific activity of 20 μmol/min/mg of protein after a 30,000-fold purification. The enzyme was not homogeneous in polyacrylamide gel electrophoresis performed either at pH 4.5 or with sodium dodecyl sulphate. Pure ChAc from rat brain would have a specific activity of approximately 100 μmol/min/mg of protein.     

Characterization and purification of the neuronal sodium-ion-coupled 5-hydroxytryptamine transporter.

The selective 5-hydroxytryptamine (5-HT) uptake inhibitor, [3H]paroxetine, has been used as a specific probe in a series of experiments aimed at characterizing the substrate and 5-HT uptake inhibitor binding domains on the neuronal sodium-ion-coupled 5-HT transporter. In addition, an affinity-chromatographic protocol which provided a greater than 3000-fold purification of this neuronal transporter protein is outlined.     

Characterisation of monoclonal antibodies which specifically recognise the human erythrocyte glucose transport protein

Two monoclonal antibodies (mabs) of subclass IgG1 have been raised against the human erythrocyte glucose transport protein. The mabs bound to the purified glucose transporter in both its membrane-bound and detergent-solubilised forms. However, they exhibited little or no binding to the detergent-solubilised nucleoside transport protein, which is present as a minor contaminant in the glucose transport protein preparation. Both mabs inhibited the binding of cytochalasin B to the glucose transport protein, reducing the affinity of this binding by greater than 2-fold. Each mab labelled the transporter polypeptide on Western blots both before and after treatment of the protein with endoglycosidase F, indicating that the epitopes recognised were located on the protein moiety of the glycoprotein. However, the mabs did not bind to the large fragments produced by tryptic or chymotryptic digestion of the native protein, although both mabs were shown to bind to sites on the cytoplasmic surface of the erythrocyte membrane.