Erik Mascher

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.

The human red cell glucose transporter in octyl glucoside. High specific activity of monomers in the presence of membrane lipids.

Human red cell membranes were stripped of peripheral proteins and partially solubilized with 50-260 mM octyl glucoside at 2-14 mg protein/ml, to find conditions that afford a high concentration of active glucose transporter after purification on DEAE-cellulose. Transporter-egg yolk phospholipid vesicles were prepared by gel filtration. The specific D-glucose equilibrium exchange activities increased with increasing dilution of the glucose transporter. At 260 mM octyl glucoside the glucose transporter became partially denaturated. At 225 mM detergent the DEAE-cellulose chromatography showed one main and one minor fraction of active glucose transporter. Nucleoside transport activity was enriched in the minor fraction. Solubilization with 75 mM octyl glucoside at 8 mg protein/ml gave a maximal concentration of purified transporter, 0.8 mg/ml, probably corresponding to complete solubilization. The phospholipids were partially retarded on the DEAE-cellulose. The specific D-glucose equilibrium exchange was high, up to 200 nmol glucose/micrograms transporter in two min at 50 mM glucose. High performance gel filtration in octyl glucoside indicated that the transporter formed dimers during the fractionation. These eluted at Mr 125,000, partially separated from the phospholipids, which appeared at Mr 55,000 (cf. Mascher, E. and Lundahl, P. (1987) J. Chromatogr. 397, 175-186). The D-glucose transport activity was low in the main fraction and high in the transporter-phospholipid fraction. Mixing of these fractions did not increase the activity. The glucose transporter is probably dependent on one or more specific membrane lipid(s). Presumably the transporter dimerizes and loses activity upon removal of these lipids.

Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography in the presence of octyl glucoside and phosphatidylserine or phosphatidylcholine

The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyl glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric immediately after the ion-exchange purification: the Mr of the Glut 1 polypeptide was estimated to be 49,000 +/- 6000 by TSKgel G3000SW chromatography monitored by low-angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut 1, which was measured to be 1.13 +/- 0.03 ml mg-1 cm-1 at 280 nm, referring to the polypeptide concentration. The Mr value is consistent with the cDNA-deduced Mr 54,117 of the very similar HepG2 glucose transporter polypeptide. At 2 degrees C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane lipids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluent lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyl glucoside and PC was nearly as effective as PS.

Structure of dodecyl sulfate-protein complexes at subsaturating concentrations of free detergent.

Earlier neutron small-angle scattering experiments had revealed the low resolution structure of the complex between sodium dodecyl sulfate (SDS) and the single polypeptide (452 amino acid residues) of a water-soluble enzyme. The saturated complex consists of three globular micelles which are connected by short flexible polypeptide segments. New experiments, described here, were performed at subsaturating concentrations of free SDS in equilibrium with the complex. The data show a decrease in stoichiometry from one bound dodecyl sulfate (DS) anion per two amino acid residues near the critical micelle concentration (CMC) to one per four residues at half the CMC. At 0.3 CMC, a two-micelle complex is formed by the recombination of the small amino-terminal micelle with the middle one; and the center-to-center distance between the carboxyl-terminal micelle and the middle one decreases from 7.5 to 6.2 nm. These structural data allow us to better understand earlier results obtained with high-performance agarose gel chromatography of the same SDS-protein complexes.

High performance agarose gel chromatography in sodium dodecyl sulfate of integral membrane proteins from human red cells, with special reference to the glucose transporter

Integral membrane proteins from human red cells were fractionated in sodium dodecyl sulfate solutions by high performance gel filtration on the small-bead cross-linked agarose gel Superose 6. The components were identified by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The combination of Superose chromatography with electrophoresis afforded high resolution. As expected the gel filtration elution volumes depended essentially on the molecular mass, but the elution volumes decreased stepwise as the detergent concentration was increased from 0.6 to 100 mM, with the largest decrease for the glucose transporter. The resolution increased as the flow rate was decreased from 60 to 1 ml X cm-2 X h-1. The Mr values for the anion and glucose transporters as estimated by Superose 6-chromatography at 50 mM detergent were 75-80% of the corresponding Mr values obtained by electrophoresis. At 50 mM dodecyl sulfate the proteins were resolved into four fractions (a-d) which mainly contained: (a) dimer and (b) monomer of the anion transporter, (c) the glucose transporter and (d) components of Mr below 40 000. Monoclonal antibodies that possibly are directed against the glucose transporter (Lundahl, P., Greijer, E., Cardell, S., Mascher, E. and Andersson, L. (1986) Biochim. Biophys. Acta 855, 345-356) interacted only with part of the 4.5-material in fraction c in immunoblotting (Western blotting). Superose 6-chromatography of red cell glucose transporter that had been partially purified on DEAE-cellulose and Mono Q resolved one major and two minor fractions. Electrophoretic analysis showed that components of Mr 90,000, 50,000, and 25,000 had been separated from the major Mr-55,000-4.5-material and revealed size heterogeneity within the major chromatographic fraction. Heating of the glucose transporter in the presence of dodecyl sulfate caused an unexpected retardation of monomeric transporter on Superose 6. The apparent Mr decreased from 44,000 to 29,000.

Water-soluble proteins do not bind octyl glucoside as judged by molecular sieve chromatographic techniques

It is well known that the non-ionic detergent octyl glucoside (1-O-n-octyl-beta-D-glucopyranoside) solubilizes biological membrane components. It forms complexes with membrane-spanning proteins by hydrophobic interactions and it forms mixed micelles with membrane lipids. In contrast, non-ionic detergents usually do not bind to water-soluble proteins. According to a recent report, substantial and cooperative binding of octyl glucoside to several water-soluble proteins does occur near the critical micelle concentration. However, data have been obtained that contradict this report. No decrease was found in the elution volumes of five water-soluble proteins on molecular sieve chromatography on two Superose columns in tandem when 35 mM octyl glucoside was included in the eluent. No binding of the detergent to these proteins was observed at 20 or 22.5 mM octyl glucoside on molecular sieve chromatography on a TSK SW guard column as determined by differential refractometry and UV spectrophotometry of the proteins in the absence or presence of octyl glucoside. The experiments were done with the same buffer system and with six of the proteins used in the reported study. It is concluded that, as expected, there is no binding of octyl glucoside to water-soluble proteins above the detection limit (0.1 g detergent/g protein) of the refractometric method. The binding of, on average, 1.3 +/- 0.2 g of detergent per gram of water-soluble protein that was observed at 20 mM octyl glucoside in the reported study is not consistent with the present results.

High-performance agarose gel chromatography in octyl glucoside of integral membrane proteins from human red cells, with special reference to the glucose transporter

Integral membrane proteins and lipids from human red cells were fractionated in the presence of octyl glucoside by high-performance gel chromatography on a 22-ml column of the small-bead cross-linked agarose gel Superose 6, at 5 degrees C, pH 7.6 and 30-50 mM detergent. To avoid aggregation a relatively high flow-rate, 9 ml/h, was chosen. At low ionic strength four main fractions were resolved, which contained anion transporter multimers(I), glycophorin oligomers(II), glucose transporter dimers(III) and phospholipids(IV). In 0.5 M sodium chloride the resolution was lower but the yield of the glucose transporter was markedly higher, and chromatography of partially purified glucose transporter gave a protein recovery of about 90%. The apparent Mr values for the octyl glucoside complexes of the main components were: anion transporter, 900,000; glycophorin A, 210,000-360,000, dependent on ionic strength; glucose transporter, 110,000-160,000; lipids, 70,000. Some components aggregated with time: at a flow-rate of 1 ml/h mainly glycophorins and the glucose transporter were eluted, but no anion transporter, and fractionation performed 20 h after solubilization showed extensive aggregation of proteins. Superose-6 chromatography of glucose transporter and membrane lipids that had been isolated on DEAE-cellulose partially resolved the transporter and the phospholipid fractions. In this case, the resolution was better with 50 than with 30 mM detergent. The maximum glucose transport activity was approximately one-tenth of that observed before fractionation and appeared in two main fractions, at the main transporter fraction as well as at the overlap between the transporter and the lipids. The activity level was the same in both fractions, although the protein concentration was much lower in the second one. Addition of 2 mM egg-yolk phospholipids to the eluent did not increase the activity. This strongly indicates that the glucose transporter needs some specific membrane lipids to retain high transport activity. At the concentration of ca. 0.3 mg/ml used, the glucose transporter was probably eluted as a dimer in the absence of phospholipids and as a monomer in their presence.

Rapid reversed-phase high-performance liquid chromatography method for quantitation, at high pH, of the recombinant apolipo-protein A-IMilano in Escherichia coli fermentation broth

An automated reversed-phase high-performance liquid chromatography method for quantitative determination of recombinant apolipoprotein A-IMilano (r-Apo A-IM) in E. coli fermentation broth has been developed and evaluated. The use of a unique matrix (Poros IIR/H) makes it possible to achieve rapid separation and good resolution at high pH. The r-Apo A-IM-containing fraction is well separated from other proteins allowing a reliable quantification. The automation and high sample throughput of this method makes it very useful for routine determination of r-Apo A-IM in fermentation broth and in eluates from the various purfication steps. With suitable modifications and adaptions this method is likely to be useful for similar rapid analytical determination of recombinant proteins in complex solutions.