Sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase chromatography of unfolded proteins

Sequential sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography on a fluorocarbon packing (poly F column, DuPont) provide fully denatured but highly purified protein, which is free of low-molecular-weight substances and directly amenable to structural analysis. Conditions for the preparative elution of four test proteins (bovine serum albumin, carbonic anhydrase, myoglobin, cytochrome c) blotted to Immobilon membranes have been optimized. Phenol saturated with 50 mM Tris-HCl (pH 8.25) and containing 2% SDS and 1% 2-mercaptoethanol is able to elute proteins that have been blotted to Immobilon membranes, stained with Coomassie R-250, and stored as dry sheets, largely irrespective of their molecular mass. Polypeptides that are not degraded by exposure to low pH can also be efficiently extracted directly from stained gels with 70% formic acid. If further separation of polypeptides is not needed, a simple run of a protein sample dissolved in 1-2% SDS on the poly F column will efficiently remove low-molecular-weight substances, including SDS.     

Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Solubilisation of human erythrocyte band 4.1 protein in the non-ionic detergent Tween 20

Extraction of spectrin-depleted erythrocyte membranes with the non-ionic detergent Tween 20, in a 0.1 M glycine-NaOH buffer (pH 9.8) leads to the solubilization of band 4.1 and the sialoglycoproteins. The comigration of band 4.1 with the sialoglycoproteins in gel filtration and detergent-free electrophoresis indicated that these proteins may be associated as complexes of high molecular weight. Although treatment of intact membranes with Tween 20 under the same conditions does not lead to direct solubilization of proteins, severe disruption of the membranes was observed under phase contrast microscopy. Suspension of the treated membranes in 5 mM phosphate buffer (pH 8.0) leads to the solubilization of band 4.1, spectrin, actin and the sialoglycoproteins. High molecular weight complexes of band 4.1 and the sialoglycoproteins were isolated from these extracts, suggesting a possible interaction between band 4.1 and sialoglycoproteins which may be important for linking the cytoskeleton to the membrane.     

The stereospecific d-glucose transport activity of cholate extracts from human erythrocyte membranes

The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at ?70°C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high d-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90 000, 23% of the polypeptides of molecular weight around 55 000, 30% of the phospholipids and at least 6% of the stereospecific d-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4°C the activity of the extrac decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.     

Alterations of mouse embryo cells during in vitro aging

Trichocysts from the ciliated protozoan Paramecium aurelia have been solubilized in aqueous solution at neutral pH by heating to temperatures of 70 °C for 10 min. The product of such solubilization appears identical to that achieved by the previous method using trichocysts solubilized in sodium dodecyl sulfate (SDS) when examined by SDS polyacrylamide disc gel electrophoresis. While the solubilization of the trichocyst is virtually total in distilled water, no more than 20% of the total protein solubilizes in the presence of salts even in amounts as low as 0.05 M. Heat solubilized trichocysts show two principal components when run on standard 10% polyacrylamide disc gels and two bands when run on SDS polyacrylamide disc gels. The relationship between the two bands seen on standard gels and the two bands seen on SDS gels is unknown at this time. These results represent the first successful solubilization of this organelle in aqueous solution at neutral pH as opposed to previous reports requiring strong denaturants such as SDS, guanidine hydrochloride, and pH extremes. While the solubilization of trichocysts by heat most probably results in denaturation of the constituent proteins, it does occur in aqueous solution without the use of strong denaturants or pH extremes allowing the use of standard analytical procedures not possible in the presence of these reagents.     

Identification and partial purification of the insulin-responsive glucose transporter from 3T3-L1 adipocytes

The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte [3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside [1.0% (w/v) + asolectin] solubilized over 90% of the [3H]NBMPR-binding sites, with near-complete retention of [3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating [3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.     

Studies on toxin of Aspergillus fumigatus. XXII. Fashion of binding of Asp-hemolysin to human erythrocytes and Asp-hemolysin-binding proteins of erythrocyte membranes

The fashion of binding of Asp-hemolysin to human erythrocytes and the isolation of Asp-hemolysin-binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp-hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp-hemolysin showed diffuse, ring-like or cap-like staining by the peroxidase-labeled antibody method under the light microscope. The distribution of Asp-hemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate-0.1 M Tris-HC1 buffer (pH 7.5) containing 0.2 M NaCl and 1 mM EDTA, and the extract was chromatographed on an affinity column consisting of Asp-hemolysin attached to activated thiol-Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol-Sepharose 4B and eluted with 50 mM cysteine as toxin-membrane components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.     

The spore wall and polar tube proteins of the microsporidian Nosema grylli: the major spore wall protein is released before spore extrusion

Incubation of Nosema grylli spores in alkaline--saline solution (10 mM KOH, 170 mM KCl) leads to solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year) in water or 0.02% sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting. IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40 under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral solution leads to their discharge. Subsequent wash of discharged spores with 1-3% SDS, 9 M urea and treatment by 100% 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular weights about 46, 34, 21 and 15 kDa.     

The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes

The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus. On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin (3/1). In the bovine erythrocytes and their ghosts, the increase by 40-50% or the decrease by 10-23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40-50%, respectively. The depletion of ATP (less than 0.9 mg ATP/100 ml packed erythrocytes) enhanced K+ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mM MgCl2 alone or in the presence of 1 mM MgCl2 and 1 mM CaCl2. Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mM CaCl2 up to 81% of total activity, without appreciable K+ leakage and hot or hot-cold hemolysis. These results suggest that the presence of cholesterol or phosphatidylethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B. cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes.     

Solubilization of lipids from hamster bile-canalicular and contiguous membranes and from human erythrocyte membranes by conjugated bile salts.

We have demonstrated in vitro the efficacy of the taurine-conjugated dihydroxy bile salts deoxycholate and chenodeoxycholate in solubilizing both cholesterol and phospholipid from hamster liver bile-canalicular and contiguous membranes and from human erythrocyte membrane. On the other hand, the dihydroxy bile salt ursodeoxycholate and the trihydroxy bile salt cholate solubilize much less lipid. The lipid solubilization by the four bile salts correlated well with their hydrophobicity: glycochenodeoxycolate, which is more hydrophobic than the tauro derivative, also solubilized more lipid. All the dihydroxy bile salts have a threshold concentration above which lipid solubilization increases rapidly; this correlates approximately with the critical micellar concentration. The non-micelle-forming bile salt dehydrocholate solubilized no lipid at all up to 32 mM. All the dihydroxy bile acids are much more efficient at solubilizing phospholipid than cholesterol. Cholate does not show such a pronounced discrimination. Lipid solubilization by chenodeoxycholate was essentially complete within 1 min, whereas that by cholate was linear up to 5 min. Maximal lipid solubilization with chenodeoxycholate occurred at 8-12 mM; solubilization by cholate was linear up to 32 mM. Ursodeoxycholate was the only dihydroxy bile salt which was able to solubilize phospholipid (although not cholesterol) below the critical micellar concentration. This similarity between cholate and ursodeoxycholate may reflect their ability to form a more extensive liquid-crystal system. Membrane specificity was demonstrated only inasmuch as the lower the cholesterol/phospholipid ratio in the membrane, the greater the fractional solubilization of cholesterol by bile salts, i.e. the total amount of cholesterol solubilized depended only on the bile-salt concentration. On the other hand, the total amount of phospholipid solubilized decreased with increasing cholesterol/phospholipid ratio in the membrane.     

Detergent-resistant membranes in human erythrocytes and their connection to the membrane-skeleton

In cell membranes, local inhomogeneity in the lateral distribution of lipids and proteins is thought to existin vivo in the form of lipid ‘rafts’, microdomains enriched in cholesterol and sphingolipids, and in specific classes of proteins, that appear to play specialized roles for signal transduction, cell-cell recognition, parasite or virus infection, and vesicular trafficking. These structures are operationally defined as membranes resistant to solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). This definition appears to be necessary and sufficient, although additional manoeuvres, not always described with sufficient detail, may be needed to ensure isolation of DRMs, like mechanical homogenization, and changes in the pH and/or ionic strength of the solubilization medium. We show here for the human erythrocyte that the different conditions adopted may lead to the isolation of qualitatively and quantitatively different DRM fractions, thus contributing to the complexity of the notion itself of lipid raft. A significant portion of erythrocyte DRMs enriched in reported lipid raft markers, such as flotillin-1, flotillin-2 and GM₁, is anchored to the spectrin membrane-skeleton via electrostatic interactions that can be disrupted by the simultaneous increase in pH and ionic strength of the solubilization medium. An erratum to this article is available at .     

Membrane-associated phosphoproteins in Plasmodium berghei-infected murine erythrocytes

Normal and Plasmodium berghei (NYU-2 strain)-infected murine erythrocytes display substantially different patterns of plasma membrane phosphoproteins phosphorylation. Intact erythrocytes (normal and parasite infected) incubated with 32Pi and isolated washed erythrocyte plasma membranes incubated with gamma-32P-ATP were analyzed for phosphoproteins by SDS PAGE and autoradiography. Two new phosphoproteins of molecular weight 45,000 (pp45) and 68,000 (pp68), which are absent in normal erythrocyte membranes, are associated with the membranes of infected erythrocytes subjected to both intact-cell and isolated-membrane phosphorylation conditions. Two-dimensional gel electrophoresis indicates that pp45 and pp68 are of parasite origin. Partial or complete proteolytic digestion reveals that pp45 is phosphorylated at similar amino acid residues both in intact cells and in isolated membranes. The pp45 phosphoprotein can be detected at as low as 3% parasitemia and its phosphorylation is not affected by 10 microM cAMP, 1 mM Ca2+, or 5 mM EGTA. Extraction of isolated washed plasma membranes with 0.5% Triton X-100 or 0.1 M NaOH indicates that pp45 is detergent insoluble and only partially extractable with NaOH, suggesting that pp45 is closely associated with the host erythrocyte plasma membrane.     

Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland.

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.     

Type B nucleoside-diphosphatase of rat brain. Purification and properties of an enzyme with high thiamin pyrophosphatase activity

Type B nucleoside-diphosphatase was purified from membranes of rat brain by solubilization with a non-ionic detergent and successive column chromatographies on DEAE-cellulose DE-52, concanavalin-A-Sepharose, Bio-Gel HT, blue-Sepharose CL-6B, chelating Sepharose 6B, Ultrogel AcA44 and TSK gel G3000 SW. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis and its molecular mass was estimated to be 75 kDa. It hydrolyzed thiamin diphosphate as well as GDP, IDP and UDP. Thiamin diphosphate (TPP) was hydrolyzed twice as efficiently as nucleoside diphosphates in the presence of Mn2+ at pH 7.4. The Km values for TPP, GDP, IDP and UDP were 0.66, 0.40, 0.54 and 1.06 mM respectively. ATP, ADP and pyridoxal 5'-phosphate inhibited thiamin-pyrophosphatase activity competitively and their Ki values were 2.3 mM, 1.0 mM and 0.59 mM respectively. The optimum pH of thiamin-pyrophosphatase activity was 7.4 in the presence of Mn2+ and that of GDP-hydrolytic activity was 6.5 in the presence of Mg2+.     

Chemical modification of the anion selectivity of the PhoE porin from the Escherichia coli outer membrane

The PhoE porin of Escherichia coli is induced by phosphate deprivation and when purified, forms moderately anion-selective channels in lipid bilayer membranes. To further investigate the basis of anion selectivity, PhoE was chemically acetylated with acetic anhydride. Acetylation modified the mobility and staining characteristics of the PhoE porin on SDS-polyacrylamide gel electrophoresis but the acetylated protein was still found in its normal trimeric state after solubilization in SDS at low temperatures. Furthermore, the acetylated PhoE porin retained its ability to reconstitute into lipid bilayer membranes and the single channel conductance in 1 M KCl was unaltered. Zero-current potential measurements demonstrated that whereas the native PhoE porin was anion-selective, a 30-40-fold increase in preference for cations upon acetylation resulted in the acetylated PhoE porin being cation-selective. Increasing the pH of KCl solutions bathing lipid bilayer membranes from pH 3 to pH 6 caused symmetrical 4-fold increases in the selectivity of both the native and acetylated PhoE proteins for cations. In contrast, increasing the pH from 7 to 9 caused a 2.5-fold increase in selectivity only for the native PhoE porin. These results suggest that the basis of anion selectivity in the native PhoE porin is fixed protonated amino groups (possibly on lysines) in or near the channel, and furthermore indicate that deprotonated carboxyl groups have a strong influence on ion selectivity.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extraction and purification of the chief glycoprotein of the erythrocyte membrane

The major glycoprotein of the human erythrocyte membrane has been released from ghosts, by the non-ionic detergent Tween 20 at pH 8,5 and at low ionic strength and further purified by successive passages through columns of DEAE Sephadex at pH 6,8 and CM Sephadex at pH 5 or by hydroxyapatite chromatography at pH 6,8. The purified glycoprotein thus obtained represents about 1 % of the membrane proteins, and shows two major bands upon polyacrylamide gel electrophoresis. These bands designed as PAS 1 and PAS 2 are the dimer and the monomer of the glycoprotein. Several other minor bands can also appear on SDS gels, according to experimental conditions of solubilization and purification and are probably oligomers of PAS 1 and PAS 2. This glycoprotein possess inhibitory activity against various phytohemagglutinins.