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.     

Structure of the cytochrome c oxidase complex of rat liver. 2. Topological orientation of polypeptides in the membrane as studied by proteolytic digestion and immunoblotting

The orientation of the thirteen polypeptides of rat-liver cytochrome c oxidase in the inner mitochondrial membrane was studied by proteolytic digestion of mitoplasts and sonicated particles. After separation by sodium dodecylsulfate gel electrophoresis proteins were transferred on nitrocellulose, and individual polypeptides were identified by incubation with polypeptide-specific antisera, followed by fluorescein-isothiocyanate-conjugated protein A. The three catalytic polypeptides I-III and seven nuclear coded polypeptides (IV, Vb, VIa, VIc, VIIa, VIIb and VIII) were found accessible to proteases from the cytoplasmic phase. Polypeptides II, IV, Va, Vb and VIa were accessible from the matrix phase, indicating a transmembraneous orientation of polypeptides II, IV, Vb and VIa. Together with data on cross-linking and on cytochrome-c-protected labeling of polypeptides, a model of the cytochrome c oxidase complex was developed. It is suggested that the cytochrome c binding site on polypeptide II is surrounded by several nuclear-coded polypeptides, which may modulate the affinity of the enzyme towards cytochrome c.     

Two-dimensional protein mapping by gold stain and immunoblotting

A method is described for mapping individual polypeptides on a two-dimensional separation of a complex mixture of proteins. Polypeptides are identified by immunoblotting on nitrocellulose prestained for total protein by a colloidal gold stain which does not inhibit antibody binding. The same polypeptides can subsequently be identified on gold-stained blots run under identical conditions, without the need for immunoblotting.     

Ionophore properties of peptide fragments of brain Na+,K+-Atpase

Polypeptide fragments from Na+, K+-ATPase of cattle brain are obtained by the bromocyan-treatment of the protein and subsequent gel filtration via sephadexes; the fragments were reconstructed into bilayer lipid membranes (BLM). Polypeptides of fractions I and II induce cationic and polypeptides of fraction IV--cationic-anionic conductivity of BLM. Neither sodium nor potassium selectivity of BLM modified by protein fragments of fractions I and II was observed. Fluctuations of the modified membranes current are of spasmodic character, ATP and inhibitors of the sodium pump do not affect them. The induction of current fluctuations peculiar to channels into BLM is supposed to be a character of polypeptides obtained after the ATPase splitting but not of the cation-transport system of the sodium pump.     

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins

We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.     

Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels

A protein which inhibited complement channel formation was isolated from extracts of papain-digested human erythrocyte membranes using DEAE-Sephacel, Bio-Gel A0.5m column chromatographies, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and elution with 2% NP-40 solution. The purified protein showed a molecular weight of 18 kDa, and efficiently inhibited hemolysis of EC5-7 cells with C8 and C9, but did not show any decay-accelerating activity to C5 convertase. Immunochemical analysis of native membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the antibody against this protein gave a single band having the same mobility as this protein; papain did not eliminate a significant portion of this protein.     

The erasable Western blot

A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a 10-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70 degrees C in 2% (w/v) sodium dodecyl sulfate containing 100 mM beta-mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed.     

Protein organization of rat synaptic plasma membranes and synaptic vesicles: A one- and two-dimensional study

The protein organization of rat brain synaptic plasma membranes (SPM) and synaptic vesicles (SV) was investigated by surface iodination and one- and two-dimensional electrophoresis. Polypeptides of molecular weights (MWs, in Kilodaltons) 170 K, 135 K, 96-86 K, 68-64-61 K, 56 K, 52 K, 38 K, 35-33 K, and 18 K are predominantly or exclusively exposed on the extracellular side of synaptosomes. Several polypeptides of MW between 70 K and 40 K are exclusively exposed on the cytoplasmic side of SPM. The use of two-dimensional electrophoresis allowed to recognize that, for some classes of MW, there are polypeptides of nearly the same MW and different isoelectric points exposed on both sides of SPM. The synaptosomal membrane shows a predominance of acidic proteins on the extracellular side and more neutral and basic proteins on the cytoplasmic side. With respect to SPM, SV are particularly enriched with polypeptides of MW 71 K, 56 K, 39-38 K, 32 K, 16 K, and 15 K. One of them, a doublet of MW 39-38 K, is the most highly labeled species upon surface iodination and is similar, but not identical, with a doublet located on the cytoplasmic side of SPM.     

Sub-set characteristics of DNA sequences involved in tight DNA/polypeptide complexes and their homology to nuclear matrix DNA

Polypeptides co-isolating with DNA induce the binding of a fraction of native DNA fragments to nitrocellulose filters. Southern analysis reveals a high intensity of self-hybridization of the DNA sequences retained on nitrocellulose filters. Consistently, the DNA fraction passing the filters shows only weak hybridization when probed with DNA retained on filters. This indicates that the DNA/polypeptide complexes reside on a non-random sub-set of DNA sequences. Moreover, a high degree of homology was found between residual nuclear matrix DNA sequences and the DNA sequences retained on nitrocellulose filters. This indicates that the DNA sequences associated with tightly bound polypeptides originate from sites where the genome is salt-stably anchored in the nuclear matrix.     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.     

A two-dimensional electrophoretic analysis of the proteins and glycoproteins of liver plasma membrane domains and endosomes. Implications for endocytosis and transcytosis.

1. Polypeptides of liver plasma membrane fractions enriched in three surface domains of hepatocytes, blood-sinusoidal, lateral and bile canalicular, were analysed by isoelectric focusing (IEF) and non-equilibrium pH gel electrophoresis (NEPHGE) across a wide pH range, followed by SDS/PAGE. The overall Coomassie Blue-stained polypeptide patterns in the fractions were different. lateral plasma membrane fractions contained a characteristically higher number of polypeptides focusing at the basic pH range, whereas few basic polypeptides were present in sinusoidal plasma membrane fractions. The glycoproteins in these plasma membrane fractions stained by a lectin overlay technique with radio-iodinated concanavalin A, wheat-germ agglutinin and a slug lectin, were also different. 2. The polypeptides and glycoproteins of 'early' and 'late' endosome fractions were also compared by two-dimensional electrophoresis. Their composition was shown by Coomassie Blue staining, lectin overlay staining and in membranes metabolically labelled with [35S]methionine to be generally similar. The glycoproteins of sinusoidal plasma membranes and early and late endosomes were generally similar, but major differences in polypeptides of molecular mass 20-50 kDa, pI 7.5-8.5, in plasma membranes and endosomes were demonstrated, with a specific population of basic (pI 8-9) low-molecular-mass polypeptides being present at highest levels in 'late' endosomal fractions (shown by Coomassie Blue staining). 3. Analysis of the distribution of three specific membrane glycoproteins identified by using immunoblotting techniques showed that the asialoglycoprotein and the divalent-cation-sensitive mannose 6-phosphate receptors were present in sinusoidal plasma membrane and in early and late endocytic fractions: they were not detected in canalicular plasma membrane fractions. In contrast, 5'-nucleotidase was detected in all fractions examined. The role of the endocytic compartment in regulating trafficking pathways between the plasma membrane domains of the hepatocyte is discussed.     

A high molecular weight topoisomerase I from Xenopus laevis ovaries

DNA topoisomerase I has been purified from homogenates of mature Xenopus laevis ovaries. The initial stages in purification of the native enzyme employed a rapid series of three chromatographic steps, followed by gel filtration performed in the presence of sodium dodecyl sulfate. Polypeptides that might represent topoisomerase I were identified by specific labeling of the topoisomerase species with radioactive DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of topoisomerase I radiolabeled with DNA identified three polypeptides with mobilities consistent with sizes of 165, 125, and 88 kDa. All three polypeptides were found to possess topoisomerase activity following elution from the gel and renaturation. Partial proteolytic digestion of the radiolabeled 165-, 125-, and 88-kDa polypeptides with Staphylococcus aureus V8 endoproteinase resulted in identical autoradiographic patterns. This suggests that the 125-kDa and 88-kDa polypeptides may be degradation products of the 165-kDa species. The 165-kDa topoisomerase I exhibited the same sensitivity to camptothecin as the total, native topoisomerase I fraction.     

Binding of zona pellucida proteins to a boar sperm polypeptide of Mr 53,000 and identification of zona moieties involved

Experiments have been carried out to identify proteins on boar spermatozoa that bind to components of the zona pellucida. Polypeptides in sodium deoxycholate extracts of boar spermatozoa and in whole seminal plasma have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose sheet by electroblotting and probed with 125I-labelled heat-solubilized zona pellucida from pig oocytes or ovulated eggs. Zona proteins bound avidly and consistently to a polypeptide of Mr 53,000 on blots of capacitated and noncapacitated sperm and weakly to polypeptides of Mr 67,000, 38,000 and 18,000. On blots of seminal plasma the 125I-labelled probes bound to two polypeptides of Mr 65,000 and 19-24,000. Identification of the zona proteins that were binding to the aforementioned proteins on blots showed that all the major zona pellucida glycoproteins were involved, including those acquired from oviduct secretions. Binding of 125I-ovulated zona pellucida to the polypeptide of Mr 53,000 also occurred in extracts of testicular and epididymal boar spermatozoa. The results are discussed in relation to sperm-egg recognition in the pig.     

Direct binding of F-actin to ponticulin, an integral plasma membrane glycoprotein

We have developed an 125I-labeled F-actin blot overlay assay for the identification of F-actin-binding proteins after transfer to nitrocellulose from SDS-polyacrylamide gels. Two major F-actin-binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F-actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin-binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J., [1987]: J. Cell Biol. 105:1741-1751). The binding of F-actin to ponticulin on blots is specific because unlabeled F-actin competes with 125I-labeled F-actin and because G-actin does not bind. Nitrocellulose-bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F-actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125-I-labeled F-actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2-D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F-actin in proportion to their abundance. Thus the actin-binding activies of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin-binding activity of nitrocellulose-bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I-labeled F-actin blot overlay assay also may enable us to identify F-actin-binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.     

LIGHT-HARVESTING COMPLEX AF'OPROTEINS IN CYTOPLASMIC VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Cells of Chlamydomonas reinhardtii Dangeard strain cw15arg7A contain electron-opaque material, often in the form of large granules, within cytoplasmic vacuoles. Immunoelectron microscopy with antibodies to polypeptide 11, a component of the major light-harvesting chlorophyll (Chl) a/b-protein complex (LHCII,) of thylakoid membranes, revealed the presence of LHCII Polypeptides within the chloroplast and in vacuolar material in cells grown in the light. Vacuolar material was also heavily immunodecorated in dark-grown cells that did not synthesize Chl. Accumulation of LHCII polypeptides was further studied in greening and light-grown cells of a pale green mutant, deficient in LHCII, that was derived from cu15arg7A by insertional mutagenesis. Light-grown cells of this mutant strain contained relatively few thylakoid membranes and synthesized LHCII polypeptides at a low rate. However, cytoplasmic vacuoles were immunoreactive. Appearance of mature-sized LHCII polypeptides in vacuoles suggested that these proteins were partially translocated across the envelope but not retained by the chloroplast without assembly of LHCII.     

Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after "Western" blotting

Sections of nitrocellulose containing bound 32P-labeled polypeptides were excised from "Western" blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the 32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.     

Low molecular mass GTP-binding proteins of adrenal chromaffin cells are present on the secretory granule

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS-polyacrylamide gel electrophoresis and GTP-binding proteins detected using [alpha-32P]GTP binding to nitrocellulose blots. Four GTP-binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP-binding polypeptides co-sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP-binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP-binding proteins could be involved in exocytosis in adrenal chromaffin cells.     

Polypeptides of the Golgi Apparatus of Neurons from Rat Brain

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

High-performance gel permeation chromatography of proteins and peptides on columns of TSK-G2000-SW and TSK-G3000-SW: a volatile solvent giving separation based on charge and size of polypeptides

Trifluoroacetic acid (0.1% w/v) is an excellent solvent for polypeptides, is volatile, and has a low absorbance of ultraviolet light of low wavelength. Polypeptides subjected to chromatography on columns of TSK-G2000-SW or TSK-G3000-SW in this solvent were eluted as sharp peaks. Retention volume was dependent not only on molecular weight but also on the number of formal charges per molecule. For polypeptides with a similar molecular weight, that with the highest proportion of basic amino acid residues was eluted earliest. For the TSK-G2000-SW column, the degree of deviation from a linear relationship between elution volume and logarithm of molecular weight was directly proportional to the molecular weight to the power 2/3 divided by the number of positive charges per molecule. Inclusion of 0.25 M sodium chloride in the solvent increased both the upper and lower limits of the molecular weight range over which separation occurred. The use of 0.1% trifluoroacetic acid as the mobile phase is thus particularly applicable to the separation of peptides of low molecular weight.