Resolution of microsomal membranes into fractions differing in polypeptide composition

Microsomes from rat liver, prepared by gel filtration, were subjected to centrifugation in a continuous sucrose density gradient containing a low concentration of deoxycholate. The membranes were subfractionated into five bands differing in appearance and equilibrium density. Each band, when analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, displayed a characteristic population of membrane proteins.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate

The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg(2+) during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.     

Separation and characterization of two distinct hemagglutinins contained in purified leukocytosis-promoting factor from Bordetella pertussis.

1. The leukocytosis-promoting factor of Bordetella pertussis was found to contain two hemagglutinins with different susceptibilities to papain and separable from each other by agarose gel filtration with Tris - HCl buffer containing 1 M NaCl. 2. One hemagglutinin, referred to as hemagglutinin HA, had a high hemagglutinating activity, but neither leukocytosis-promoting nor histamine-sensitizing activity. The other hemagglutinin, referred to as hemagglutinin LPF appeared to be identical with the leukocytosis-promoting factor and possessed a low hemagglutinating and high leukocytosis-promoting and histamine-sensitizing activities. 3. The hemagglutinating activity of hemagglutinin HA was highly sensitive to papain. The hemagglutinating, leukocytosis-promoting, and histamine-sensitizing activities of hemagglutinin LPF were fairly resistant to the enzyme. 4. The two hemagglutinins were distinct from each other in immunological and chemical properties. 5. Morphologically, hemagglutinin HA showed itself to be filamentous molecules of approx. 2 X 40 nm, while hemagglutinin LPF comprised of spherical molecules of approx. 6 nm diameter. 6. The molecular weight values of hemagglutinin HA estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and sucrose density gradient centrifugation were approx. 126 000 and 133 000, respectively. Those of hemagglutinin LPF estimated by polyacrylamide gel electrophoreis at pH 4.5, sucrose density gradient centrifugation and gel filtration on a 10% agarose column were 107 000, 103 000 and 30 000, respectively. A possible reason for obtaining such a low molecular weight value by gel filtration is discussed.     

Association of three chicken proteins with the 34 kD target of rous sarcoma virus tyrosine kinase

Transformation of chicken embryo fibroblasts by avian retroviruses induces the tyrosine phosphorylation of a 34-39 kD cellular protein (p34). In vitro, p34 isolated from intestine interacts with F-actin in a Ca2+-dependent manner. We report here that, in the absence of Ca2+ chelators, three proteins co-purified with p34 extracted from a cytosolic or membrane fraction of chicken embryo fibroblasts; these two fractions account respectively for 10-20% and 50% of the total cellular p34. Isolated from the cytosoluble fraction of fibroblasts by sucrose gradient centrifugation and hydrophobic chromatography, p34 and the other proteins behaved as a homogeneous species upon non-denaturing gel electrophoresis, gel filtration, and CsCl density gradient centrifugation, thus indicating a strong association. Moreover, an analysis by electron microscopy following uranyl acetate staining revealed particles with a raspberry-like shape. This association was always disrupted by the calcium-chelating agent, EGTA.     

Nematode chromosomal proteins--I. Isolation of chromatin and preliminary characterization of the chromosomal proteins of Caenorhabditis elegans.

1. A procedure is described which gives clean chromatin preparations from the free-living nematode Caenorhabditis elegans. It involves homogenization using glass beads, collection of the precipitate from a low speed centrifugation, removal of cell membranes with Triton X-100, several washes with 0.14 M NaCl, sucrose density gradient centrifugation, a cycle of extraction and reprecipitation using dilute Tris buffer and 0.14 M NaCl respectively, and final extraction of the purified deoxyribonucleoprotein in 10 mM Tris-HCl (pH 8). 2. Acidic urea gel electrophoresis of the histones from C. elegans yielded 4 main groups which were preliminary identified as H1, H2a (+ H3?), H2b, H4 and moved on the gels in that order of increasing mobility. the coincidence of histone H3 with H2a was putative, but its presence was firmly suggested by the generation of a dimeric form in oxidizing conditions. 3. By SDS-Tris-glycine gel electrophoresis of the non-histone chromosomal proteins of C. elegans, about 18 proteins were distinguished with molecular weights ranging from 15,000 to 100,000 daltons.     

Isolation of the intercellular glycoproteins of desmosomes

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.     

Isolation of Fc receptor shed from pig lymphocytes by a temperature shift

Lymphocytes obtained from pig blood by gradient centrifugation were subjected to a temperature shift (4 to 37 degrees C). The proteins released from the plasma membrane were fractionated by affinity chromatography using immunoglobulin G immobilized on fine polyamide particles. The main component liberated from the adsorbent by diethylamine buffer (pH 11.5) exhibited an apparent Mr of 18000-20000 in SDS-polyacrylamide gel electrophoresis. This crude receptor preparation possessed a substantially higher affinity to immobilized immunoglobulin G than to immobilized Fab fragment and inhibited significantly the binding of labeled immunoglobulin G to pig lymphocytes.     

Isolation and chemical characterization of two distinct "link proteins" from bovine nasal cartilage proteoglycan complex

Bovine nasal cartilage proteoglycan aggregates have been dissociated and separated by dissociative density gradient centrifugation into proteoglycan sub-units and "link fraction". The latter contained mainly the two "link proteins" as shown by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two "link proteins" were then separated by preparative gel electrophoresis under dissociative conditions. Molecular weight and amino acid composition of both proteins are presented.     

Analysis of rat serum apolipoproteins by isoelectric focusing. I. Studies on the middle molecular weight subunits.

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.     

Isolation and Characterization of Outer and Cytoplasmic Membranes from Spheroplasts of Acetobacter aceti

The spheroplast membrane of Acetobacter aceti IFO 3284 was separated into outer and cytoplasmic membranes by alkaline sucrose density gradient centrifugation after treatment of the cells with lysozyme in sucrose-EDTA, pH 8.0. The cytoplasmic membrane, which was transparent and red colored, showed a specific gravity of 1.15 g/ml, and a number of protein components. High contents of heme b and heme c, and high enzyme activities of various membrane-bound primary dehydrogenases, which are characteristics of acetic acid bacteria, were found in the cytoplasmic membrane fraction. On the other hand, the outer membrane, which was white and turbid when homogenized, exhibited a high content of 2-keto-3-deoxyoctonate, and only four major polypeptides were observed on SDS-polyacrylamide gel electrophoresis. The outer membrane showed a specific gravity of 1.25 g/ml due to its high lipopolysaccharide content. A predominant species of the outer membrane proteins, tentatively designated as AI, was found to be heat-modifiable in SDS solution. The Al peptide on SDS-polyacrylamide gel showed varied migration, from a position corresponding to 31,000 daltons to one of 37,000 daltons, when heated at over 60°C and then subjected to gel electrophoresis.     

Dengue virus-induced modifications of host cell membranes.

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.     

Isolation ofSpiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis

This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction, which consisted essentially of extended sheets of membranes, exhibited membrane-boundpara-nitrophenylphosphatase specific activity 1.5-fold over that of the whole-cell lysate. Only traces of soluble NADH oxidase were present in the membrane preparation. The latter fraction appeared homogeneous upon sorbitol density gradient centrifugation and banded at an equilibrium density of 1.107 g/ml. The plasma membrane proteins were then analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 40 different proteins were detected in the membrane preparations. By comparison with the patterns obtained for whole-cell extracts and cytoplasmic fractions, a protein map ofS. citri could be established in which membrane and cytoplasmic proteins were identified.     

Origin and Structure of the Group-Specific, Complement-Fixing Antigen of Rickettsia rickettsii

Rickettsia rickettsii was treated with ether and examined by negative-contrast electron microscopy. Group-specific complement-fixing antigen was seen to be originating from the cell wall. The antigen was composed predominately of round particles 10 to 60 nm in diameter. Intact R. rickettsii and antigen from ether-treated organisms were purified by density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. The whole rickettsial cell was composed of a minimum of 30 proteins which ranged in molecular weight from about 23,000 to 155,000. The "soluble" antigen contained nine proteins ranging in molecular weight from about 28,000 to 150,000.     

DNA- and RNA-binding proteins of chromatin from Escherichia coli

The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sj?stad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.     

Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.     

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.     

小麦小花高纯度线粒体的分离及其蛋白质双向电泳体系建立

应用差速离心和Percoll不连续密度梯度法分离纯化小麦三核期小花线粒体. 在裂解液选择、IPG胶条pH值范围、SDS-PAGE胶浓度及蛋白质上样量等方面对线粒体蛋白质双向电泳体系进行探索和优化,确立了一套适用于小麦小花高纯度完整线粒体的分离方法及其蛋白质双向电泳的技术体系. 结果表明,采用20%、24%和40% Percoll密度梯度和28% Percoll自形成密度高速离心体系,获得了有活性、高纯度且较完整的线粒体;经TCA-丙酮法提取蛋白,以7 mol/L尿素,2 mol/L硫脲,4% CHAPS(W/V),65 mmol/L DTT,0.5% IPG缓冲液(V/V),0.001% 溴酚蓝(W/V)裂解液溶解蛋白,采用17 cm,pH 4～7 IPG胶条和11% SDS-PAGE分离胶,上样量为160 μg,硝酸银染色法,更适合小麦小花线粒体蛋白质组双向电泳分离. 经PDQuest 2DE 8.0.1软件包统计分析,在2-DE图谱上分辨出约150个蛋白点,蛋白点清晰呈圆形,无横条纹干扰,这为利用双向电泳技术在亚细胞水平对线粒体进行蛋白质组学研究与分析奠定了基础,更为进一步分析研究线粒体与雄性不育的关系提供了理论与技术支撑.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.     

A study of the intracellular and secreted forms of the MUC2 mucin from the PC/AA intestinal cell line.

In this study we present data on the entire population of MUC2 molecules secreted from and within the cell layer of an intestinal cell line. The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin. Oligomerized forms of the mucin were found in both the cell layer and medium; however, precursor forms were confined to the cell layer. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Three different populations of MUC2 were identified: one at low density (>1.3 g/ml) containing the N-glycosylated, non-O-glycosylated polypeptide; a second at intermediate density (1.3-1.35 g/ml) which may represent partially O-glycosylated intermediates; and a third at high density (1.36-1.48 g/ml) containing the mature MUC2 mucins. Rate-zonal centrifugation and agarose electrophoretic analysis of the low-density fraction indicated that the N-glycosylated MUC2 polypeptide was present as putative monomer and dimer/oligomer species. The combination of isopycnic density gradient centrifugation with agarose electrophoresis provides a new and simple approach that allows us to follow the MUC2 gene product from polypeptide through to the mature glycosylated mucin.     

Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea-polyacrylamide gel electrophoresis (AU-PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU-PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3-10. AU-PAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)-PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient's serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were alpha-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS-PAGE or AU-PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis.