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Showing papers on "Gel electrophoresis published in 1980"


Journal ArticleDOI
TL;DR: These experiments involving three highly specific serine proteases support the conclusion that the triplet observed on polyacrylamide gels is factor VIII.
Abstract: Factor VIII has been purified approximately 300000-fold from bovine plasma by ammonium sulfate fractionation, glycine precipitation, DEAE-Sephadex column chromatography, sulfate--Sepharose column chromatography, Sephadex G-200 gel filtration, and factor X--Sepharose column chromatography. The highly purified preparation migrated as a triplet on sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis with apparent molecular weights of 93000, 88000, and 85000. The coagulant activity of the purified preparations was inhibited by antibodies raised in rabbits against either the purified factor VIII protein or a preparation of factor VIII/von Willebrand factor. Antibodies to the purified protein also inhibited the coagulant activity of factor VIII/von Willebrand factor preparations. The purified factor VIII contained no platelet-aggregating activity, as measured in human platelet-rich plasma. The purified preparation of factor VIII was required for the activation of factor X in the presence of factor IXa, calcium, and phospholipid. It was activated about 30-fold by thrombin or factor Xa plus calcium and phospholipid, and each of these reactions was accompanied by a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the protein. Factor VIII was rapidly inactivated by bovine-activated protein C in a reaction requiring calcium and phospholipid. This reaction was also associated with a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the highly purified protein. These experiments involving three highly specific serine proteases support the conclusion that the triplet observed on polyacrylamide gels is factor VIII.

470 citations


Journal ArticleDOI
TL;DR: Quantitation of 99 abundant polypeptides (acidic and basic) in pulse- labeled and long-term labeled cells revealed that the relative amount (i.e., the rate of synthesis) of most polypePTides remains constant throughout the cell cycle.
Abstract: The polypeptides synthesized during the cell cycle of HeLa cells were analyzed by means of two-dimensional gel electrophoresis followed by fluorography under conditions in which the position of 700 polypeptides (acidic and basic) could be reproducibly assessed. Mitotic cells obtained by mechanical detachment and synchronized cells in other stages of the cell cycle were labeled with [35S]methionine for 30-min pulses or for long terms starting at the beginning of each phase. Visual comparison of the polypeptide maps obtained in the different stages of the cell cycle showed that these were strikingly similar, and there was no indication that the synthesis of any of the detected polypeptides was confined to only one of the cell cycle phases. Quantitation of 99 abundant polypeptides (acidic and basic) in pulse-labeled and long-term labeled cells revealed that the relative amount (i.e., the rate of synthesis) of most polypeptides, including total actin, alpha-actinin, 6 abundant basic nonhistone proteins, and 13 major acidic proteins present in Triton cytoskeletons, remains constant throughout the cell cycle. Among the few variable polypeptides (markers), we have identified alpha- and beta-tubulin (increase in M), the subunit of the 100-A filament protein "fibroblast type" (decreases in M), and a 36,000 mol wt acidic cytoarchitectural protein that increases in S. A few other unidentified polypeptides have also been found to vary in M and in M and G2, but no marker was found in G1.

435 citations


Journal Article
TL;DR: Rabbit TNF has been purified 2000-fold by a series of salt precipitations, gel filtrations, ion exchange chromatography, and lectin affinity chromatography to a single species on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Abstract: Rabbit TNF has been purified 2000-fold by a series of salt precipitations, gel filtrations, ion exchange chromatography, and lectin affinity chromatography to a single species on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). TNF activity could be recovered from nondenaturing gel systems and has been shown to be an alpha-globulin with an isoelectric point of 5.1. The m.w. was estimated to be 68,000 d by SDS-PAGE, 55,000 by gel filtration, and 52,000 by glycerol gradient centrifugation. TNF activity was stable over the pH range of 6 to 10 and was relatively heat stable, not being inactivated at 70 degrees C for 1 hr. TNF activity was pronase sensitive, but relatively trypsin resistant. Neuraminidase and phospholipase C treatment did not destroy TNF activity. Partially purified TNF was still capable of eliciting hemorrhagic necrosis in susceptible tumors. Crude TNF serum had an interferon titer of 3000 U, whereas the partially purified sample had a titer of <30 U.

376 citations


Journal ArticleDOI
TL;DR: Triton gel electrophoresis provides rapid analysis of very small amounts of haemoglobin, and permits examination of globin chain composition as well as globin synthetic ratios.
Abstract: Separation of globin chains by electrophoresis provides a simple and rapid method for the determination of the G gamma/A gamma ratio in human fetal haemoglobin, and of biosynthetic rates of the globin chains. Whole haemolysates were analysed by electrophoresis on polyacrylamide gels in urea, acetic acid and Triton X-100. Electrophoresis of haemolysates from newborn infants led to four bands: A gamma, G gamma, beta and alpha. The identity of these bands was indicated by examination of haemoglobins of known globin chain composition. In 15 samples, the % G gamma was similar by Triton gels and by amino acid analysis of the gamma CB-3 peptide. Some mutant globin chains were also separable with the electrophoretic technique. Triton gel electrophoresis provides rapid analysis of very small amounts of haemoglobin, and permits examination of globin chain composition as well as globin synthetic ratios.

375 citations


Journal ArticleDOI
TL;DR: A number of enzymes, including amylases, dehydrogenases, and proteases, were shown to be renaturable after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

289 citations


Book ChapterDOI
TL;DR: This chapter describes the techniques for the recovery of DNA from gels, the methods of purification and concentration of recovered DNA, and the choice of method.
Abstract: Publisher Summary This chapter describes the techniques for the recovery of DNA from gels. Agarose or polyacrylamide gel electrophoresis is widely used as a high resolution technique for fractionation of DNA molecules by size. The basic methods for recovery of DNA from gel slices are (1) electroelution, (2) elution by diffusion, (3) gel dissolution, and (4) extrusion of DNA by gel compression. Choice of a method depends on a number of factors such as the type of gel matrix, gel concentration, DNA size, scale of the procedure, and the level of gel contamination that can be tolerated in the recovered DNA solution. With agarose gels, contamination of recovered DNA with varying amounts of agarose is a certainty, and in many cases this interferes with subsequent enzymatic treatments or analysis. Methods of agarose removal include equilibrium density gradient centrifugation, extraction, and chromatographic separation. The chapter describes the methods of DNA recovery, the methods of purification and concentration of recovered DNA, and the choice of method.

267 citations


Journal ArticleDOI
TL;DR: The phosphocellulose fraction against astrocytes and that against Schwann cells co-migrated in native gel electrophoresis at pH 4.5, providing strong evidence that the same molecule acts on both cell types.

246 citations


Book ChapterDOI
TL;DR: This chapter discusses the analysis of nucleic acids in gels using glyoxal and acridine orange, a powerful reagent for rapid determination of gross nucleic acid structure after gel electrophoresis.
Abstract: Publisher Summary This chapter discusses the analysis of nucleic acids in gels using glyoxal and acridine orange. To determine the molecular weights of nucleic acids using gel electrophoresis techniques, the molecules must have equivalent conformations. This is achieved by removing native secondary and tertiary structure with a variety of chemical denaturants, thus reducing the electrophoretic mobility to a simple function of polynucleotide molecular weight, gel composition, and voltage gradient applied. The chapter describes the method for denaturing RNA or DNA molecules with glyoxal, followed by electrophoresis through either polyacrylamide- or agarose-containing slab gels in a low ionic strength buffer. Using this method reliable molecular weight estimates for RNA and DNA molecules of varying sizes and G + C contents were obtained; glyoxalated DNA and RNA molecules were shown to lie on the same log molecular weight versus mobility curve, or on very similar curves. The chapter describes the use of the metachromatic stain, acridine orange, for the visualization of nucleic acids in gels. When intercalated between the stacked bases of double-helical nucleic acids, acridine orange manifests a green fluorescence. Its metachromasy visually confirms glyoxal denaturation and makes it a powerful reagent for rapid determination of gross nucleic acid structure after gel electrophoresis.

228 citations


Journal ArticleDOI
TL;DR: The results indicate that C2 toxin is constructed with two separate protein components, which are not covalently held together, and that its toxicity is elicited by cooperation of the two components.
Abstract: Two dissimilar proteins, designated as components I and II, of botulinum C2 toxin elaborated by strain 92-13 were purified to a homogeneous state. The molecular weights determined by sodium dodecyl sulfate gel electrophoresis were 55,000 for component I and 105,000 for component II. Whereas each component showed no or feeble toxicity even after being treated with trypsin, the toxicity was elicited when these two components were mixed and trypsinized. The toxicity of the mixture of components I and II at a ratio of 1:2.5 on a protein basis was 2.2 X 10(4) mouse intraperitoneal 50% lethal doses per mg of protein and increased by 2,000 times or more when treated with trypsin. These results indicate that the molecular characteristics of botulinum C2 toxin differ from those of the toxin of Clostridium botulinum types A through F in that C2 toxin is constructed with two separate protein components, which are not covalently held together, and that its toxicity is elicited by cooperation of the two components.

201 citations


Journal ArticleDOI
06 Mar 1980-Nature
TL;DR: Cloned DNA complementary to the messenger RNAs for four immunologically distinct VSGs are cloned and hybridised these complementary DNAs with restriction digests of T. brucei nuclear DNA and infer that activation of a VSG gene involves the production of an expression-linked copy of that gene.
Abstract: Pathogenic African trypanosomes evade the immune system of their mammalian hosts by the sequential expression of alternative cell-surface glycoproteins (reviewed in refs 1,2). Variant surface glycoproteins (VSGs) purified from cloned variants of Trypanosoma brucei have similar molecular weights (about 60,000), but differ in amino acid composition, N-terminal amino acid sequence and C-terminal structure. We have cloned DNA complementary to the messenger RNA's for four immunologically distinct VSGs and hybridised these complementary DNAs (cDNAs) with restriction digests of T. brucei nuclear DNA, fractionated by gel electrophoresis and transferred to nitrocellulose strips. Each cDNA recognises a unique set of fragments and this basic set is present unaltered in the nuclear DNAs from the four variants. In addition, each probe recognises an extra fragment only in nuclear DNA isolated from cells expressing the VSG corresponding to the cDNA probe. We infer that activation of a VSG gene involves the production of an expression-linked copy of that gene.

195 citations


Journal ArticleDOI
TL;DR: Immunofluorescence microscopy reveals that calmodulin is present in the microvilli prior to biochemical fractionation of intestinal cells and thus is not bound artifactually during the isolation procedure, consistent with the hypothesis that the 110,000 protein is the majorCalmodulin-binding protein of the core filament structure.

Journal ArticleDOI
TL;DR: The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N ePSilon B 29-di(azid Obenzoic acid)ins insulin (DAB-Insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidOBenzoIC acid.
Abstract: The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.

Journal ArticleDOI
TL;DR: A human osteosarcoma cell line, U‐2 OS, cultured under serumfree conditions, was shown to produce a growth factor (osteosarComa‐derived growth factor, ODGF) for human‐cultured glial cells, fibroblasts, and other cells.
Abstract: A human osteosarcoma cell line, U-2 OS, cultured under serumfree conditions, was shown to produce a growth factor (osteosarcoma-derived growth factor, ODGF) for human-cultured glial cells, fibroblasts, and other cells. ODGF, collected from the spent medium of 2 OS cultures, was purified by a sequence involving heparin-Sepharose chromatography, hydrophobic chromatography, gel chromatography, and preparative gel electrophoresis in SDS. Purified ODGF, at a concentration of 3 ng/ml, elicited a mitogenic response in human glial cells equivalent to 50% of that afforded by human serum at a final concentration of 1%. The preparation was estimated to be > 50% pure. The biological activity of ODGF resided in a cationic, relatively heat-resistant, reduction-susceptible protein with a molecular weight of 30,000 (by gel chromatography and SDS-gel electrophoresis). The electrophoretic behaviour of radioiodinated ODGF suggested that the protein was composed of two different polypeptide chains (about 13,000-14,000 and 16,000-17,000 daltons, respectively) linked via disulphide bonds. The molecular makeup of ODGF was thus similar to that of platelet-derived growth factor. 125I-ODGF could be precipitated by an antibody to platelet-derived growth factor, indicating that the two factors were immunologically related. Resemblance with platelet-derived growth factor was also indicated by the finding that the latter (but not, e.g., fibroblast growth factor or epidermal growth factor) competed with 125I-ODGF for binding to human-cultured glial cells.

Journal ArticleDOI
01 Feb 1980-Science
TL;DR: The purification of human fibroblast interferon has been simplified to a two-step procedure consisting of affinity chromatography on Blue Sepharose and sodium dodecyl sulfate polyacrlamide gel electrophoresis.
Abstract: The purification of human fibroblast interferon has been simplified to a two-step procedure consisting of affinity chromatography on Blue Sepharose and sodium dodecyl sulfate polyacrlamide gel electrophoresis. A preliminary amino acid composition and the sequence of the 13 amino-terminal residues of homogeneous interferon prepared by this method is reported.

Journal ArticleDOI
TL;DR: An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle and could be activated by a protein activator (FA) in the presence of ATP and Mg ions.

Journal ArticleDOI
TL;DR: Mouse and human cultured cell lines show a similar set of protein-bound fatty acid, and it is proposed that fatty acid acylation is a general cellular activity that modifies proteins destined to become membrane-bound.

Journal ArticleDOI
TL;DR: A polyacrylamide slab gel electrophoresis procedure was used to compare cellular proteins from bacterial isolates of gingival crevice floras and provided additional verification of the identity of strains characterized by conventional phenotypic tests.
Abstract: A polyacrylamide slab gel electrophoresis procedure was used to compare cellular proteins from bacterial isolates of gingival crevice floras. Isolates with identical protein patterns consistently were shown to be members of the same species. When used to screen isolates, the procedure reduced total analytical time and expense without sacrificing accuracy, and it provided additional verification of the identity of strains characterized by conventional phenotypic tests. Images

Journal ArticleDOI
TL;DR: The viral polypeptide, p63, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.
Abstract: A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.

Journal ArticleDOI
TL;DR: The results indicate that α‐tubulin is an integral vesicle membrane protein, whereas most of the β sub‐unit is peripherally attached and can be easily dissociated from the vesicles membrane with EGTA.
Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis, and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the alpha and beta subunits. In the presence of ethyleneglycolbis)aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the alpha-tubulin whereas the beta subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of alpha-tubulin than of the beta subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that alpha-tubulin is an integral vesicle membrane protein, whereas most of the beta subunit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.

Journal ArticleDOI
TL;DR: Human HLA-DR antigens were immunoprecipitated from Nonidet P-40 extracts of [35S]methionine-labeled B lymphoblastoid cell lines and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF).
Abstract: Human HLA-DR antigens were immunoprecipitated from Nonidet P-40 extracts of [35S]methionine-labeled B lymphoblastoid cell lines and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Two-dimensional (2-D) gel analyses, combining SDS-PAGE in the first dimension and IEF in the second dimension, revealed that the heavy (alpha) and light (beta) chains of each DRw specificity displays microheterogeneity of charge. However, the pattern of the heavy chain did not vary among different DRw specificities. In contrast, the light chains of different DRw types varied both in apparent size and charge distribution. Removal of sialic acids with neuraminidase or inhibition of glycosylation with tunicamycin reduced the microheterogeneity of both DR subunits. However, the heavy and light chains each still focused as two major bands, suggesting that other post-translational modifications contribute to the microheterogeneity or that there are two nonallelic DR-like molecules. After treatment with either neuraminidase or tunicamycin, the DR light chains, but not the heavy chains, were still structurally polymorphic. The DR light chains of serologically cross-reactive specificities displayed similar 2-D gel patterns suggesting that the structural polymorphism of the DR light chains is the basis for the serologically detected polymorphism of the HLA-DR antigens. Two additional polypeptides were observed in immunoprecipitates of DR antigens. These proteins, designated M1 and M2, both had a basic isoelectric point and were invariant among different cell lines. The protein M1 may be intracellular because it can not be immunoprecipitated from the cell surface.

Journal ArticleDOI
TL;DR: Results suggested that the calmodulin-dependent phosphodiesterase from bovine brain has a subunit structure of alpha2, which indicates that the stoichiometry of the complex is cal modulin2 alpha2.

Journal ArticleDOI
TL;DR: Data suggest that Aleutian disease virus is a nondefective parvovirus, which has a single non-complementary strand with a molecular weight of about 1.4 X 10(6).
Abstract: We characterized a strain of Aleutian disease virus adapted to growth in Crandall feline kidney cells at 31.8 degrees C. When purified from infected cells, Aleutian disease virus had a density in CsCl of 1.42 to 1.44 g/ml and was 24 to 26 nm in diameter. [3H]thymidine could be incorporated into the viral genome, and the viral DNA was then studied. In alkaline sucrose gradients, Aleutian disease virus DNA was a single species that cosedimented at 15.5S with single-stranded DNA from adeno-associated virus. When the DNA was analyzed on neutral sucrose gradients, a single species was again observed, which sedimented at 21S and was clearly distinct from 16S duplex adeno-associated virus DNA. A similar result was obtained even after incubation under annealing conditions, implying that the bulk of Aleutian disease virus virions contained a single non-complementary strand with a molecular weight of about 1.4 X 10(6). In addition, two major virus-associated polypeptides with molecular weights of 89,100 and 77,600 were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virus purified from infected cultures labeled with [35S]methionine. These data suggest that Aleutian disease virus is a nondefective parvovirus.

Journal ArticleDOI
TL;DR: Two 3-hydroxyacyl-CoA dehydrogenase enzymes in rat liver, one in mitochondria, and another in peroxisomes, were purified and compared for their properties to be completely different enzymes.

Journal ArticleDOI
TL;DR: The findings suggest that the cell surface topography of this bacterium may be very irregular, and it is speculated that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents.
Abstract: Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents.

Journal ArticleDOI
TL;DR: The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis to clearly follow the distribution of specific proteins during nuclear extraction.
Abstract: The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis. The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction. In the nuclear washes and chromatin, we observed five classes of proteins: (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; (c) proteins of 94,000, 25,000, and 20,500 mol wt specific to the nuclear washes; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix; and (e) two proteins of 68,000 mol wt present only in the final chromatin. The major 65,000-75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the second major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA-bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNp particles and vary in the degree to which they share different intracellular compartments.

Journal ArticleDOI
TL;DR: It is suggested that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly.
Abstract: The separation of DNA fragments by electrophoresis at high temperature in a denaturing gradient is independent of the length of the fragments. We have suggested that the basis of fragment separation is that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly. This model is consistent with the observation that fragments of lambda phage DNA cleaved by different restriction endonucleases reach the same final depth in the gel if they contain the same least-stable sequence. A unique set of bands is produced from the electrophoresis of randomly fragmented DNA; this would be expected if there were a limited number of melting centers occupying discrete genetic loci. An intact DNA molecule penetrates about as deeply into the gel as the uppermost band after fragmentation; this would be expected only if the least-stable sequence controls the final depth of the whole molecule.

Journal ArticleDOI
TL;DR: A schematic model was developed to explain the structure of the large noncovalently bound complexes based on their molecular weight and observed component fragments, which supports the two-stranded half-staggered overlap model as the basic unit of fibrin structure.
Abstract: Crosslinked fibrin was digested by plasmin, and three soluble complexes larger than DD/E were purified and characterized. After gel filtration chromatography, the purified complexes were shown to have molecular weights of 465,000, 703,000, and 850,000, as determined by equilibrium sedimentation. Each of the complexes was dissociated into two or more fragments by SDS-polyacrylamide gel electrophoresis. The structure of these subunit fragments was deduced from determinations of their molecular weights and polypeptide chain composition and from known sites of plasmin cleavage of fibrin. Fragments larger than DD have been identified that contain intact gammagamma crosslinks as well as fragments resulting from cleavages at or near this site. The former include DY (mol wt 247,000), YY (mol wt 285,000), DXD (mol wt 461,000), and YXD (mol wt 500,000); and the latter include fragments XD (mol wt 334,000) and XY (mol wt 391,000). A schematic model was developed to explain the structure of the large noncovalently bound complexes based on their molecular weight and observed component fragments. Our scheme supports the two-stranded half-staggered overlap model as the basic unit of fibrin structure, in which each complex consists of fragments from two adjacent complementary antiparallel fibrin strands. The smallest derivative, complex 1, is the DD/E complex; complex 2 contains apposed DY and YD fragments, and complex 3 consists of fragments DXD and YY. Complex 4 is less well-characterized, but its intact structure is projected to consist of YXD and DXY fragments from adjacent fibrin strands. Each complex is heterogeneous in subunit composition, reflecting additional plasmin cleavages within and/or adjacent to its theoretical boundaries. Since most of the protein initially released into solution from degrading fibrin is as complexes larger than DD/E, the derivatives described in this report are likely to be major circulating degradation products of crosslinked fibrin in vivo.

Journal ArticleDOI
TL;DR: Results of cross-linking experiments strongly suggest that two of the major proteins of the gonococcal outer membrane (proteins 1 and 3) form a hydrophobically associated trimeric unit in situ which can be stabilized by selective cross- linking reagents.
Abstract: The proteins of the outer membrane of Neisseria gonorrhoeae play an important role in the serotyping system defined by K. H. Johnston et al. (J. Exp. Med. 143:741–758, 1976). This study attempted to delineate the molecular arrangement of the major proteins of the outer membrane of the gonococcus by using three approaches. First, natural protein-protein relationships were demonstrated by symmetrical, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, proteins exposed on the surface of outer membrane vesicles were cross-linked by using the bifunctional reagents dimethyl-3,3′-dithiobispropionimidate and dithiobis[succinimidyl propionate]. Third, specific antigen-antibody interactions on the surface of membrane vesicles were analyzed by radioautographic techniques. The major proteins of the outer membrane of the gonococcus were defined, and a nomenclature was devised to take into account the effects of heat and reducing agents on the resolution of these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of cross-linking experiments strongly suggest that two of the major proteins of the gonococcal outer membrane (proteins 1 and 3) form a hydrophobically associated trimeric unit in situ which can be stabilized by selective cross-linking reagents. Results substantiated that these proteins are responsible for imparting serotypic specificity.

Journal ArticleDOI
TL;DR: Primary structure studies have shown AA to be a single chain protein of 76 residues, and SAA, therefore, appears to contain a peptide of 33 amino acids that is missing from AA, and it is calculated from quantitative COOH-terminal analyses that SAA is of 11,000-11,900 mol wt.
Abstract: Serum amyloid A proteins (SAA), presumed precursors of the tissue amyloid A proteins (AA) characteristic of secondary amyloidosis, have been isolated from the plasma high-density lipoproteins (HDL) of normals after etiocholanolone-induced inflammation and from patients with Wegener's granulomatosis, systemic lupus erythematosis, juvenile rheumatoid arthritis, Waldenstrom's macroglobulinemia, and Goodpasture's syndrome. At least six polymorphic forms of SAA wer identified among the low molecular weight proteins of HDL, and these comprosed up to 27% of the total HDL protein. Gel and ion-exchange chromatography permitted isolation of the SAA polymorphs in homogeneous form. Their amino acid compositions were very similar, they were indistinguishable in cationic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems, and each had the terminal sequency COOH-Tyr-Lys-Phe-. Charge heterogeneity in anionic-urea polyacrylamide gel electropherograms was unaffected by neuaminidase treatment, and none of the SAA protein bands stained with the periodate-Schiff reagent. The two major SAA polymorphs, designated SAA4 and SAA5 according to their order of elution from DEAE-cellulose, had different NH2-terminal sequences. Manual Edman degradation demonstrated NH2-arg-ser-phe-phe- for SAA4 and NH2-ser-phe-phe- for SAA5. This NH2-terminal heterogeneity corresponds to that most frequently reported for AA and suggests that microheterogeneity in SAA may underlie that already documented in AA. Sufficient quantitites of the other SAA polymorphs were not available for similar analyses, but the amino acid compositions do not indicate that NH2-terminal heterogeneity accounts for all of the observed polymorphism. Artifactual polymorphism also appears unlikely, and the heterogeneiy of SAA may reflect origin from more than one cell type with or without posttranslational modificaton. We calculate from quantitative COOH-terminal analyses that SAA is of 11,000-11,900 mol wt. Primary structure studies have shown AA t be a single chain protein of 76 residues, and SAA, therefore, appears to contain a peptide of 33 amino acids that is missing from AA.

Journal Article
TL;DR: Binding of 125I-labeled lectins to total cellular proteins on polyacrylamide gels following electrophoresis showed that the major wheat germ agglutinin-binding components of F1 cells were altered in Wa-4 cells, suggesting a possible basis for the glycosylation change.
Abstract: Glycoproteins of a metastasizing line of B16 mouse melanoma and a poorly metastasizing wheat germ agglutinin-resistant clone were compared. Cell surface proteins and glycoproteins were isotopically labeled by lactoperoxidase-catalyzed iodination and by NaB 3 H 4 reduction after oxidation by periodate or galactose oxidase and subsequently analyzed by gel electrophoresis and autoradiography. Differences were observed in the relative mobilities of several major cell surface components. Binding of 125 I-labeled lectins to total cellular proteins on polyacrylamide gels following electrophoresis showed that the major wheat germ agglutinin-binding components of F 1 cells were altered in Wa-4 cells. Similar differences were not observed in concanavalin A-binding components. Total cellular glycopeptides were analyzed after separation into structurally distinct classes. The acidic “complex” N -glycosidic glycopeptides from the resistant cells were of lower molecular weight than those from the parent cells. No differences were observed among the mannose-rich N -glycosidic glycopeptides or the alkali-labile O -glycosidic oligosaccharides. Structural studies involving methylation analysis revealed that in the altered glycopeptides of the resistant cells the amount of neuraminic acid residues was decreased to one-half, concomitant with an increase in the amount of fucose. The lost sialic acid was bound to C-3 of galactose, whereas the increased fucose was found on C-3 of 4-substituted N -acetylglucosamine. A possible basis for the glycosylation change and its relation to the biological behavior are discussed.