Showing papers on "Gel electrophoresis published in 1970"
TL;DR: A wide variety of proteins have been shown to bind identical amounts of an amphiphile, sodium dodecyl sulfate, on a gram per gram basis at monomer equilibrium concentrations above 0.5 mM.
Abstract: A wide variety of proteins have been shown to bind identical amounts of an amphiphile, sodium dodecyl sulfate, on a gram per gram basis at monomer equilibrium concentrations above 0.5 mM. The binding is independent of ionic strength and primarily hydrophobic in nature. Only the monomeric form of the amphiphile binds to proteins, not the micellar form. The application of these results to models for biological membranes and to gel electrophoresis in sodium dodecyl sulfate is discussed.
642 citations
TL;DR: The bacterial superoxide dismutase, which catalyzes the disproportionation of univalently reduced oxygen, has now been purified from Escherichia coli and was found to contain manganese.
534 citations
TL;DR: Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions, and one of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope.
Abstract: Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions. The fraction with the higher density was enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes, and the similar morphology of the fragments and intact cell walls. The less-dense fraction was enriched in vesicles derived from the cytoplasmic membrane, as indicated by the enrichment of cytochromes, the enzymes lactic and succinic dehydrogenase and nitrate reductase, and the morphological similarity of the vesicles to intact cytoplasmic membrane. Both fractions were rich in phospholipid. The protein composition was compared by mixing the cytoplasmic membrane-enriched fraction from a (3)H-labeled culture with the cell wall-enriched fraction from a (14)C-labeled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved; of these, 27 were increased two- to fourfold in the cytoplasmic membrane-enriched fraction, whereas 6 were similarly increased in the cell wall-enriched fraction. One of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope. This protein accounted for 70% of the total protein of the cell wall, and its occurrence in the envelope from spheroplasts suggests that it is a structural protein of the outer membranous component of the cell wall.
401 citations
TL;DR: The behavior of macromolecules in gel filtration and gel electrophoresis may be predicted from Ogston's model for a random meshwork of fibers to apply to nonspherical molecules and to several gel types.
Abstract: Unified theory for gel electrophoresis and gel filtration: The behavior of macromolecules in gel filtration and gel electrophoresis may be predicted from Ogston's model for a random meshwork of fibers. This model has been generalized to apply to nonspherical molecules and to several gel types. The model provides equations for inter-relationships between mobility, partition coefficient, gel concentration, and molecular radius; it gives a non-Gaussian distribution of pore sizes as a function of gel concentration. The theory defines conditions for optimal separation and optimal resolution in gel filtration and gel electrophoresis. The difference in resolving power between the two fractionation methods is accounted for by the fact that gel filtration is a form of partition chromatography.
392 citations
TL;DR: Two-dimensional gel electrophoresis separates all of the component proteins of the ribosomal subunits of Escherichia coli into 21 and 34 proteins in the 50S, subunit.
Abstract: Two-dimensional gel electrophoresis separates all of the component proteins of the ribosomal subunits of Escherichia coli. This method shows 21 proteins in the 30S, and 34 proteins in the 50S, subunit.
272 citations
TL;DR: The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum membranes, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids.
Abstract: Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b5 as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences.
203 citations
TL;DR: Commercial preparations of yeast hexokinase are contaminated with a trace of at least one proteolytic enzyme, and a reliable molecular weight can be obtained by SDS-polyacrylamide gel electrophoresis only when specific steps are taken to prevent this proteolysis.
169 citations
TL;DR: The protein composition of purified T4 phage, phage heads and capsids was examined by sodium dodeoyl sulfate-acrylamide gel electrophoresis and found that two of the eight proteins are partially released from heads by freeze-thaw or osmotic shock treatment and are probably internal proteins.
135 citations
TL;DR: In this paper, bovine adrenal chromaffin granules contained most of the cholesterol and phospholipids of the particle and 22% of the total protein, the protein/lipid ratio was about 0.45 (w/w).
Abstract: Washed membranes of bovine adrenal chromaffin granules contained most of the cholesterol and phospholipids of the particle and 22% of the total protein. The protein/lipid ratio was about 0.45 (w/w). Dopamine(3,4-dihydroxyphenethylamine)β-hydroxylase, Mg2+-activated nucleoside triphosphatase and cytochrome b-559 activities were present in the membrane. ATP was the best substrate for the nucleoside triphosphatase, whose pH optimum was 6.4, Km 7×10−4m and Vmax. 1.8μmol/h per mg of protein. Treatment of the membranes with various detergents caused a preferential solubilization of protein compared with lipids. Membranes dissolved in sodium dodecyl sulphate or phenol–acetic acid–urea were subjected to polyacrylamide-gel electrophoresis at alkaline and acid pH respectively. The electrophoretic patterns given by the proteins of the chromaffin granule membrane were distinct from those given by the chromogranins, and from those given by mitochondrial and microsomal membrane proteins.
127 citations
TL;DR: Molecular weights of the isolated proteins from the 30S and 50S ribosomal subunits of Escherichia coli were determined by two independent methods: polyacrylamide gel electrophoresis with sodium dodecylsulphate and equilibrium sedimentation.
Abstract: Molecular weights of the isolated proteins from the 30S and 50S ribosomal subunits of Escherichia coli were determined by two independent methods: polyacrylamide gel electrophoresis with sodium dodecylsulphate and equilibrium sedimentation. The values for the molecular weights determined by gel electrophoresis range from 10,900 to 65,000 for the proteins of the 30S subunit and from 9,600 to 31,500 for those of the 50S subunit, with number averages of 19,000 and 16,300, respectively, in agreement with those obtained by equilibrium sedimentation.
113 citations
TL;DR: The purification procedure provides an over-all yield of about 25% from cell cake to crystals, and is adaptable to large scale isolation of the enzyme.
TL;DR: Double-stranded RNA preparations obtained from cytoplasmic polyhedrosis virus of silkworm, rice dwarf virus and reovirus have been analyzed by polyacrylamide gel electrophoresis and ultraviolet scanning and electron microscopic observation of the size distribution of these RNA preparations confirms the results.
TL;DR: A high molecular weight, rapidly-labelled ribosomal RNA precursor in the pearoot tip and in artichoke-tuber tissue is described and the method of synthesis of ribosome synthesis in plants is discussed in relation to that in animals.
TL;DR: Phage λ dissociates in boiling SDS into at least three major and three minor proteins, which separate from each other in gel electrophoresis, and by purifying the resulting complete phage particles it was possible to identifyThree of the proteins as constituents of the head and three of the tail.
TL;DR: The purification to homogeneity of the membrane adenosine triphosphatase from Streptococcus faecalis is described and a model of the enzyme with a formula α6β6 is proposed, consisting of a hexagonal arrangement of 6 globules, each of which is made up of an α and a β subunit.
TL;DR: The rate of amino acid incorporation into a specific uterine protein isolated by gel electrophoresis has been shown to be markedly stimulated within an hour after estrogen administration, and its synthesis would appear to be initiated as soon as the estrogen-receptor complex reaches the nucleus.
Abstract: The rate of amino acid incorporation into a specific uterine protein (induced protein band) isolated by gel electrophoresis has been shown to be markedly stimulated within an hour after estrogen administration. Injection of actinomycin D (8 mg/kg) prior to estrogen blocks the synthesis of induced protein. The accumulation of the product of the actinomycin D-sensitive step (induced protein band RNA) is significant 15 minutes after estrogen, and its synthesis would appear to be initiated as soon as the estrogen-receptor complex reaches the nucleus. Blocking protein synthesis with puromycin or cycloheximide did not affect the accumulation of induced protein band RNA, indicating that this is one of the earliest macromolecular synthetic events to occur after estrogen administration.
TL;DR: SDS-Polyacrylamide gel electrophoresis of human fibrinogen orfibrin revealed two α-chain species which differed in molecular weight by 3000, which may be responsible for the minor α (A)-chain component found in “native” fibr inogen.
TL;DR: Hundred-fold purification of intact microtubules from homogenates of rat brain is reported, and the purified fractions show a major band which migrates like purified tubulin in the SDS gel electrophoresis system.
Abstract: Hundred-fold purification of intact microtubules from homogenates of rat brain is reported. The success of purification depends on stabilizing the microtubule structure by the combined effects of hexylene glycol, acidic Ph, and low temperature. A practical, negative stain, electron microscopic assay is used to study purity and stability of microtubule fractions. The purified fractions show a major band which migrates like purified tubulin in the SDS gel electrophoresis system.
TL;DR: The molecular weights of the subunits of spinach ribulose diphosphate carboxylase have been estimated as 55, 800 and 12, 000 by SDS-acrylamide gel electrophoresis and it is suggested that the native enzyme has 8 heavy catalytic subunits and 8–10 light structural or regulatory subunits.
TL;DR: Analysis of the carbohydrate content revealed 1 residue of terminal sialic acid, 10 to 11 residues of N-acetyl glucosamine, 5 to 6 residues of galactose, and 15 to 16 residues of mannose per molecule of lactoferrin, demonstrating that bovine lact oferrin is composed of a single polypeptide chain.
TL;DR: It would appear that this solubilizing rat-liver plasma membrane proteins by the use of the detergents sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 will have general usefulness in the study of membrane enzymes.
TL;DR: The membrane adenosine triphosphatase from Streptococcus faecalis has been purified by heat treatment, gel filtration through Agarose, and repeated chromatography on diethylaminoethyl cellulose and appears to be homogeneous.
TL;DR: Pulse-chase experiments combined with differential extraction indicated that conversion of P23 into A-protein and alteration of the protein coded by gene 22 (P22) appeared to be vital steps in formation of normal capsids.
Abstract: Radioisotopically labeled T4-proteins extracted from purified capsids and phage and from infected cells were separated by gel electrophoresis in the presence of sodium dodecyl sulfate and a reducing reagent. They were identified by autoradiography and by counting of the fractionated gels. Four major protein bands (F, A, D, and E) were detected in capsid and phage. These accounted for more than 90% of the total capsid protein and 70% of the phage protein (60% of the total capsid protein was in A-band). Coelectrophoresis of [14C]proteins from capsids and [3H]proteins from phage-infected cells indicated that the protein coded by gene 23 (P23) was a peptide chain approximately 25% longer than A-protein. Pulse-chase experiments combined with differential extraction indicated that conversion of P23 into A-protein and alteration of the protein coded by gene 22 (P22) appeared to be vital steps in formation of normal capsids. Mutations in several other genes known to prevent normal capsid formation inhibited conversion of P23 to A-protein and alteration of P22.
TL;DR: Fingerprints of tryptic digests of phenylalanyl-tRNA synthetase of Escherichia coli NP 3 suggest that the smallest identical subunit is one fourth of the size of the native protein.
Abstract: Fingerprints of tryptic digests of phenylalanyl-tRNA synthetase of Escherichia coli NP 3 suggest that the smallest identical subunit is one fourth of the size of the native protein (mol. wt. 181000). Determination of the subunit size by gel electrophoresis reveals a molecular weight of 43000 ± 2000. By the criteria employed the enzyme therefore seems to be composed of four identical subunits.
Phenylalanyl-tRNA synthetase which has been denatured by treatment with 8 M urea is able to reform active enzyme indistinguishable by its size from the native protein. Again, a size of about 45000 was determined for the subunits active in the association reaction.
TL;DR: A polyacrylamide gel electrophoresis of sulfitolyzed fibrinogens is described, separating the S-sulfopolypeptides into three main groups, and the α(A) fraction is further resolved into a doublet.
TL;DR: Electrophoretic work further elucidated or confirmed taxonomic relations between species obtained previously by well known classical methods of geographical, morphological, cytological, and hybridization analyses.
Abstract: Polyacrylamide gel electrophoresisof seed proteins offers a biochemical approach to the evolutionary aspects of plant speciation. The background and theory of polyacrylamide gel electrophoresis were thoroughly discussed by Ornstein (1964), and the method and application of the technique to analyze and compare human serum proteins were presented by Davis (1964). Amino acid changes within a protein, due to mutational changes, can result in altered protein migration rates when the proteins are compared in the matrix system of polyacrylamide. Therefore, since species differ genetically at many loci, the individuality of each plant species can usually be expressed according to its protein banding pattern. Steward et al. (1965), Boulter et al. (1966), and Sastry and Virupaksha (1967) modified the polyacrylamide gel electrophoretic technique so as to detect protein changes in developing and differentiating plant seedlings and to examine protein content of seeds. Many workers believe proteins from seed or other dormant tissue from plants represent a more stable reflection of the genomic state in a given species than that obtained from developing seedlings. Subsequently, their electrophoretic work further elucidated or confirmed taxonomic relations between species obtained previously by well known classical methods of geographical, morphological, cytological, and hybridization analyses. For example, Fox et al. (1964) with Leguminoseae, Vaughan
TL;DR: T4 thioredoxin was purified from an extract of Escherichia coli B infected from T4 am 122 in essentially pure form as judged from ultracentrifugation data, gel electrophoresis, and from the stoichiometry of its reaction with NADPH.
TL;DR: Transfer RNA was purified from pea root and soybean hypocotyl by selective dissolution in 3·0 M sodium acetate and subsequent DEAE-cellulose chromatography, and the reaction was further characterized by determining the kinetics of aminoacylation with increasing (but always limiting) tRNA conc.
TL;DR: The enzyme which catalyzes the transfer of l-alanine from l-alanyl transfer RNA to UDP-N-acetylmuramyl-l-Ala-d-Glu- l-Lys-d.Ala has been purified to homogeneity, as shown by polyacrylamide gel electrophoresis, from an extract of Lactobacillus viridescens.