DEAE-Sephadex Column Research Articles

Heterogeneity of tubulin subunits.

Tubulin, the subunit protein of microtubules, is a dimer that sediments at 6 S and has a molecular weight of 110,000. Using high resolution polyacrylamide gel electrophoresis, we have demonstrated the presence of two peptide chains, of molecular weight 56,000 and 53,000, in tubulin purified from brain. Two peptide chains of similar molecular weight were identified in each of the A- and B-tubulins isolated from flagella of sea urchin sperm. In all cases, the protein concentrations in the bands were equal. Each of the subunits ran as a single band when eluted from the gel and electrophoresed again in the same type of gel. Chromatography of purified brain tubulin on DEAE-Sephadex columns gave only a single peak containing both subunits in equal amounts. Cyanogen bromide peptides were prepared from each of the bands after elution from polyacrylamide gel. While certain of the peptides appear to be common to both subunits, substantial differences exist between them. The tubulin dimer is composed of two nonidentical subunits.

5S RNA III, a new nucleus-specific 5S RNA

A new species of 5S RNA, referred to as 5S RNA III, has been found in the Novikoff hepatoma ascites cell nuclei. This RNA, which composes 28% of total nuclear 5S RNA and co-migrates with ribosomal 5S RNA I and 5S RNA II on 8% polyacrylamide gel electrophoresis, has been isolated by chromatography on DEAE Sephadex columns. The nucleotide composition is A 24, U 31, G 24 and C 20; its A+U G+C ratio is 1.24. T 1 RNase digestion produced the following unique olig o nucleotides from 5S RNA III: (U 2C 2)Gp, (CUA)Gp, (C 4A 3U 8)Gp, [(UmU) (C 2A 3U 5) (AGmC)], (CAU 2)Gp, (C 2AU 2)Gp and (C 3A 3U 2)Gp. Similarly, unique oligonucleotides were produced by complete pancreatic RNase digestion of 5S RNA III, including UmUp, A-A-Up, A-A-Cp, (AG)Cp, A-Gm-Cp, (A 2G)Up, A-A-A-Up and (A 3G)-Up.

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue

Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.

Multiple molecular forms of cytoplsmic glycerol-3-phosphate dehydrogenase in rat liver

Two major zones of cytoplasmic l -glycerol-3-P dehydrogenase (EC 1.1.1.8) activity appeared in polyacrylamide gel isoelectric focusing analysis of homogenates of livers from rats weighing 300–350 g. At least three other minor zones of activity were also observed. The more anionic of the two major zones of activity does not appear in homogenates of livers from smaller rats but can be generated in vitro by storing the homogenates overnight at 4°. Liver homogenates could be resolved into three zones of activity by DEAE-Sephadex column chromatography. Two of these isoenzymic forms of the enzyme were isolated at a high level of purity by a series of steps including a final column of DEAE-Sephadex and were characterized by amino acid analysis. One of these two forms was indistinguishable from the single major form of the enzyme isolated and characterized from livers of smaller rats and corresponded to the more cationic of the two major forms of the enzyme observed by gel electrofocusing in this work. The second form isolated corresponded to the most cationic of the minor forms of the enzyme observed in gel electrofocusing. Alternative methods of smaple introduction into electrofocusing gels were employed including internal layering and injection, in addition to the usual polymerization of the sample throughout the gel.

Arginine Racemase of Pseudomonas graveolens: I. PURIFICATION, CRYSTALLIZATION, AND PROPERTIES

Abstract Arginine racemase has been purified approximately 5400-fold and crystallized from an extract of Pseudomonas graveolens. The purification procedure consists of sonic disruption, ammonium sulfate fractionation, treatment with 1-butanol, DEAE-cellulose and DEAE-Sephadex column chromatography, and Sephadex G-150 gel filtration, followed by crystallization. The purified enzyme is homogeneous by the criteria of ultracentrifugation (s020,w = 5.2 S) and disc gel electrophoresis. The molecular weight is 167,000, assuming a partial specific volume of 0.74. The enzyme is yellow in solution and exhibits an absorption maximum at 420 mµ. The absorption spectrum is shifted neither by varying pH of the enzyme nor by addition of arginine. One mole of pyridoxal 5'-phosphate is tightly bound per 42,000 g of enzyme. The apoenzyme was obtained by dialysis against phenylhydrazine solution. Addition of pyridoxal 5'-phosphate caused return of the 420 mµ absorption peak and reconstitution of activity. Reduction of arginine racemase with sodium borohydride shifted the 420 mµ peak to about 315 mµ, and destroyed the racemase activity. The enzyme catalyzes racemization of lysine, arginine, e-N-acetyllysine, ornithine, 2,3-diaminopropionate, homoarginine, 2,4-diaminobutyrate, ethionine, citrulline, homocitrulline, δ-N-acetylornithine, theanine, glutamine, and methionine in this order, when examined at pH 10.0. Maximum activities for arginine, lysine, theanine, and ornithine are found in the pH regions of 9.0 to 10.6, 7.5 to 10.6, 7.0 to 10.0, and 6.5 to 9.0, respectively. The following Michaelis constants were determined: d-arginine, 1.0 x 10-3 m, and pyridoxal 5'-phosphate, 4.0 x 10-7 m. The enzyme activity was inhibited by hydroxylamine and d- and l-penicillamine.

Isolation and fractionation of glycopeptides from porcine thyroglobulin.

1. Glycopeptides were isolated by gel filtration on Sephadex G-25 and Sephadex G-50 from a Pronase digest of porcine thyroglobulin. 2. Isolated glycopeptides were separated into five main fractions on a column of DEAE-Sephadex A-25. Of these fractions I to III were further purified by SE-Sephadex C-25 or DEAE-Sephadex A-25 column chromatography. Several of the purified glycopeptides were homogeneous on paper electrophoresis. 3. Based on the chemical composition and molecular weight of the fractionated glycopeptides, two distinct types of heterosaccharide chain were demonstrated. 4. One type of the heterosaccharide unit consisted of four to eight residues of mannose and two residues of glucosamine and had a molecular weight of 1000-1700. The other type of unit contained sialic acid, fucose and galactose in addition to mannose and glucosamine and had a molecular weight of about 3600. 5. Mild alkaline treatment of the glycopeptide did not result in the destruction of threonine and serine. 2-Acetamido-1-N-(4-l-aspartyl)-2-deoxy-beta-d-glucopyranosylamine was isolated from partial acid hydrolysates.

Étude du méthionine-tARN dans la glande mammaire de brebis. Hétérogénéité et purification

Summary The heterogeneity of methionine-tRNA in sheep mammary gland has been studied by several chromatographic techniques. Three acceptors have been demonstrated; only one among them is acylated by methionyl-tRNA synthetase from E. coli , and may be purified by successive chromatography on columns of BD-cellulose and DEAE-Sephadex A 50. The mixture of the two other isoacceptors was purified by phenoxyacetylation, after enrichment by means of the chromatographic techniques mentioned above. The specific activity of the samples obtained was greater than 1,200 pmoles of methionine per unit of absorbance in both cases. Methionyl-tRNA of E. coli ( 14 C) and of mammary gland ( 3 H) were hydrolysed by RNase T 1 ; by means of chromatography of the hydrolysates on a DEAE-cellulose column in the presence of urea, it is possible to differenciate the 3′-terminal oligonucleotides corresponding to the tRNA Met m E. coli and tRNA Met II M. G. . The partial separation of the 3 H and 14 C radioactivities in the case of the oligonucleotides derived from methionyl tRNA Met f E. coli and tRNA Met I M. G. argues in favour of a difference in the 3′-terminal sequence of these two tRNAs.

Comparison of the carbohydrate portion of membrane H-2 alloantigens isolated from spleen cells and tumor cells

[ 3H]Fucose- and [ 3H]glucosamine-labeled glycopeptides were obtained from H-2 transplantation alloantigen glycoproteins purified by specific antisera from spleen cells of two strains of inbred mice, C57BL/6 (H-2 b) and BALB/c (H-2 d). Upon Sephadex G-50 column chromatography, these radiolabeled glycopeptides were found to have an elution volume corresponding to a molecular weight of approx. 3300. This value is identical to the molecular size of glycopeptides from H-2 alloantigens isolated from a fibrosarcoma (Meth-A) tumor cell line derived from the BALB/c mouse and a lymphoma (E.L. 4) cell line derived from the C57BL/6 mouse. Upon DEAE-Sephadex column chromatography, the H-2 alloantigen glycopeptides of spleen cells also showed properties almost identical to the glycopeptides of antigen isolated from tumor cells. These findings confirm that the properties of the glycopeptides of H-2 alloantigens from normal spleen and tumor sources were not different by the methods used for comparison.

Human erythrocyte acetylcholinesterase I. Resolution of activity into two components

1. 1. Human erythrocyte acetylcholinesterase solubilized by Triton X-100 has been resolved into two components by DEAE-Sephadex column chromatography. The two partially purified enzymes have apparently identical molecular weights with different electrophoretic mobility. The K m values of the two components were shown not to differ significantly. 2. 2. The molecular weight of Triton-solubilized acetylcholinesterase was estimated by gel filtration, in the presence of Triton X-100, to be approx. 420 000. Following the removal of the detergent, the molecular weight of the enzyme was shown to be greater than 2·10 6.

Structural Studies of Bovine Liver Rhodanese: I. ISOLATION AND CHARACTERIZATION OF TWO ACTIVE FORMS OF THE ENZYME

Crystalline bovine liver rhodanese, prepared by ammonium sulfate and pH precipitation, has been shown to be comprised of two fully active components present in approximately equal amounts which are separable by polyacrylamide gel electrophoresis and by ion exchange chromatography. The two rhodanese forms, designated A and B on the basis of their order of elution from columns of DEAE-Sephadex, are not in equilibrium nor do they represent free enzyme and an enzyme-substrate complex. Furthermore, the two rhodanese species are identical with respect to kinetic parameters, amino acid composition, NH2-terminal amino acid, sulfhydryl content, tryptic peptide maps, and molecular weight. Both forms exhibit equal activity toward β-mercaptopyruvate and utilize this sulfur donor at an efficiency of about 1% that of thiosulfate. Although no chemical or physical basis for the difference between the two rhodanese forms has been found as yet, a new, milder method for the preparation of the enzyme yields a preponderance of rhodanese A (85 to 90%). This, considered together with the elution characteristics of rhodaneses A and B, suggests that rhodanese B may arise during the course of the purification by deamidation of the A form.

Studies on the active principle from Abrus precatorius L. Leguminosae seed kernels

Abrin, a highly toxic protein, has been purified and crystallized with a yield of 0.15 per cent from the kernels of Abrus precatorius seeds. The purification involves extraction with 5 per cent acetic acid, ammonium sulfate fractionation and DEAE Sephadex column chromatography. The crystalline abrin is homogeneous with respect to sedimentation criterion. No hemagglutinating activity was detected in crystalline abrin. Based upon sedimentation equilibrium analysis, a molecular weight of 65 × 10 3 was calculated for the protein while amino acid and tryptic peptide map analysis indicates that the crystalline protein contains two peptide chains.

Occurrence of a New Enzyme Reducing Metmyoglobin in Dolphin Muscle

A new enzyme has been obtained in a crystalline state from the muscle of blue white dolphin. This enzyme resembles to methemoglobin reductase from erythrocyte with respect to (a) elution pattern of DEAE-Sephadex column chromatography, (b) absorption spectra, (c) molecular weight and (d) activity of reducing methemoglobin, metmyoglobin and ferric cytochrome c. However, distinct differences can be observed between two enzymes with regard to (a) sedimentation coefficient, (b) diffusion coefficient, (c) frictional ratio, (d) pH-mobility curve and (e) specific activity of reducing the above three substrates. It is advocated that enzyme is termed metmyoglobin reductase.

Regulation of DNA synthesis in mouse macrophages: I. Sources, action and purification of the macrophage growing factor (MGF)

Summary Mouse peritoneal macrophages in vitro can be induced to proliferate when incubated in medium conditioned by other cells. The stimulating activity of the macrophage growing factor (MGF) is highly species-specific and the release of MGF seems to be restricted to cells of fibroblastic type, lymphoblastic lines being non-producers. MGF can be purified and concentrated on QAE-or DEAE-Sephadex column, from which it elutes as a strong acidic component.

Isolation and properties of human α-lactalbumin

1. 1. A simple chromatographic procedure is presented for isolation of α-lactalbumin from human whey on columns of DEAE-Sephadex and Sephadex G-100. 2. 2. The protein resembles bovine and guinea pig α-lactalbumins in size and amino acid composition, but differs in end groups and immunological specificity.

Hemoglobin J Kaohsiung: β59 Lys → Thr

Hb J Kaohsiung was found, in 1969, in one Taiwanese male resident of Kaohsiung City, Taiwan, during a population survey for hemoglobin variants. From the results of the present chemical structural analyses it is concluded that the site of the anomaly in this singly-fast variant is the β-59 or βE 3 position where a threonyl group replaces the lysyl group normally present. From the chromatographic separations of the mixture of Hb A/J using a DEAE Sephadex column it was found that the relative amounts of Hb A 0 and Hb J Kaohsiung were 53 and 47%, respectively. Hb J Kaohsiung represents the third singly-fast or J-type hemoglobin variant found thus far in Chinese subjects in Taiwan. Previously, Hb J Meinung and Hb J Taichung were reported to have the respective anomalies of β56 (βD7) Gly → Asp and β129 (βH 7) Ala → Asp.

Identification of the two 3H-GTP binding proteins in [formula omitted] supernatant as Tu-Ts and Tu FACTORS

Of the two 3H-GTP-binding components separated on DEAE-Sephadex column chromatography of an E. coli supernatant fraction, the one was identified as Tu-Ts complex and the other as Tu. This assignment was based on the observation that the former rapidly binds with 3H-GDP whereas the latter binds with it only slowly. This was further confirmed by elution pattern of the two components from Sephadex G-IOO column. Molecular weight of Tu, Ts and Tu-Ts was estimated to be approximately 5 × 10 4, 5 × 10 4, and 1 × 10 5 respectively.

Studies on Extracellular Ribonuclease from Yeast

An extracellular ribonuclease of Rhodotorula glutinis was purified about 50-fold from the culture broth, by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex column chromatography, acetone fractionation and Sephadex G-100 gel filtration. The purified enzyme was homogeneous when examined by the ultracentrifuge and disc electrophoresis. The molecular weight was calculated to be 58,900 and 56,000 by the sedimentation-diffusion method and the sedimentation equilibrium method, respectively.

Purification and Properties of the Thrombin-Like Principle of Agkistrodon acutus Venom and Its Comparison with Bovine Thrombin

SummaryBy means of DEAE-Sephadex column chromatography, Agkistrodon acutus venom was separated into 12 fractions. The thrombin-like activity was concentrated in Fr.10. This fraction was rechromatographed on Sephadex G-200. A single band was found on microzone electrophoresis at pH 7.4. A single boundary with S20, w of 3.82 S, which was almost symmetrical, was observed by ultracentrifugation. The estimated molecular weight is 33,500. The chemical analysis shows that the thrombin-like principle of the venom is a glycoprotein. The specific activity is 13 times higher than that of the crude venom. The optimal pH value of the thrombin-like principle (pH 7.5) of the venom is almost identical with that of the bovine thrombin (pH 7.2). The thrombinlike principle of the venom is not affected by heparin, while the clotting activity of the bovine thrombin is inhibited by heparin. The thrombin-like principle of the venom is much more heat stable than the bovine thrombin.No clot retraction was found with the thrombin-like principle of the venom, while the marked clot retraction occurred after plasma was coagulated with thrombin.Thrombin can activate factor XIII (Fibrin stabilizing factor), while the thrombinlike principle can not.

The preparation and assay of sedoheptulose-7-phosphate

The enzymic synthesis of sedoheptulose-7-phosphate from ribose-5-phosphate in the presence of ribose-5-phosphate ketol-isomerase, ribulose-5-phosphate-3-epimerase, and transketolase was investigated. The reaction was driven in the direction of heptulose formation by the addition of NAD, glyceraldehyde phosphate dehydrogenase, and arsenate. These three enzymes could be replaced by a liver supernatant. The sedoheptulose-7-phosphate was purified by barium precipitation and separated from arsenate by gradient elution from a column of DEAE-Sephadex A-25. The product was assayed colorimetrically with cysteine-sulfuric acid and enzymically with transaldolase and by a new procedure. The new enzymic procedure involved the, phosphorylation of the monophosphate to sedoheptulose-1,7-diphosphate by ATP and phosphofructokinase and the cleavage of the diphosphate by aldolase to erythrose-4-phosphate and dihydroxyacetone phosphate. In the presence of glycerol-1-phosphate dehydrogenase the latter was reduced to glycerol-1-phosphate with the oxidation of an equivalent amount of NADH.

The quantitative relationship of urinary oestrogen conjugates after injection of tritiated oestradiol

Following the injection of tritiated oestradiol-17β to 6 pregnant women, the urinary oestrogen conjugates were separated by DEAE-Sephadex columns into 3-glucosiduronates, 16(or 17)-glucosiduronates and sulphates. These fractions were further divided into oestrone, oestradiol-17β and oestriol by Brown's method. In the first 24-hr specimens following the injection, oestrone glucosiduronate was quantitatively the most important fraction, followed by oestriol-3-glucosiduronate and oestriol-16-glucosiduronate, and 17β-oestradiol-17-glucosiduronate and 17β-oestradiol-3-glucosiduronate. The quantitative relationship of these 5 oestrogen conjugates changed in the second and third 24-hr urine specimens in which the proportion of the gluco-siduronates of oestrone and 17β-oestradiol fell progressively, while the percentage of the two oestriol glucosiduronates increased.