Purification of UDP-N-acetylenolpyruvoylglucosamine reductase from Escherichia coli by affinity chromatography, its subunit structure and the absence of flavin as the prosthetic group

1979 ◽  
Vol 57 (2) ◽  
pp. 188-196 ◽  
Author(s):  
Rashid A. Anwar ◽  
Mariana Vlaovic
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Absorption Spectrum ◽  
Absorption Band ◽  
Affinity Chromatography ◽  
Subunit Structure ◽  
Prosthetic Group ◽  
A Value ◽  
No Absorption

The enzyme UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) was purified to homogeneity from Escherichia coli by affinity chromatography on a NADP-agarose column. The evidence suggests that the enzyme (molecular weight 35 000) is composed of two nonidentical subunits of molecular weight 21 500 and 13 500, respectively. The absorption spectrum of the purified enzyme shows no absorption band around 450 nm and thus does not support the previous suggestions that the enzyme is a flavoprotein. However, the A280: A260 ratio gives a value of 0.86 which suggests the presence of tightly bound nucleotide. A quantitative transfer of tritium from 1,4-[4-3H]NADPH to UDP-N-acetylenolpyruvoylglucosamine to form UDP-N-[3H]acetylmuramic acid was also observed, which clearly shows that the enzyme is not a flavoprotein.

scholarly journals Thiol reduction of human α2-macroglobulin. The subunit structure

1972 ◽  
Vol 127 (1) ◽  
pp. 187-197 ◽  
Author(s):  
J. M. Jones ◽  
J. M. Creeth ◽  
R. A. Kekwick
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Experimental Conditions ◽  
A Value ◽  
Α2 Macroglobulin ◽  
A Subunit ◽  
Normal Human ◽  
Ph Range

1. Human α2-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s020,w. 3. The dissociation that occurs in the pH range 4.5–2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

scholarly journals l-Phenylalanyl-tRNA Synthetase of Escherichia coli K-10. A Reinvestigation of Molecular Weight and Subunit Structure

1974 ◽  
Vol 43 (3) ◽  
pp. 601-607 ◽  
Author(s):  
Till Hanke ◽  
Peter Bartmann ◽  
Hauke Hennecke ◽  
Heinz M. Kosakowski ◽  
Rainer Jaenicke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Trna Synthetase ◽  
Subunit Structure

The Three Dimensional Structure of NADP + Specific Isocitrate Dehydrogenase of Escherichia Coli

1975 ◽  
Vol 33 ◽  
pp. 670-671
Author(s):  
Louis T. Germinario
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Isocitrate Dehydrogenase ◽  
Tertiary Structure ◽  
Carbon Film ◽  
Positive Staining ◽  
Subunit Structure ◽  
E Coli ◽  
Thin Carbon ◽  
Citrate Phosphate

The NADP+ specific isocitrate dehydrogenase (IDH) three-Ds - isocitrate: NADP+: oxidoreductase (decarboxylating), E.C. 1.1.1.42. of Escherichia coli was recently purified to electrophoretic homogeneity. The enzyme has a molecular weight of 83,000 daltons, exists as a catalytically active dimer with identical subunits of a molecular weight of 42,000 daltons and an isoelectric point of pH 5. The native enzyme crystallizes in the form of a regular octahedron ranging in size from 5 to 30 um. In this investigation the subunit structure (tertiary Structure) and quaternary structure of E. coli IDH is described along with functional implications.Electrophoretically pure E. coli (F 143/KL259) IDH (.43 mg/ml, specific activity 30.5 enzyme units/mg protein) was maintained in 0.0125 M citrate-phosphate (pH 5.5) containing 20% (v/v) glycerol and 2mM MgCl2 at 4°C. Fenestrated 400 mesh copper grids were pre-coated with gold before overlaying a thin carbon film (∼30Å thick). Enzyme was deposited on the support film by lightly touching a drop of enzyme-buffer solution, followed by positive staining with 1% uranyl acetate (pH 4.7), rinsed gently in the citrate-phosphate buffer and air dried. Denaturation of IDH enzyme complex into its constituent subunits was effected by treating grids containing enzyme with 10M urea and stained and rinsed as indicated above.

scholarly journals The subunit structure of horse spleen apoferritin: the molecular weight of the oligomer and its stability to dissociation by dilution (Short Communication)

1973 ◽  
Vol 131 (4) ◽  
pp. 855-857 ◽  
Author(s):  
Robert R. Crichton ◽  
Robert Eason ◽  
Allan Barclay ◽  
Charles F. A. Bryce
Keyword(s):  
Molecular Weight ◽  
Short Communication ◽  
Sedimentation Equilibrium ◽  
Subunit Structure ◽  
Subunit Dissociation ◽  
A Value

The oligomer molecular weight of horse spleen apoferritin was determined by sedimentation-equilibrium techniques and a value of 443000 found. It is concluded that the apoferritin molecule consists of 24 subunits. At concentrations as low as 0.01μm there is no evidence of subunit dissociation.

scholarly journals Inosine 5′-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5′-monophosphate

1979 ◽  
Vol 183 (3) ◽  
pp. 481-494 ◽  
Author(s):  
H J Gilbert ◽  
C R Lowe ◽  
W T Drabble
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Competitive Inhibitor ◽  
Subunit Structure ◽  
Imp Dehydrogenase ◽  
Finger Printing ◽  
Monospecific Antisera

Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic ‘finger-printing’ demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli.

Tryptophan synthase: purification, and some properties of an inhibitor from pea roots

1971 ◽  
Vol 49 (6) ◽  
pp. 821-832 ◽  
Author(s):  
James Chen ◽  
W. G. Boll
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Absorption Spectrum ◽  
Neurospora Crassa ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Ultraviolet Absorption Spectrum ◽  
Tryptophan Synthase ◽  
Shoot Tissue ◽  
Pea Roots

An undialyzable, thermostable inhibitor of tryptophan synthase from pea plants, Escherichia coli, and Neurospora crassa occurs in the filtrate from acetone extraction of pea tissue. A procedure is described for purification of the inhibitor extracted from roots. The final, purified preparation is homogeneous as shown by disc electrophoresis on acrylamide gel. The molecular weight, estimated by gel filtration on Sephadex G-200, is about 18 000. The inhibitor is stable even on heating at pH 7.0 in a water bath at 100 °C and has a characteristic ultraviolet absorption spectrum. The inhibitor shows some protein-like properties and acid hydrolysates were found to contain substances sensitive to ninhydrin. However, the hydrolysates also contain substances, presumably sugars, sensitive to benzidine reagent, appreciable sugar in galactose equivalents, and some fluorescent substances. The inhibitor was also found in shoot tissue. In both root and shoot the inhibitor increases, on a protein basis, with age of the tissue.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

Evaluation of Stemness Maintenance Properties of the Recombinant Human Laminin α2 LG1-3 Domains in Human Mesenchymal Stem Cells

2019 ◽  
Vol 26 (10) ◽  
pp. 785-791
Author(s):  
Ji-Eun Kim ◽  
Hye-Jin Seo ◽  
SuJin Lee ◽  
Jun-Hyeog Jang
Keyword(s):  
Escherichia Coli ◽  
Stem Cells ◽  
Molecular Weight ◽  
Mesenchymal Stem Cells ◽  
Cell Adhesion ◽  
Human Mesenchymal Stem Cells ◽  
Adhesion Assay ◽  
Proliferation Assay ◽  
Undifferentiated State ◽  
Laminin Α2

Background: Laminin, a member of the Extracellular Matrix (ECM), is a glycoprotein that is used as a factor that affects cell adhesion, proliferation, survival, and differentiation. Of these, five globular domains (LG domains) of the alpha chain play an important role in influencing the cell by binding to the integrin. Objective: This study aimed to evaluate the ability of globular domains 1-3 of laminin alpha2 (rhLAMA2LG1-3) in maintaining the pluripotency of human Mesenchymal Stem Cells (hMSCs), which are widely used in regenerative medicine. Methods: hMSCs were grown in the medium supplemented with rhLAMA2LG1-3, then the effect of the protein on hMSCs were confirmed through cell adhesion assay, proliferation assay and RTPCR. Results: rhLAMA2LG1-3 expressed in Escherichia coli has a molecular weight of 70 kDa, at 1 µg/ml concentration of rhLAMA2LG1-3, the attachment and proliferation of hMSCs were approximately 3.18-fold and 1.67-fold, respectively, more efficient than those of untreated controls. In addition, the undifferentiated state and degree of stemness of hMSCs were measured, on the basis of CD90 and CD105 levels. In the rhLAMA2LG1-3-treated hMSCs, the expression levels of CD90 and CD105 increased by 2.83-fold and 1.62-fold, respectively, compared to those in untreated controls. Conclusion: rhLAMA2LG1-3 can be potentially used in stem cell therapy to improve the viability and maintain the undifferentiated state of hMSCs.

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