scholarly journals Characterization of phospholipid methylation in rat brain myelin

1995 ◽  
Vol 307 (1) ◽  
pp. 239-244 ◽  
Author(s):  
V Tsvetnitsky ◽  
L Auchi ◽  
A Nicolaou ◽  
W A Gibbons
Keyword(s):  
Rat Brain ◽  
Affinity Purification ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Methyl Transferase ◽  
Sds Page ◽  
Triton X ◽  
Predominant Product

Highly purified rat brain myelin was solubilized in Triton X-100 and myelin phospholipid N-methyltransferase was characterized. The enzyme activities were separated by isoelectric focusing and ion-exchange chromatography. The phospholipid methyl-transferase has shown at least four peaks of activity with pIapp. values of 4.5, 5.2, 6.2 and 8.4. After affinity purification each of these activities revealed a close set of bands of approx. 65 kDa on SDS/PAGE. These data together with those from preparative SDS/PAGE separations suggested that rat brain myelin contains three acidic and at least one basic phospholipid-methylating isoenzymes and that the major isoenzyme in each case is approx. 65 kDa in size. While the predominant product of the reaction catalysed by all detected isoforms was monomethylated phosphatidylethanolamine, the least acidic isoform (pIapp. 6.2) also formed about 20% phosphatidylcholine, suggesting that these isoenzymes may play different roles in vivo.

scholarly journals Biochemical Characterization of a Thiol-Activated, Oxidation Stable Keratinase from Bacillus pumilus KS12

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Rinky Rajput ◽  
Richa Sharma ◽  
Rani Gupta
Keyword(s):  
Ion Exchange ◽  
Bacillus Pumilus ◽  
Biochemical Characterization ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amidolytic Activity ◽  
Alkaline Serine Protease ◽  
Sds Page ◽  
Temperature Optima

An extracellular keratinase from Bacillus pumilus KS12 was purified by DEAE ion exchange chromatography. It was a 45 kDa monomer as determined by SDS PAGE analysis. It was found to be an alkaline, serine protease with pH and temperature optima of 10 and 60C, respectively. It was thiol activated with two- and eight-fold enhancement in presence of 10 mM DTT and β-mercaptoethanol, respectively. In addition, its activity was stimulated in the presence of various surfactants, detergents, and oxidizing agents where a nearly 2- to 3-fold enhancement was observed in presence of H2O2 and NaHClO3. It hydrolyzed broad range of complex substrates including feather keratin, haemoglobin, fibrin, casein,and α-keratin. Analysis of amidolytic activity revealed that it efficiently cleaved phenylalanine → leucine → alanine- p-nitroanilides. It also cleaved insulin B chain between Val2- Asn3, Leu6-Cys7 and His10-Leu11 residues.

scholarly journals Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

2018 ◽  
Author(s):  
Bingyu Ye ◽  
Wenlong Shen ◽  
Minglei Shi ◽  
Yan Zhang ◽  
Cunshuan Xu ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Clinical Investigation ◽  
Affinity Purification ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Radioprotective Activity ◽  
Backbone Cyclization

Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Ni-chelating affinity purification, the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.

scholarly journals ISOLASI DAN KARAKTERISASI ENZIM b-1,3-ENDOGLUKANASE DARI TANAMAN KUBIS (Brassica oleracea cv. Capitata L.)

2010 ◽  
Vol 15 (2) ◽  
pp. 99-105
Author(s):  
Y. Sri Manuhara
Keyword(s):  
Ion Exchange ◽  
Brassica Oleracea ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibition Zone ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Optimum Ph

Isolation and characterization of β-1,3-endoglucanase from cabbage (Brassica oleracea var. capitata L.) have been done. It showed 40° C of optimum temperature, and optimum pH is 7. After the purification with hydrophobic interaction chromatography and ion exchange chromatography, it’s activity was increased. Based on SDS-PAGE analysis, β-1,3-endoglucanase have molecular weight around 48 kD. Antifungal activity of β-1,3-endoglukanase show that it has best inhibition zone on Fusarium solanii at extract from ion exchange chromatography.

scholarly journals Purification and characterization of phosphatidylinositol synthase from human placenta

1994 ◽  
Vol 297 (3) ◽  
pp. 517-522 ◽  
Author(s):  
B E Antonsson
Keyword(s):  
Human Placenta ◽  
Microsomal Fraction ◽  
Enzyme Reaction ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Sds Page ◽  
Phosphatidylinositol Synthase ◽  
Triton X

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C.

scholarly journals Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

2018 ◽  
Author(s):  
Bingyu Ye ◽  
Wenlong Shen ◽  
Minglei Shi ◽  
Yan Zhang ◽  
Cunshuan Xu ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Clinical Investigation ◽  
Affinity Purification ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Radioprotective Activity ◽  
Backbone Cyclization

Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Ni-chelating affinity purification, the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

Post-proline endopeptidase, further characterization of the enzyme from pig kidneys

1984 ◽  
Vol 49 (8) ◽  
pp. 1846-1853 ◽  
Author(s):  
Karel Hauzer ◽  
Tomislav Barth ◽  
Linda Servítová ◽  
Karel Jošt
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Relative Molecular Mass ◽  
Enzyme Molecule ◽  
Thiol Compounds ◽  
Modified Method ◽  
N Terminus

A post-proline endopeptidase (EC 3.4.21.26) was isolated from pig kidneys using a modified method described earlier. The enzyme was further purified by ion exchange chromatography on DEAE-Sephacel. The final product contained about 95% of post-proline endopeptidase. The enzyme molecule consisted of one peptide chain with a relative molecular mass of 65 600 to 70 000, containing a large proportion of acidic and alifatic amino acids (glutamic acid, aspartic acid and leucine) and the N-terminus was formed by aspartic acid or asparagine. In order to prevent losses of enzyme activity, thiol compounds has to be added.

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

Proteolysis in Heterocyst-Forming Cyanobacteria: Characterization of a Further Enzyme with Trypsin-Like Specificity, and of a Prolyl Endopeptidase from Anabaena variabilis

1994 ◽  
Vol 49 (1-2) ◽  
pp. 70-78 ◽  
Author(s):  
Ulrike Strohmeier ◽  
Christian Gerdes ◽  
Wolfgang Lockau
Keyword(s):  
Ion Exchange ◽  
Proteolytic Enzymes ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Anabaena Variabilis ◽  
Prolyl Endopeptidase ◽  
Cyanobacterium Anabaena

Soluble extracts of the cyanobacterium Anabaena variabilis ATCC 29413 and an engineered mutant that lacks an intracellular protease cleaving after Lys and Arg (Maldener, Lockau, Cai, and Wolk, Mol. Gen. Genet. 225, 113-120 (1991)) were separated by ion exchange chromatography, and protease profiles determined using azocasein, Nα-benzoyl-ᴅ,ʟ arginine- 4-nitroanilide and N-carbobenzoxy-glycyl-ʟ-proline-4-nitroanilide as substrates. A second enzyme cleaving at the carboxyl side of lysine and arginine, and a prolyl endopeptidase were detected, enriched and characterized. Both proteolytic enzymes appear to be located in the periplasm.

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