scholarly journals Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.

1995 ◽  
Vol 131 (1) ◽  
pp. 125-135 ◽  
Author(s):  
S Parpal ◽  
J Gustavsson ◽  
P Strålfors
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Nitrous Acid ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Subcellular Fractionation ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Surface Proteins ◽  
Preparative Scale

A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related to the carbohydrate moiety of phosphatidylinositol glycan anchors of many cell surface proteins. Herein we demonstrate that [32]phosphate in freshly isolated adipocytes and [3H]galactose in cultured hepatoma cells (H4IIE) labeled the same set of three different glycolipids. With all three, the radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We isolated the three phospholipase C-released substances. One of them was susceptible to nitrous acid deamination, indicative of a hexosamine with a free amino group. This phosphooligosaccharide structure had an apparent molecular mass between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and interconvertible species. Adipocytes stimulated with insulin accumulated the nitrous acid sensitive phosphooligosaccharide: after stimulation the intracellular level of free phosphooligosaccharide increased threefold within 5 min, fell off during the next few minutes and then remained at a slightly elevated level. After insulin stimulation the intracellular concentration of free phosphooligosaccharide was > 1,000-fold higher than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insulinomimetic effects on protein phosphorylation of insulin target proteins in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [32P]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [32P]phosphooligosaccharide was found also in the microsomal fraction.

scholarly journals Structure Characterization and Otoprotective Effects of a New Endophytic Exopolysaccharide from Saffron

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 749 ◽  
Author(s):  
Juan Li ◽  
Guimei Wu ◽  
Cuiying Qin ◽  
Wuhai Chen ◽  
Gang Chen ◽  
...  
Keyword(s):  
Cell Damage ◽  
Gel Filtration ◽  
2D Nmr ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Structure Characterization ◽  
Crocus Sativus ◽  
Zebrafish Model ◽  
Cochlear Hair Cells

Saffron, a kind of rare medicinal herb with antioxidant, antitumor, and anti-inflammatory activities, is the dry stigma of Crocus sativus L. A new water-soluble endophytic exopolysaccharide (EPS-2) was isolated from saffron by anion exchange chromatography and gel filtration. The chemical structure was characterized by FT-IR, GC-MS, and 1D and 2D-NMR spectra, indicating that EPS-2 has a main backbone of (1→2)-linked α-d-Manp, (1→2, 4)-linked α-d-Manp, (1→4)-linked α-d-Xylp, (1→2, 3, 5)-linked β-d-Araf, (1→6)- linked α-d-Glcp with α-d-Glcp-(1→ and α-d-Galp-(1→ as sidegroups. Furthermore, EPS-2 significantly attenuated gentamicin-induced cell damage in cultured HEI-OC1 cells and increased cell survival in zebrafish model. The results suggested that EPS-2 could protect cochlear hair cells from ototoxicity exposure. This study could provide new insights for studies on the pharmacological mechanisms of endophytic exopolysaccharides from saffron as otoprotective agents

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

scholarly journals The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein

1997 ◽  
Vol 322 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Philemon PAPANASTASIOU ◽  
Malcolm J. McCONVILLE ◽  
Julie RALTON ◽  
Peter KÖHLER
Keyword(s):  
Specific Surface ◽  
Acyl Chain ◽  
Giardia Duodenalis ◽  
Compositional Analysis ◽  
Surface Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Proteins ◽  
Phase Partitioning ◽  
Chromatography Analysis

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis(syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardiaisolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is post-translationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of [3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.

Purification and Characterization of Laccase fromChaetomium thermophilium and Its Role in Humification

1998 ◽  
Vol 64 (9) ◽  
pp. 3175-3179 ◽  
Author(s):  
Benny Chefetz ◽  
Yona Chen ◽  
Yitzhak Hadar
Keyword(s):  
Municipal Solid Waste ◽  
Solid Waste ◽  
Laccase Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Type I ◽  
Light Spectrum ◽  
Solid Media ◽  
Wide Range

ABSTRACT Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process.C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals Conditions Contributing to the Stability of Factor VIII Coagulant Activity

1977 ◽  
Author(s):  
T. Exner ◽  
K.A. Rickard ◽  
H. Kronenberg
Keyword(s):  
Factor Viii ◽  
High Purity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Buffer System ◽  
Factor Viii Activity ◽  
Coagulant Activity ◽  
The Stability ◽  
Degree Of Purification

Factor VTII tends to become less stable the greater its degree of purification. The loss of factor VIII during preparation of high activity concentrates makes such processes uneconomical. Conditions contributing to the stability of factor VIII were investigated.High purity factor VIII was incubated with plasma components fractionated by gel filtration and by anion exchange chromatography. Factor VIII activity was assessed initially and after several hours incubation. Several fractions destroying factor VIII activity were clearly resolved. Fractions stabilizing factor VIII were associated only with albumin.Various buffer systems were investigated similarly. A non-chelating buffer system containing albumin was found to give optimal factor VIII stability.

Identification of Glucagon-Like Peptide-1(7–36) Amide in Rat Brain

1989 ◽  
Vol 26 (2) ◽  
pp. 169-171 ◽  
Author(s):  
S Yoshimoto ◽  
M Hirota ◽  
C Ohboshi ◽  
K Shima
Keyword(s):  
Rat Brain ◽  
Anion Exchange ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Experimental Conditions ◽  
Specific Radioimmunoassay ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
The Brain

Acid-urea extract of rat brain was examined by glucagon-like peptide-1 (GLP-1) specific radioimmunoassay. A single peak was observed which co-eluted with GLP-1(7–36)amide on gel filtration and anion exchange chromatography. In contrast, GLP-1(1–37) was not detected under our experimental conditions. The fact that GLP-1 (7–36)amide, but not GLP-1(1–37), was present in rat brain suggests that preproglucagon was processed in the brain in the same manner as in the intestine and not as in the pancreas.

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