scholarly journals Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein

1995 ◽  
Vol 312 (2) ◽  
pp. 543-548 ◽  
Author(s):  
P E Golstein ◽  
A Sener ◽  
R Beauwens
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Sodium Cholate ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Iodide Uptake ◽  
Protein Insertion ◽  
Set Up

A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.

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.

Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11 691

2021 ◽  
Author(s):  
Ken-ji Yokoi ◽  
Sosyu Tsutsui ◽  
Gen-ya Arakawa ◽  
Masakazu Takaba ◽  
Koichi Fujii ◽  
...  
Keyword(s):  
High Performance ◽  
Culture Medium ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Reading Frame ◽  
Exclusion Chromatography ◽  
Chain Lengths

Abstract Information about the inulosucrase of non-lactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0–9.0 and 50 °C–55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multi-angle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3,806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3–27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

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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