The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator

2014 ◽  
Vol 92 (6) ◽  
pp. 564-575 ◽  
Author(s):  
Simon Baturin ◽  
Jamie J. Galka ◽  
Hadeesha Piyadasa ◽  
S. Gajjeraman ◽  
Joe D. O’Neil
Keyword(s):  
Membrane Protein ◽  
Protein Interactions ◽  
Acyl Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Thermal Dissociation ◽  
Sodium Dodecyl ◽  
Integral Membrane Protein ◽  
Head Group ◽  
Glycerol Facilitator ◽  
The Stability

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein–protein interactions are discussed.

scholarly journals Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide

1981 ◽  
Vol 196 (2) ◽  
pp. 471-479 ◽  
Author(s):  
A P Thomas ◽  
A P Halestrap
Keyword(s):  
Membrane Protein ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Approximate Amount ◽  
Specific Labelling

1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.

scholarly journals Bis(β-lactosyl)-[60]fullerene as novel class of glycolipids useful for the detection and the decontamination of biological toxins of the Ricinus communis family

2014 ◽  
Vol 10 ◽  
pp. 1504-1512 ◽  
Author(s):  
Hirofumi Dohi ◽  
Takeru Kanazawa ◽  
Akihiro Saito ◽  
Keita Sato ◽  
Hirotaka Uzawa ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Protein Interactions ◽  
Colloidal Solution ◽  
Ricinus Communis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cycloaddition Reaction ◽  
Azide Group ◽  
Red Color ◽  
Sds Page

Glycosyl-[60]fullerenes were first used as decontaminants against ricin, a lactose recognition proteotoxin in the Ricinus communis family. A fullerene glycoconjugate carrying two lactose units was synthesized by a [3 + 2] cycloaddition reaction between C60 and the azide group in 6-azidohexyl β-lactoside per-O-acetate. A colloidal aqueous solution with brown color was prepared from deprotected bis(lactosyl)-C60 and was found stable for more than 6 months keeping its red color. Upon mixing with an aqueous solution of Ricinus communis agglutinin (RCA120), the colloidal solution soon caused precipitations, while becoming colorless and transparent. In contrast, a solution of concanavalin A (Con A) caused no apparent change, indicating that the precipitation was caused specifically by carbohydrate–protein interactions. This notable phenomenon was quantified by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results were discussed in terms of detection and decontamination of the deadly biological toxin in the Ricinus communis family.

scholarly journals Use of G-protein fusions to monitor integral membrane protein–protein interactions in yeast

10.1038/80274 ◽  
2000 ◽  
Vol 18 (10) ◽  
pp. 1075-1079 ◽  
Author(s):  
Kathleen N. Ehrhard ◽  
Jörg J. Jacoby ◽  
Xin-Yuan Fu ◽  
Reinhard Jahn ◽  
Henrik G. Dohlman
Keyword(s):  
Membrane Protein ◽  
G Protein ◽  
Protein Interactions ◽  
Integral Membrane Protein ◽  
Protein Protein Interactions

scholarly journals Calcium-dependence of insulin receptor phosphorylation

1983 ◽  
Vol 214 (2) ◽  
pp. 361-366 ◽  
Author(s):  
W E Plehwe ◽  
P F Williams ◽  
I D Caterson ◽  
L C Harrison ◽  
J R Turtle
Keyword(s):  
Membrane Protein ◽  
Insulin Receptor ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Beta Subunit ◽  
Receptor Phosphorylation ◽  
32P Incorporation ◽  
Isolated Rat

Phosphorylation of the insulin receptor of isolated rat adipocytes in response to insulin has been studied. Immunoprecipitation of adipocyte membrane protein demonstrated increased incorporation of 32P after exposure to insulin for 15 min, but this was dependent on the presence of physiological concentrations of Ca2+ and Mg2+. Autoradiography of solubilized immunoprecipitated membrane protein after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that most of the 32P incorporation occurred in a band corresponding to Mr 95 000, which has been identified previously as the beta-subunit of the insulin receptor. 32P incorporation was inhibited by 2,4-dinitrophenol and trifluoperazine. It is suggested that insulin-receptor phosphorylation is an energy-requiring process that is Ca2+-dependent and may be modulated by calmodulin. Phosphorylation may proceed independently of glucose transport.

scholarly journals Cell-surface discoidin in aggregating cells of Dictyostelium discoideum

1982 ◽  
Vol 204 (3) ◽  
pp. 787-794 ◽  
Author(s):  
I C Madley ◽  
M J Cook ◽  
B D Hames
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Electrophoretic Analysis ◽  
Integral Membrane Protein ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Surface Receptors

Both discoidin I and discoidin II have been detected on the surface of aggregating (10 h developmental stage) cells of Dictyostelium discoideum NC4 by radioiodination of the cell-surface followed by immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis. Approx. 92% of cell-surface discoidin I and 72% of cell-surface discoidin II can be eluted with 0.5 M-galactose, showing that most of each endogenous lectin is not present as integral membrane protein but rather is bound to cell-surface discoidin receptors. Two-dimensional polyacrylamide-gel-electrophoretic analysis of discoidin I suggests that the native tetramer may be a hetero-multimer composed of both Ia and Ib subunits. Cell-surface discoidin I also contains both types of subunit, but it is not clear whether both subunits have corresponding cell-surface receptors.

Stability of Secretory Granules Containing Growth Hormone and Prolactin from Bovine Anterior Pituitary Gland

1974 ◽  
Vol 52 (4) ◽  
pp. 327-335 ◽  
Author(s):  
André Lemay ◽  
Fernand Labrie ◽  
Denis Drouin
Keyword(s):  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Inhibitory Effect ◽  
High Yield ◽  
Maximal Effect ◽  
Enzymatic Markers ◽  
The Stability

Bovine adenohypophyseal secretory granules were purified by a technique giving a high yield of large granules containing 70–90% of prolactin (PRL) and 10–20% of growth hormone (GH). Purity of the preparation was checked by electron microscopy and enzymatic markers. Stability of the isolated secretory granules was studied by measurement of the optical density of the granule suspensions and by measurement of GH and PRL release by disc gel electrophoresis at pH 8.9. At 0 °C, granules suspended in an hypotonic medium are stable at pH 7.4. At room temperature, they are particularly stable at acidic pH but are almost completely solubilized at a pH ranging from 7.0 to 10.0, a half-maximal effect on PRL release being observed at pH 7.5. Lysolecithin has a marked solubilizing effect at a pH of 3.0–6.0 whereas 0.15% sodium dodecyl sulfate does not affect granule stability at a pH below 5. ATP (0.5 mM) inhibits GH and PRL release to, respectively, 65 and 27% of the control rates during a 60 min incubation at 37 °C and pH 7.4. A concentration of 2 mM Ca2+ has a marked inhibitory effect on both GH and PRL release whereas 2 mM Mg2+ inhibits PRL release but does not affect the release of GH. EGTA increases PRL release by 30% but does not significantly affect GH release. As evidenced by polyacrylamide gel electrophoresis, no degradation of GH or PRL occurred during a 1 h incubation of secretory granules at 37 °C and pH 7.4. These data suggest a role of Ca2+- and Mg2+-ATPase activities on the stability and possibly the formation of GH- and PRL-containing secretory granules.

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