A Mild Purification Method for Polysaccharide Binding Membrane Proteins: Phase Separation of Digitonin Extracts to Isolate the Hyaluronate Synthase fromStreptococcussp. in Active Form

1996 ◽  
Vol 7 (4) ◽  
pp. 343-346 ◽  
Author(s):  
Sabine Prehm ◽  
Volker Nickel ◽  
Peter Prehm
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Active Form ◽  
Purification Method ◽  
Hyaluronate Synthase

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

1991 ◽  
Vol 280 (3) ◽  
pp. 745-751 ◽  
Author(s):  
N M Hooper ◽  
A Bashir
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Aqueous Phase ◽  
Hydrophobic Membrane ◽  
Rich Phase ◽  
Glycosyl Phosphatidylinositol ◽  
Ionic Detergent ◽  
Membrane Spanning ◽  
Triton X ◽  
Insoluble Pellet

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

scholarly journals The hyaluronate synthase from a eukaryotic cell line

1993 ◽  
Vol 290 (3) ◽  
pp. 791-795 ◽  
Author(s):  
L Klewes ◽  
E A Turley ◽  
P Prehm
Keyword(s):  
Monoclonal Antibodies ◽  
Phase Separation ◽  
Affinity Chromatography ◽  
Cell Line ◽  
Aqueous Phase ◽  
Plasma Membranes ◽  
Eukaryotic Cell ◽  
Molecular Masses ◽  
Triton X ◽  
Hyaluronate Synthase

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

scholarly journals Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39509 ◽  
Author(s):  
Jane A. English ◽  
Bruno Manadas ◽  
Caitriona Scaife ◽  
David R. Cotter ◽  
Michael J. Dunn
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Post Mortem ◽  
Targeted Analysis ◽  
Post Mortem Brain

Properties of Inosinic Acid Dehydrogenase from Bacillus subtilis. I. Purification and Physical Properties

1973 ◽  
Vol 51 (10) ◽  
pp. 1380-1390 ◽  
Author(s):  
Tai-Wing Wu ◽  
K. G. Scrimgeour
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Active Form ◽  
Kinetic Properties ◽  
Purification Method ◽  
Molecular Weights ◽  
Inosinic Acid ◽  
Apparent Homogeneity ◽  
Catalytically Active ◽  
Membrane Bound

IMP (inosinic acid or inosine-5′-phosphate) dehydrogenase has been purified to apparent homogeneity from Bacillus subtilis. The purification method yields an enzyme preparation that retains a constant level of inhibition by guanosine 5′-phosphate. The enzyme is membrane bound, and can be removed from membrane material after treatment either with detergents or with phospholipase A. Both the membrane-bound and solubilized forms of IMP dehydrogenase have similar kinetic properties. The soluble enzyme can occur in a number of oligomeric forms, with molecular weights that are multiples of 100 000 daltons. Although both the tetramer and the dimer appear to be catalytically active, no conclusions can yet be drawn about the quaternary structure of the enzymically active form(s).

Divide and conquer: How phase separation contributes to lateral transport and organization of membrane proteins and lipids

2020 ◽  
Vol 233 ◽  
pp. 104985
Author(s):  
Ethan J. Miller ◽  
Amanda M. Ratajczak ◽  
Autumn A. Anthony ◽  
Matthew Mottau ◽  
Xaymara I. Rivera Gonzalez ◽  
...  
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Divide And Conquer ◽  
Lateral Transport

scholarly journals The rapid “teabag” method for high-end purification of membrane proteins

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jenny Hering ◽  
Julie Winkel Missel ◽  
Liying Zhang ◽  
Anders Gunnarsson ◽  
Marie Castaldo ◽  
...  
Keyword(s):  
Applied Sciences ◽  
Membrane Proteins ◽  
Size Exclusion ◽  
Purification Method ◽  
New Approach ◽  
Purification Time ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Processing Steps

Abstract Overproduction and purification of membrane proteins are generally challenging and time-consuming procedures due to low expression levels, misfolding, and low stability once extracted from the membrane. Reducing processing steps and shortening the timespan for purification represent attractive approaches to overcome some of these challenges. We have therefore compared a fast “teabag” purification method with conventional purification for five different membrane proteins (MraY, AQP10, ClC-1, PAR2 and KCC2). Notably, this new approach reduces the purification time significantly, and the quality of the purified membrane proteins is equal to or exceeds conventional methods as assessed by size exclusion chromatography, SDS-PAGE and downstream applications such as ITC, crystallization and cryo-EM. Furthermore, the method is scalable, applicable to a range of affinity resins and allows for parallelization. Consequently, the technique has the potential to substantially simplify purification efforts of membrane proteins in basic and applied sciences.

scholarly journals The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination.

1980 ◽  
Vol 86 (1) ◽  
pp. 292-303 ◽  
Author(s):  
W A Muller ◽  
R M Steinman ◽  
Z A Cohn
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Active Form ◽  
Electrophoretic Analysis ◽  
Molecular Weights ◽  
Novel Proteins ◽  
Novel Method ◽  
External Face ◽  
Vacuolar System ◽  
Generating System

A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable, enzymatically active form. The addition of LPO-latex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membrane-derived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrates both acid phosphatase and lactoperoxidase activities. The exposure of LPO-latex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4 degrees C leads to incorporation of label into TCA-precipitable material. The incorporated cel-associated label was present as monoiodotyrosine, and negligible amounts were found in lipids. Cell viability remained > 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical.

Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins

Methods ◽  
2011 ◽  
Vol 54 (4) ◽  
pp. 396-406 ◽  
Author(s):  
Rommel A. Mathias ◽  
Yuan-Shou Chen ◽  
Eugene A. Kapp ◽  
David W. Greening ◽  
Suresh Mathivanan ◽  
...  
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Mouse Liver ◽  
Microsomal Membrane ◽  
Isolation And Purification ◽  
Triton X
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