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 Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114. Application to subcellular fractions of the adrenal medulla

1986 ◽  
Vol 233 (2) ◽  
pp. 525-533 ◽  
Author(s):  
J G Pryde ◽  
J H Phillips
Keyword(s):  
Endoplasmic Reticulum ◽  
Phase Separation ◽  
Membrane Proteins ◽  
Low Temperature ◽  
Membrane Glycoproteins ◽  
Chromaffin Granule ◽  
Rich Phase ◽  
Granule Membrane ◽  
Separation Of Proteins ◽  
Triton X

After solubilization with the detergent Triton X-114, membrane proteins may be separated into three groups: if the membrane is sufficiently lipid-rich, one family of hydrophobic constituents separates spontaneously at low temperature; warming at 30 degrees C leads to separation of a detergent-rich phase and an aqueous phase. Using the chromaffin-granule membrane as a model, we found that many intrinsic membrane glycoproteins are found in the latter phase, probably maintained in solution by adherent detergent. They precipitate, however, when this is removed by dialysis, leaving in solution those truly hydrophilic proteins that were originally adhering to the membranes. We have used this method with mitochondria, and with Golgi- and rough-endoplasmic-reticulum-enriched microsomal fractions: it has proved to be a rapid and convenient method for effecting a partial separation of proteins from a variety of different membranes.

Purification and immunocytochemical localization of neuraminidase from Tritrichomonas foetus

Parasitology ◽  
1999 ◽  
Vol 118 (1) ◽  
pp. 17-25 ◽  
Author(s):  
B. P. DIAS FILHO ◽  
M. BENCHIMOLI ◽  
A. F. B. ANDRADE ◽  
J. ANGLUSTER ◽  
W. DE SOUZA
Keyword(s):  
Phase Separation ◽  
Aqueous Phase ◽  
High Performance ◽  
Polyclonal Antibodies ◽  
Previous Treatment ◽  
Peripheral Region ◽  
Tritrichomonas Foetus ◽  
Neuraminidase Activity ◽  
Ionic Detergent ◽  
Insoluble Pellet

Lysis of Tritrichomonas foetus with a solution of the non-ionic detergent Triton X-114 at 0 °C, followed by low-speed centrifugation, resulted in a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 °C into a detergent-rich phase and an aqueous phase. Neuraminidase activity was mostly located in the detergent-insoluble pellet. When the parasites were incubated with bacterial phosphatidylinositol phospholipase C (PI–PLC) prior to detergent solubilization and phase separation neuraminidase activity was predominantly recovered in aqueous phase, rather than in the pellet and detergent phase. The molecular mass determined by gel permeation in high performance liquid chromatography (HPLC) and SDS–PAGE was 80000 Da. Indirect immunofluorescence microscopy using polyclonal antibodies raised in rabbits against the purified neuraminidase, indicated that the enzyme is exposed on the cell surface. Previous treatment of the cells with PI–PLC significantly reduced antibody binding. Incubation of cryo-sections with the antibodies followed by detection using gold-labelled anti-rabbit IgG confirmed the presence of neuraminidase in the plasma membrane enclosing the cell body and flagella and in the membrane of vesicles preferentially located at the peripheral region of the protozoan.

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 Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation

1988 ◽  
Vol 255 (2) ◽  
pp. 463-470 ◽  
Author(s):  
L P Belzunces ◽  
J P Toutant ◽  
M Bounias
Keyword(s):  
Phase Separation ◽  
Apis Mellifera ◽  
Aqueous Phase ◽  
Sucrose Gradient ◽  
Electrophoretic Analysis ◽  
High Salt ◽  
Sedimentation Analysis ◽  
Low Salt ◽  
S Peak ◽  
Triton X

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion.

scholarly journals The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N

1985 ◽  
Vol 231 (2) ◽  
pp. 445-449 ◽  
Author(s):  
R Matsas ◽  
S L Stephenson ◽  
J Hryszko ◽  
A J Kenny ◽  
A J Turner
Keyword(s):  
Phase Separation ◽  
Membrane Protein ◽  
Total Activity ◽  
Aminopeptidase N ◽  
The Other ◽  
Aminopeptidase Activity ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Rich Phase ◽  
Triton X

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 × 10(−7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 × 10(−6) M) than for the other two forms (IC50 = 1.6 × 10(−5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 × 10(−5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 × 10(−4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.

Endogenous glycosylphosphatidylinositol-specific phospholipase C releases renal dipeptidase from kidney proximal tubules in vitro

2001 ◽  
Vol 353 (2) ◽  
pp. 339-344 ◽  
Author(s):  
Sung Wook PARK ◽  
Kyong CHOI ◽  
Cheol KIM ◽  
Hwang Hee Blaise LEE ◽  
Nigel M. HOOPER ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Phase Separation ◽  
Phospholipase C ◽  
Aqueous Phase ◽  
Proximal Tubules ◽  
Gpi Anchor ◽  
Ph Optimum ◽  
Hydrophilic Nature ◽  
Triton X

Spontaneous enzymic release of renal dipeptidase (RDPase; EC 3.4.13.19), a glycosylphosphatidylinositol (GPI)-linked ectoenzyme, was observed in vitro during incubation of porcine proximal tubules at 37°C. Triton X-114 phase separation of the released RDPase showed that the majority of the enzyme activity partitioned into the aqueous phase, indicating its hydrophilic nature. Immunoblot analyses using an antibody against the cross-reacting determinant (CRD) inositol 1,2-cyclic monophosphate, the epitope formed by phospholipase C (PLC) cleavage of the GPI anchor on a protein, detected the released RDPase. Reprobing the immunoblot with an anti-RDPase serum showed the RDPase band co-migrating with the CRD band. The release of RDPase from the proximal tubules was a Ca2+-dependent process and had a pH optimum of 9.0. These results indicate that RDPase is released from the proximal tubules by the action of a distinct endogenous GPI-specific PLC.

Enrichment of hydrophobic membrane proteins using Triton X-114 and subsequent analysis of their N-glycosylation

2016 ◽  
Vol 1860 (8) ◽  
pp. 1710-1715 ◽  
Author(s):  
Tamara Pavić ◽  
Ivan Gudelj ◽  
Toma Keser ◽  
Maja Pučić-Baković ◽  
Olga Gornik
Keyword(s):  
Membrane Proteins ◽  
Hydrophobic Membrane ◽  
Triton X

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

scholarly journals The role of the Tim8p–Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

2002 ◽  
Vol 158 (6) ◽  
pp. 1017-1027 ◽  
Author(s):  
Sean P. Curran ◽  
Danielle Leuenberger ◽  
Einhard Schmidt ◽  
Carla M. Koehler
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Intermembrane Space ◽  
Hydrophobic Membrane ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Mitochondrial Carrier Family ◽  
Pass Through ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p–Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p–Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p–Tim13p complex is not dependent on zinc, and the purified Tim8p–Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.

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