Analysis of toxoplasma gondii proteins after Triton X-114 solubilization and hidropholic chromotograhy

The distribution of the surface proteins of toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Twopolypeptides with 15,000 and 70,000 distributed on both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities.two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of t. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.

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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

Biochemical Journal ◽

10.1042/bj2800745 ◽

1991 ◽

Vol 280 (3) ◽

pp. 745-751 ◽

Cited By ~ 55

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.

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Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography

Journal of Chromatography A ◽

10.1016/j.chroma.2012.05.041 ◽

2012 ◽

Vol 1245 ◽

pp. 143-149 ◽

Cited By ~ 26

Author(s):

Yuesheng Dong ◽

Fan Zhang ◽

Zhiming Wang ◽

Li Du ◽

Aiyu Hao ◽

...

Keyword(s):

Human Serum Albumin ◽

Pichia Pastoris ◽

Serum Albumin ◽

Human Serum ◽

Hydrophobic Interaction ◽

Hydrophobic Interaction Chromatography ◽

Phase System ◽

Two Phase ◽

Extraction And Purification ◽

Recombinant Human Serum Albumin

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N-F TRANSITION OF HUMAN SERUM ALBUMIN: A PROBE BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY

Affinity Chromatography and Biological Recognition ◽

10.1016/b978-0-12-166580-7.50080-3 ◽

1983 ◽

pp. 489-494 ◽

Cited By ~ 2

Author(s):

Eugene Sulkowski ◽

Margaret Madajewicz ◽

Darrell Doyle

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Hydrophobic Interaction ◽

Hydrophobic Interaction Chromatography

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

Biochemical Journal ◽

10.1042/bj2310445 ◽

1985 ◽

Vol 231 (2) ◽

pp. 445-449 ◽

Cited By ~ 84

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.

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

Biochemical Journal ◽

10.1042/bj2330525 ◽

1986 ◽

Vol 233 (2) ◽

pp. 525-533 ◽

Cited By ~ 98

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.

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Recovery of Active Polyphenol Oxidase and Peroxidase from Plant Tissues with High Phenolics and Chlorophylls

10.20944/preprints201810.0758.v1 ◽

2018 ◽

Author(s):

Sarana Rose Sommano

Keyword(s):

Phase Separation ◽

High Temperature ◽

Enzymatic Activity ◽

Polyphenol Oxidase ◽

Plant Tissues ◽

Rich Phase ◽

Post Harvest ◽

Membrane Bound ◽

Triton X ◽

High Enzymatic Activity

The present protocol described extraction of active polyphenol oxidase and peroxidase from a plant rich in phenolics and chlorophylls in the post-harvest browning syndrome of B. myrtifolia.  Initially, general optimisation using conventional enzyme extractions was performed.  However, along with membrane-bound proteins, chlorophylls and phenols were also released with Triton X (TTX).  With a view to obtaining high enzymatic activity, removal of the released chlorophylls and phenols by formation of TTX-114 micelles in the detergent rich phase after high-temperature induced phase separation was tested.

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Hydrophobic interaction chromatography of human serum α1-antitrypsin and α1-acid glycoprotein

Journal of Chromatography B Biomedical Sciences and Applications ◽

10.1016/s0378-4347(00)00232-2 ◽

2000 ◽

Vol 744 (1) ◽

pp. 73-79 ◽

Cited By ~ 3

Author(s):

Sándor Oláh ◽

Tibor Kremmer ◽

Mariann Boldizsár

Keyword(s):

Human Serum ◽

Hydrophobic Interaction ◽

Hydrophobic Interaction Chromatography ◽

Acid Glycoprotein

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Purification of Glycerol Kinase by "Dye-Ligand" Chromatography and Hydrophobic Interaction Chromatography on Bead-Cellulose Derivatives

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19930445 ◽

1993 ◽

Vol 58 (2) ◽

pp. 445-451 ◽

Cited By ~ 7

Author(s):

Vladimír Žúbor ◽

Albert Breier ◽

Marta Horváthová ◽

Dagmar Hagarová ◽

Peter Gemeiner ◽

...

Keyword(s):

Hydrophobic Interaction ◽

Specific Activity ◽

Hydrophobic Interaction Chromatography ◽

Glycerol Kinase ◽

Enzymic Activity ◽

Cellulose Derivatives ◽

Long Term Storage ◽

Bead Cellulose ◽

Term Storage

The crude extract of cytosole enzymes was obtained from homogenized cells of Saccharomyces cerevisiae by partition. The enzyme was then isolated from the lower aqueous phase displaying higher glycerol kinase activity by dye-ligand chromatography on Cibacron Blue (CB) or Remazol Brilliant Blue R (RB)-derivatized bead-cellulose, ATP being the eluent. The specific activity of glycerol kinase rised more than 10 and 7-times after affinity dye-ligand chromatography and hydrophobic interaction chromatography, respectively. Glycerol kinase obtained by the latter method was purified by CB-bead cellulose. The final preparation maintained its enzymic activity without noticeable losses during a long-term storage at 4 °C in dark.

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