Triton X-100-4 M urea as an extraction medium for membrane proteins. II. Molecular properties of pure cytochrome b559. Lipoprotein containing small polypeptide chains and a limited lipid composition

Lipid composition in cellular membranes plays an important role in maintaining the structural integrity of cells and in regulating cellular signaling that controls functions of both membrane-anchored and cytoplasmic proteins. ATP-dependent ABC and P4-ATPase lipid transporters, two integral membrane proteins, are known to contribute to lipid translocation across the lipid bilayers on the cellular membranes. In this review, we will highlight current knowledge about the role of cholesterol and phospholipids of cellular membranes in regulating cell signaling and how lipid transporters participate this process.

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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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[17] Isolation of integral membrane proteins by phase partitioning with triton X-114

Methods in Enzymology - Aqueous Two-Phase Systems ◽

10.1016/0076-6879(94)28019-3 ◽

1994 ◽

pp. 182-193 ◽

Cited By ~ 101

Author(s):

John S. Brusca ◽

Justin D. Radolf

Keyword(s):

Membrane Proteins ◽

Integral Membrane Proteins ◽

Phase Partitioning ◽

Triton X

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Differential partitioning of plasma membrane proteins into the triton X-100-insoluble cytoskeleton fraction during concanavalin A-induced receptor redistribution

Journal of Cell Science ◽

10.1242/jcs.92.1.85 ◽

1989 ◽

Vol 92 (1) ◽

pp. 85-91

Author(s):

W.F. Patton ◽

M.R. Dhanak ◽

B.S. Jacobson

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Cell Surface ◽

Concanavalin A ◽

Temporal Order ◽

Surface Glycoprotein ◽

Plasma Membrane Proteins ◽

Cell Surface Glycoprotein ◽

Extensive Cell ◽

Triton X

The plasma membrane proteins of Dictyostelium discoideum were characterized with respect to their partitioning into the Triton-insoluble cytoskeleton fraction of the cell during concanavalin A-induced capping. Two fractions of plasma membrane-associated concanavalin A were identified; one that immediately associated with the cytoskeleton fraction via cell surface glycoproteins, and one that partitioned with the cytoskeleton only after extensive cell surface glycoprotein cross-linking. Three major classes of polypeptides were found in the plasma membrane that differed with respect to their partitioning properties into the cytoskeleton fraction. The temporal order of association of the polypeptides with the cytoskeleton during concanavalin A-induced capping corresponded to the strength of their association with the cytoskeleton fraction as determined by pH and ionic strength elution from unligated cytoskeletons.

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Evidence for a second class of membrane glycoprotein involved in cell adhesion

Journal of Cell Science ◽

10.1242/jcs.93.4.631 ◽

1989 ◽

Vol 93 (4) ◽

pp. 631-640

Author(s):

W.E. Norris

Keyword(s):

Extracellular Matrix ◽

Biological Activity ◽

Cell Adhesion ◽

Membrane Proteins ◽

Soluble Fraction ◽

Biological Activities ◽

Integral Membrane Proteins ◽

Membrane Glycoprotein ◽

Transmembrane Receptors ◽

Triton X

It is believed that transmembrane relationships exist between the cytoskeleton and the extracellular matrix through integral membrane proteins, almost certainly glycoproteins, which would act as transmembrane receptors. Such receptors would include those involved in cell adhesion. I have been able to isolate a detergent-soluble fraction from chick embryo fibroblasts that is enriched in these integral membrane proteins by making use of their amphipathic character to phase-separate them in the detergent Triton X-114. Antisera raised to this fraction had biological activities interfering with cell adhesion and motility. A 45 X 10(3) Mr glycoprotein unique to this fraction appears to be responsible for this biological activity and is a candidate for a transmembrane receptor involved in cell adhesion.

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Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽

10.1182/blood.v60.4.894.894 ◽

1982 ◽

Vol 60 (4) ◽

pp. 894-904 ◽

Cited By ~ 15

Author(s):

D Pidard ◽

JP Rosa ◽

TJ Kunicki ◽

AT Nurden

Keyword(s):

Membrane Proteins ◽

Divalent Cation ◽

Human Platelet ◽

Polyacrylamide Gel Electrophoresis ◽

Indirect Evidence ◽

Human Platelets ◽

Platelet Membrane ◽

Membrane Glycoproteins ◽

Nonionic Detergent ◽

Triton X

Abstract Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

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ASSOCIATION OF LIPID WTTH THE CYTOSKELETON (CS) OF AGGREGATING PLATELETS PRELABELED WITH 3H-PALMITTC ACID

10.1055/s-0038-1643639 ◽

1987 ◽

Author(s):

A E Livne ◽

A M Packham ◽

A M Guccione ◽

F J Mustard

Keyword(s):

Electron Microscopy ◽

Membrane Proteins ◽

Palmitic Acid ◽

Protease Activity ◽

Lipid Extraction ◽

Early Time ◽

Human Platelets ◽

Dnase I ◽

Time Points ◽

Triton X

To investigate the association of lipid with the CS of platelets during aggregation, rabbit and human platelets were isolated and labeled with 3H-palmitic acid; lipid extraction showed about 80% in phospholipid. Limited aggregation was induced with ADP (undpr conditions in which release of granule contents did not occur) or thrombin, and CS isolated after lysis with 1% Triton X-1DD, 5 mM EGTA. CS from unactivated platelets had approx. 0.03% of the total platelet label, but after aggregation with ADP (2 µM) or thrombin (0.l U/ml) for 20-30 sec, 3 to 5% of the label was with the CS. Stirring was necessary and added fibrinogen enhanced aggregation and association of label with the CS; incorporation of label increased exponentially as aggregation proceeded and decreased exponentially during deaggregation. The amount of label with the CS was related more directly to the number of sites of contact than to the numbor of platelets in the aggregates. To determine how much label was bound to CS, some experiments were done in which the CS was disrupted and loss of label was assessed. In these experiments, DNase I and Ca2+ were added to the Triton X-100 lysis medium to cause actin depolymerisation, under conditions in which the Ca2+-dependent protease activity was inhibited. This treatment greatly reduced the association of label with the CS in samples taken from early time points on the aggregation curves: when aggregation proceeded further, a large proportion of label was not dissociated by this treatment. These findings, electron microscopy, and enrichment of the CS of aggregated platelets with only some membrane proteins labeled by the 125lactopproxidase method, indicated that under the conditions of limited aggregation used, the 3H-labeled lipid was mainly associated with the CS and not with trapped membrane fragments resulting from incomplete lysis. Si nee the pattern of CS 1abeli ng (3H-palmit at el and selective association of some of the membrane proteins with the cytoskeleton/1ipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents.

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Circular Dichroism Analysis of Native and Reaggregated Mycoplasma Membranes

Canadian Journal of Biochemistry ◽

10.1139/o73-079 ◽

1973 ◽

Vol 51 (5) ◽

pp. 632-636 ◽

Cited By ~ 6

Author(s):

Shlomo Rottem ◽

Leonard Hayflick

Keyword(s):

Circular Dichroism ◽

Membrane Proteins ◽

Sodium Dodecyl Sulfate ◽

Lipid Composition ◽

Sodium Dodecyl ◽

Growth Cycle ◽

Membrane Material ◽

Α Helix ◽

Low Amplitude ◽

Circular Dichroïsm

Circular dichroism analyses of Acholeplasma laidlawii membranes solubilized by sodium dodecyl sulfate showed a typical α-helix spectrum. The estimated α-helix content was of the order of 35% calculated from the ellipticity at 208 nm of membranes solubilized by 20 mM SDS. Reaggregation of the solubilized membrane material to a membrane-like structure resulted in a distorted spectrum with low amplitude and red-shifted extrema like that of the native membranes. Throughout the growth cycle the circular dichroism spectra of membrane proteins remained the same despite the marked differences in membrane densities. The optical activity of the membranes was not affected by changing the lipid composition or extraction of over 90% of the lipids, although the latter resulted in marked destabilization of the proteins.

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