Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line

2004 ◽  
Vol 32 (5) ◽  
pp. 777-779
Author(s):  
P. Pompach ◽  
P. Man ◽  
P. Novák ◽  
V. Havlíček ◽  
A. Fišerová ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Extraction Procedure ◽  
Sucrose Density Gradient ◽  
Cellular Adhesion ◽  
Jurkat Cells ◽  
Membrane Microdomains ◽  
Sucrose Density Gradient Centrifugation ◽  
Biologically Relevant ◽  
Jurkat T Cell

Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

The Role of Lipid Rafts in the Assembly and Down-Regulation of the Factor X Activating Complex on the Activated Platelet Membrane.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1010-1010 ◽  
Author(s):  
Syed S. Ahmad ◽  
Ya-Chi Su ◽  
Peter N. Walsh
Keyword(s):  
Lipid Rafts ◽  
Gradient Centrifugation ◽  
Early Time ◽  
Sucrose Density Gradient ◽  
Platelet Membrane ◽  
Membrane Microdomains ◽  
Factor X ◽  
Sucrose Density Gradient Centrifugation ◽  
Time Points ◽  
Substrate Complex

Abstract In a recent study of the role of detergent-insoluble platelet membrane microdomains (lipid rafts) in the assembly of the factor X (FX) activating complex, we have shown, contrary to expectations, that the formation of lipid rafts after incubation of platelets with the thrombin receptor activation peptide (SFLLRN, 25 μM, for 30 min) results in the down-regulation, rather than assembly, of the enzyme-cofactor-substrate complex by sequestering FVIIIa and FX in rafts and separating them from FIXa, which is excluded from raft fractions isolated by sucrose density gradient centrifugation of triton X-100 (0.25%) solubilized platelets. Since the FX-activating complex is assembled rapidly on platelets after incubation with low concentrations of either thrombin (>1 nM) or SFLLRN (25 μM), we have now examined the kinetics of FX-activation complex assembly at early time points after exposure of platelets to agonists. Washed and gel-filtered human platelets, activated with SFLLRN (25 μM) in the presence of FVIIIa (5 U/ml, 1.5 nM) and FX (125 nM) rapidly (within 2 min) developed the capacity to support maximal rates of FIXa (1 nM) catalyzed FX activation that was transient and decayed to baseline within 5 min after exposure to agonist. At these early time points (0.5, 1 and 2 min), platelets activated with SFLLRN (25 μM) in the presence of 125I-labled FVIIIa (nM), FIXa (nM) or FX (nM) and analyzed by sucrose density gradient centrifugation after solubilization in triton X-100 (0.25%) were shown to sequester within the raft fractions ~15% of FIXa, ~15% of FVIIIa and ~15% of FX, whereas at later time points (5–30 min) only FVIIIa (~25%) and FX (~45%) were localized in rafts, from which FIXa was completely excluded. These results strongly suggest that platelet membrane microdomains (lipid rafts) form rapidly after exposure of platelets to PAR-1 agonists to colocalize the enzyme-cofactor-substrate complex, which is transient since at later time points FIXa dissociates from rafts that sequester the FVIIIa-FX complex to down-regulate FX activation.

scholarly journals The αβ monomer of the insulin receptor has hormone-responsive tyrosine kinase activity

1991 ◽  
Vol 273 (1) ◽  
pp. 49-56 ◽  
Author(s):  
E R Mortensen ◽  
J G Drachman ◽  
G Guidotti
Keyword(s):  
Plasma Membranes ◽  
Kinase Activity ◽  
Gradient Centrifugation ◽  
Insulin Receptors ◽  
Sucrose Density Gradient ◽  
Erythrocyte Membranes ◽  
Protein Kinase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Adipocyte Plasma Membranes ◽  
Receptor Dimers

Insulin receptors from turkey erythrocyte membranes exist as monomers and dimers when membranes are solubilized with detergent. We examined the ability of monomers and dimers to act as protein kinases to effect both autophosphorylation of the receptor and phosphorylation of an exogenous substrate. After separation by sucrose-density-gradient centrifugation, only receptor dimers show significant basal and insulin-stimulated kinase activity, whereas material at the position of receptor monomers is not active. Partial reduction of the membrane-bound receptors with dithiothreitol, however, produces a receptor monomer containing an alpha and a beta chain which has protein kinase activity similar to that of the original dimers. With rat adipocyte plasma membranes, which in the absence of reducing agents only contain receptor dimers, reduction with dithiothreitol also produces monomers with receptor kinase activity. Receptor monomer hormone-dependent kinase activity is insensitive to receptor concentration and shows stimulation after immobilization on an affinity support.

SPECIFIC INTERACTION OF CORTICOSTEROIDS WITH BINDING SITES IN THE PLASMA MEMBRANES OF THE RAT ANTERIOR PITUITARY GLAND

1978 ◽  
Vol 79 (2) ◽  
pp. 215-222 ◽  
Author(s):  
B. KOCH ◽  
B. LUTZ-BUCHER ◽  
B. BRIAUD ◽  
C. MIALHE
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Specific Interaction ◽  
Sucrose Density Gradient ◽  
Pituitary Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Binding Characteristics ◽  
Rat Pituitary ◽  
Rat Pituitary Cells

The binding of glucocorticoids to a crude fraction of rat pituitary plasma membranes and to solubilized membrane proteins was measured. The binding characteristics were similar to those exhibited by transcortin: radioactive corticosterone was bound to a greater extent than radioactive dexamethasone and labelled corticosterone, but not labelled dexamethasone, was displaced by unlabelled corticosterone, deoxycorticosterone and progesterone. A Scatchard plot of the binding data revealed the presence of a binding material with a dissociation constant of about 3·2 nmol/l, which sedimented at 4S after sucrose density-gradient centrifugation. It was found that the number of binding sites was inversely related to the concentration of corticosterone in the circulation and was increased after long-term adrenalectomy. These data suggest that a material similar to transcortin is complexed to the plasma membrane of rat pituitary cells.

ATTACHMENT TO THE LUTEAL PLASMA MEMBRANES: AN EARLY EVENT IN THE ACTION OF LUTEINIZING HORMONE

1973 ◽  
Vol 57 (2) ◽  
pp. 199-NP ◽  
Author(s):  
HANNU RAJANIEMI ◽  
TAPANI VANHA-PERTTULA
Keyword(s):  
Plasma Membrane ◽  
Luteinizing Hormone ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Early Event ◽  
Target Tissue ◽  
Marker Enzyme ◽  
Sucrose Density Gradient Centrifugation ◽  
Initial Event

SUMMARY The distribution of 125I-labelled luteinizing hormone (LH) was studied by autoradiography of the murine pregnant ovary 10 min after intravenous administration. The grains in autoradiograms were localized around the luteal cells. The pregnant ovary showed the highest uptake of 125I-labelled LH 30 min after the injection. No similar accumulation of 125I-labelled follicle-stimulating hormone, 125I-labelled albumin or free 125I was obtained. The ribosomal fraction of the corpus luteum contained a slightly higher level of radioactivity than the other subcellular fractions after injection of 125I-labelled LH. Sucrose density gradient centrifugation of the luteal particle preparation resulted in an enrichment of radioactivity in particles containing Na+,K+-activated ATPase, a marker enzyme of the plasma membrane. These findings support the concept of plasma membrane binding as the initial event in LH action on the target tissue.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

Sign in / Sign up

Export Citation Format

Share Document