scholarly journals Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membranes of human platelets

1994 ◽  
Vol 298 (3) ◽  
pp. 739-742 ◽  
Author(s):  
P J Cullen ◽  
Y Patel ◽  
V V Kakkar ◽  
R F Irvine ◽  
K S Authi
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Specific Binding ◽  
Human Platelets ◽  
Intracellular Membranes ◽  
Specific Binding Sites ◽  
Carrier Free ◽  
Membrane Location

In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets.

scholarly journals Localization and identification of Ca2+ATPases in highly purified human platelet plasma and intracellular membranes. Evidence that the monoclonal antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in human platelets

1995 ◽  
Vol 306 (3) ◽  
pp. 837-842 ◽  
Author(s):  
S Bokkala ◽  
S S el-Daher ◽  
V V Kakkar ◽  
F Wuytack ◽  
K S Authi
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Complex Formation ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Human Platelets ◽  
Intracellular Membrane ◽  
Intracellular Membranes

The Ca2+ATPase activities of highly purified human platelet membranes prepared by high-voltage free-flow electrophoresis have been analysed by using [gamma-32P]ATP hydrolysis, recognition by antibodies and phosphoenzyme-complex formation. The Ca2+ATPase activity present in mixed membranes was found to be predominantly associated with intracellular membranes after subfractionation, with only a low level of activity associated with plasma membranes. The intracellular-membrane Ca2+ATPase activity was inhibited totally with thapsigargin (Tg), whereas the plasma-membrane Ca2+ATPase was not significantly affected, suggesting that the latter does not belong to the SERCA (sarco-endoplasmic-reticulum Ca2+ATPase) class. A monoclonal antibody, 5F10, raised to the red-cell membrane Ca2+ATPase [Cheng, Magocsi, Cooper, Penniston and Borke (1993) Cell Physiol. Biochem. 4, 31-43] recognized two bands at 135 and 150 kDa in mixed membranes and plasma membranes, and the corresponding bands in red-blood-cell membranes, confirming the Ca2+ATPase to be of the PMCA (plasma-membrane Ca2+ATPase) type. No recognition of any band was detected in intracellular membranes. Identification of the intracellular-membrane Ca2+ATPase activity was carried out with polyclonal antibodies with known specificity towards SERCA 2b (S.2b) and SERCA 3 (N89), and a monoclonal antibody, PL/IM 430, raised against platelet intracellular membranes. All of these antibodies recognized the 100 kDa Ca2+ATPase in mixed membranes and intracellular membranes, with little or no recognition of the activity in the plasma membranes. In some membrane preparations the antibody PL/IM 430 and antiserum N89 recognized similar degradation products, of 74, 70 and 40 kDa, in the intracellular-membrane fraction. The Ca2+ATPase recognized by PL/IM 430 was immunoprecipitated, and the immunoprecipitated protein was specifically recognized by the antiserum N89, but not by S.2b. Analysis of the phosphoenzyme-complex formation revealed potent phosphorylation of the 100 and 74 kDa peptides, both recognized by PL/IM 430 and N89. These studies report the presence of a PMCA in a purified plasma-membrane fraction from human platelets, and that the antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in intracellular membranes.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini

1987 ◽  
Vol 252 (4) ◽  
pp. G535-G542 ◽  
Author(s):  
N. Viguerie ◽  
J. P. Esteve ◽  
C. Susini ◽  
N. Vaysse ◽  
A. Ribet
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Specific Radioactivity ◽  
Pancreatic Acinar Cells ◽  
Nuclear Fraction ◽  
Pancreatic Acini ◽  
Specific Binding Sites ◽  
Membrane Bound ◽  
Membrane Level

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

scholarly journals Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes

1980 ◽  
Vol 191 (3) ◽  
pp. 743-760 ◽  
Author(s):  
Richard J. Pietras ◽  
Clara M. Szego
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Low Density ◽  
Marker Enzymes ◽  
Low Density Plasma ◽  
Density Plasma

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [3H]oestradiol-17β. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl2 and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[3H]oestradiol-17β to intact cells and their fractions was detemined after equilibration for 1.5h at 4°C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (ρ=1.13–1.16) and high (ρ=1.16–1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46–63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [3H]oestradiol-17β was reduced by a 200-fold molar excess of unlabelled oestradiol-17β, oestriol or diethylstilbestrol, but not by oestradiol-17α, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60°C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5–20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes

1989 ◽  
Vol 108 (2) ◽  
pp. 115-124 ◽  
Author(s):  
Miguel Trueba ◽  
Ana I. Vallejo ◽  
Isabel Rodriguez ◽  
Iñaki Ibarrola ◽  
María J. Sancho ◽  
...  
Keyword(s):  
Binding Sites ◽  
Mouse Liver ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Specific Binding Sites ◽  
Liver Plasma Membranes
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