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.

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.

EVIDENCE FOR TWO DIFFERENT Ca2+TRANSPORT SYSTEMS ASSOCIATED WITH PLASMA AND INTRACELLULAR HUMAN PLATELET MEMBRANES

1987 ◽  
Author(s):  
J Enouf ◽  
R Breadux ◽  
N Bourdeau ◽  
S Levy-Toledano
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Human Platelets ◽  
Transport Systems ◽  
Maximal Response ◽  
Potassium Oxalate ◽  
Intracellular Enzyme ◽  
Intracellular Membranes ◽  
Membrane Enzyme

The regulation of Ca2+ concentration in different cells involves two Ca2+ pumps. The presence of such mechanisms in human platelets is still controverted. We then investigated this question by using plasma and intracellular membranes obtained after simultaneous isolation by centrifugation ca 40% sucrose from a mixed 100,000 g membrane fraction.The Ca2+ uptake by the different membrane vesicles has been studied. Both membrane fractions took up Ca2+ and the Ca2+ transport systems exhibited a high affinity towards Ca 2+.However, the two associated Ca2+ transport systems showed a different time course and exhibited different oxalate sensitivity. The plasma membranes are not permeable to potassium oxalate, while the Ca2+ uptake was stimulated by potassium oxalate in intracellular membranes.Two Ca2+ activated ATPase activities are associated with the isolated membrane fractions and appeared different for the following parameters : 1) a different time course of the two enzyme activities; 2)a similar apparent affinity towards Ca2+ (10−7 M), though inhibition of the Ca2+ ATPase activity was only observed in intracellular membranes at 10−6 M Ca2+ ; 3)a different pH dependence with a maximum at pH 7 for the enzyme of intracellular membranes and pH 8 for the enzyme of plasma membranes; 4)a 10 fold difference in the ATP requirement of the enzymes, thus the maximal response was obtained with 20 uM for the intracellular membrane enzyme and with 200 uM for the plasma membrane enzyme ; 5) a different affinity for various nucleotides as energy donors with a higher specificity of the plasma membrane enzyme towards ATP ; 6) a different vanadate inhibition-dose reponse which did not exceed 60% for the plasma enzyme while it reached 100% for the intracellular enzyme.Taken together, these studies agree with the possible role of both a plasma membrane and a dense tubular system Ca2+ -ATPases in the regulation of Ca2+ concentration in human platelets.

Is there a Cl- pump?

1988 ◽  
Vol 255 (5) ◽  
pp. R677-R692
Author(s):  
G. A. Gerencser ◽  
J. F. White ◽  
D. Gradmann ◽  
S. L. Bonting
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Direct Evidence ◽  
Objective Evaluation ◽  
Chemical Processes ◽  
Transport Processes ◽  
Cl Transport ◽  
Active Ion Transport

Three universally accepted mechanisms of Cl- transport across plasma membranes exist and they are 1) anion-coupled antiport, 2) cation-coupled symport, and 3) coupling to primary active ion transport through electrical and/or chemical processes. No unequivocal direct evidence has been provided for primary active Cl- transport (Cl- pump) despite numerous reports of cellular Cl- -stimulated adenosinetriphosphatase (ATPases) and of Cl- transport that cannot be accounted for by the three well-documented Cl- transport processes. It has been demonstrated that Cl- -stimulated ATPase activity is localized to both mitochondrial and microsomal aspects of the cellular apparatus. However, one group ascribes microsomal localization of Cl- -stimulated ATPase activity to mitochondrial contamination of that membrane fraction. Therefore, no Cl- pump could ever exist naturally in any plasma membrane. The other group simply states that there is plasma membrane localization of Cl- -stimulated ATPase activity that could function as a Cl- pump. Both arguments are logically advanced and their conclusions are consistent with their respective premises. Resolution to the question Is there a Cl- pump? rests with each reader's critique and objective evaluation.

Cl- -HCO3- -stimulated ATPase in intestinal mucosa of Aplysia

1985 ◽  
Vol 248 (2) ◽  
pp. R241-R248 ◽  
Author(s):  
G. A. Gerencser ◽  
S. H. Lee
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Intestinal Mucosa ◽  
Membrane Fraction ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Disulfonic Acid ◽  
Differential Centrifugation ◽  
Optimum Ph

The serosa negative transepithelial potential difference across Aplysia intestine is generated by a Na+-independent, active electrogenic Cl- absorptive mechanism. In an attempt to clarify the Cl- absorptive mechanism an anion-stimulated ATPase was prepared from plasma membranes from Aplysia enterocytes utilizing differential centrifugation and sucrose density gradient techniques. ATPase activity, which could be activated by either Cl- or HCO3-, was found in the plasma membrane fraction. Maximal anion-ATPase activity was achieved with either 25 mM Cl- or 25 mM HCO3-. The apparent Km for Cl- activation of the ATPase was 10.3 mM, whereas apparent Km for HCO3- was 9.7 mM. ATP was the most effective nucleotide substrate for both HCO3- and Cl- -ATPase activities, whereas optimum pH for both activities was 7.8. These enzyme activities were inhibited more than 30% by thioacyanate (10 mM). Acetazolamide and vanadate were also found to strongly inhibit both Cl- and HCO3- -ATPase activities, whereas 10 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 1 mM furosemide, or 1 mM ouabain had little or no effect. These results are consistent with the hypothesis that the active Cl- transport mechanism in Aplysia intestine could be a Cl- -HCO3- -stimulated ATPase found in the enterocyte plasma membrane.

Characterization of D-[3H]cis-diltiazem binding to membrane fractions and specific binding of calcium channel blockers to isolated flagellar membranes of Chlamydomonas reinhardtii

1988 ◽  
Vol 90 (3) ◽  
pp. 457-463
Author(s):  
R. DOLLE ◽  
W. NULTSCH
Keyword(s):  
Plasma Membrane ◽  
Chlamydomonas Reinhardtii ◽  
Binding Site ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Dissociation Constants ◽  
Intracellular Membrane ◽  
Binding Studies ◽  
Plasma Membrane Fraction

Plasma membranes were separated from the intracellular membranes by using an aqueous two polymerphase system. D-[3H]cis-diltiazem was employed to characterize benzothiazepine-selective receptors in these different membrane fractions of Chlamydomonas reinhardtii. The separation revealed that one type of binding site with higher affinity (KD = 33 nm) can be attributed to the intracellular membrane fraction and a second type with lower affinity (KD = 313nm) to the plasma membrane fraction. The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments are comparable to those determined by saturation experiments. The maximum numbers of binding sites of the intracellular membrane fraction and the plasma membrane fraction are Bmax = 6.4 pmol mg−1 protein and Bmax = 19 pmol mg−1 protein, respectively. D-[3H]cis-diltiazem binding is inhibited by (±)verapamil and calcium chloride in both fractions. Moreover, nifedipine stimulates D-[3H]cis-diltiazem diltiazem binding by the intracellular membrane fraction, but shows no effect on the plasma membrane fraction. Ligand displacement binding studies with isolated flagella revealed the occurrence of a d-cisdiltiazem binding site with about the same affinity to this drug (KD = 400 nm) as the one found inthe plasma membrane fraction. The maximum number of binding sites is 4.5 pmol mg−1 protein. The apparent dissociation constants for specific[3H]nimodipine and [3H]verapamil binding to the flagella were calculated to be 8 nm and 38 nm, respectively. The corresponding Bmax values are 345f mol mg−1 protein and 1.3 pmol mg−1 protein, respectively.

scholarly journals Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes

1992 ◽  
Vol 281 (2) ◽  
pp. 325-331 ◽  
Author(s):  
E Corvazier ◽  
J Enouf ◽  
B Papp ◽  
J de Gunzburg ◽  
A Tavitian ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Intracellular Membranes ◽  
Gtp Binding ◽  
Dependent Protein

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

Spreading of a sperm surface antigen within the plasma membrane of the egg after fertilization in the rat

Development ◽  
1983 ◽  
Vol 75 (1) ◽  
pp. 259-270
Author(s):  
Stephen J. Gaunt
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Guinea Pig ◽  
Surface Antigen ◽  
Plasma Membranes ◽  
Entire Surface ◽  
Sperm Tail ◽  
Sperm Surface ◽  
Sperm Antigens

The rat sperm surface antigen 2D6, located over the entire surface of the spermatozoon, is shown by use of a monoclonal antibody in indirect immunofluorescence experiments to spread laterally over the surface of the egg after fusion of sperm and egg plasma membranes at fertilization. Freshly fertilized eggs, obtained from superovulated rats 14h after hCG injection, showed the 2D6 antigen to have spread in a gradient over a discrete fan-shaped area of the egg surface anterior to the protruding sperm tail. Eggs at a later stage of sperm incorporation, obtained 20 h after hCG injection, snowed that the spread of antigen had extended to cover most or all of their surfaces. By 40 h after hCG injection, the approximate time that fertilized eggs cleaved to form 2-cell embryos, most of the 2D6 antigen had been lost from the cell surface. Fertilized eggs, but not unfertilized eggs or 2-cell embryos, were lysed by 2D6 monoclonal antibody in the presence of guinea pig complement. A model for sperm-egg fusion is presented to account for the observed pattern of spreading shown by the 2D6 antigen. The possible role of sperm antigens on the egg surface is discussed.

scholarly journals Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

1987 ◽  
Vol 104 (5) ◽  
pp. 1239-1248 ◽  
Author(s):  
E S Sztul ◽  
D Biemesderfer ◽  
M J Caplan ◽  
M Kashgarian ◽  
J L Boyer
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Alpha Subunit ◽  
Gamma Glutamyl Transferase ◽  
Surface Distribution ◽  
Western Blots ◽  
Glutamyl Transferase

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

scholarly journals Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes

1986 ◽  
Vol 233 (3) ◽  
pp. 661-668 ◽  
Author(s):  
N Hack ◽  
M Croset ◽  
N Crawford
Keyword(s):  
Atpase Activity ◽  
Blood Platelets ◽  
Present Report ◽  
Surface Membrane ◽  
Storage Site ◽  
Intracellular Membrane ◽  
Bivalent Cation ◽  
Platelet Surface ◽  
Intracellular Membranes ◽  
Dependent Transport

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.

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