Introduction of antibody (PL/IM 430) to a 100 kDa protein into permeabilised platelets inhibits intracellular sequestration of Ca2+

1988 ◽  
Vol 8 (4) ◽  
pp. 379-388 ◽  
Author(s):  
Nashrudeen Hack ◽  
Kalwant Singh Authi ◽  
Neville Crawford
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Vesicles ◽  
Intracellular Membrane ◽  
Channel Protein ◽  
Maximum Inhibition ◽  
Ldh Release ◽  
Intracellular Binding ◽  
Specific Control ◽  
Ca2 Channels ◽  
Intracellular Sequestration

A monoclonal antibody (PL/IM 430), previously found to inhibit the uptake of Ca2+ into highly purified platelet intracellular membrane vesicles (Hack, N., Wilkinson, J. M. and Crawford, N. 1988, Biochem. J.250, 355–361) has been introduced into saponin-permeabilised platelets. At a saponin concentration (20–25 μg/ml) commensurate with total LDH release, sequestration of Ca2+ into intracellular non-mitochondrial stores is inhibited by the antibody (~50% inhibition at 20 μg/ml IgG). At higher saponin concentrations when intracellular binding of125I-labelled mAb is maximum, inhibition of Ca2+ sequestration approaches 70%. The inhibition is specific, control studies with non-platelet directed mouse IgG and mAbs which immunoblot platelet antigens other than the 100 kDa protein did not affect the Ca2+ sequestration. No effect of the antibody were observed against IP3-induced release of prestored Ca2+, either in permeabilised platelets or with isolated intracellular membrane vesicles. The mAb PL/IM 430 appears to bind only to the Ca2+ translocating channel protein associated with the intracellular membrane (Ca2++Mg2+) ATPase and not to Ca2+ channels responsive to IP3.

scholarly journals A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase

1988 ◽  
Vol 250 (2) ◽  
pp. 355-361 ◽  
Author(s):  
N Hack ◽  
J M Wilkinson ◽  
N Crawford
Keyword(s):  
Human Platelet ◽  
Blood Platelets ◽  
Membrane Vesicles ◽  
Intracellular Membrane ◽  
Dose Dependency ◽  
Intracellular Membranes ◽  
Maximum Inhibition ◽  
Igg1 Subclass ◽  
Energy Dependent ◽  
Human Blood Platelets

To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.

scholarly journals Induction of aggregation and fusion of cholesterol-containing membrane vesicles by an anti-cholesterol monoclonal antibody

2000 ◽  
Vol 41 (4) ◽  
pp. 621-628
Author(s):  
Aitziber Agirre ◽  
Shlomo Nir ◽  
José L. Nieva ◽  
Jan Dijkstra
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Vesicles

A MONOCLONAL ANTIBODY WHOSE BINDING IS INHIBITED BY DIVALENT CATIONS

1987 ◽  
Author(s):  
M de Serres ◽  
H M Reisner ◽  
D Monroe ◽  
H Roberts
Keyword(s):  
Monoclonal Antibody ◽  
Vitamin K ◽  
Divalent Cations ◽  
Factor Ix ◽  
Protein Preparation ◽  
Coagulant Activity ◽  
Maximum Inhibition ◽  
Standard Assay ◽  
Coagulation Protein ◽  
Direct Binding

Factor IX (FIX), a vitamin K dependent coagulation protein, is functionally deficient or absent in patients with hemophilia B. Binding of Ca++ by the gammacarboxyglutamic acid residues (Gla) of FIX is necessary for coagulant activity. Antibodies havebeendes-cribed which selectively bind to FIX in the presence of Ca++ and appear to interact with Ca-H- stabilized epitopes of FIX. One IgM, Kappa murine monoclonal antibody (Mab), 129-1, has been found to react preferentially with FIX in the absence of Ca-H- or with FIX having a reduction in gammacarboxylation. 129-1 was characterized by direct binding ELISA assays, the standard assay buffer (.02M Tris - .15M NaCL pH 7.5) was supplemented with Ca-H- or ED TA to final concentrations to 5mM and lOmM respectively. In the presence of Ca-H-, titers ranged from 10 to 100 as compared to 100 K to 500 K in the presence of EDTA. Control Mab's, FIX-30 and 2D521 showed no such effect. Maximum inhibition occurred ≥2.5 mM Ca-H- concentration. Mg++, Sr-H- and Mn-H- were tested with similar results. Chemically degammacarboxylated FIXa was compared to FIX and FIXa controls in the presence of Ca-H- or EDTA, to determine the importance of Gla residues. In the presence of EDTA, Mab 129-1 had essentially equal reactivity with all these proteins (titers 100 K). In the presence of Ca-H-, binding to FIX and FIXa was inhibited (500 and 1 K respectively). However, the binding to Gla modified FIXa in the presence of Ca-H- was only slightly reduced (10 K), suggesting the importance of Gla residues in the Ca-H- mediated inhibition. FIX was purified from concentrate, coumadinized plasma and culture supernatant (produced in the absence of vitamin K) from the cell line PMN45 using HPLC affinity chromatography with Mab 2D521. Mab 129-1 binding to FIX immunopurified from concentrate and a control biochemically purified FIX protein preparation was significantly higher in the presence of EDTA (titers 500 and 5 K) than in Ca-H- (titers 10 and 10). FIX purified from coumadinized plasma or PMN45 cells showed equal low reactivity with 129-1 in the presence or absence of Ca-H- (titer 10 in all cases). FIX-30 and 2D521 controls showed no such reduction in binding. Therefore, Gla residues may play a role in the binding of 129-1 to FIX.

Omegasomes: PI3P platforms that manufacture autophagosomes

2013 ◽  
Vol 55 ◽  
pp. 17-27 ◽  
Author(s):  
Rebecca Roberts ◽  
Nicholas T. Ktistakis
Keyword(s):  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Membrane Vesicles ◽  
Double Membrane ◽  
Survival Pathway ◽  
Membrane Compartments ◽  
Specific Control ◽  
Phosphatidylinositol 3

Autophagy is a conserved survival pathway, which cells and tissues will activate during times of stress. It is characterized by the formation of double-membrane vesicles called autophagosomes inside the cytoplasm. The molecular mechanisms and the signalling components involved require specific control to ensure correct activation. The present chapter describes the formation of autophagosomes from within omegasomes, newly identified membrane compartments enriched in PI3P (phosphatidylinositol 3-phosphate) that serve as platforms for the formation of at least some autophagosomes. We discuss the signalling events required to nucleate the formation of omegasomes as well as the protein complexes involved.

Luminal Ca2+ Regulation of RyR1 Ca2+ Channel Leak Activation and Inactivation in Sarcoplasmic Reticulum Membrane Vesicles

2020 ◽  
Author(s):  
Chris Palahniuk ◽  
Mark Mutawe ◽  
James Gilchrist
Keyword(s):  
Membrane Vesicles ◽  
Ruthenium Red ◽  
Sarcoplasmic Reticulum Membrane ◽  
Channel Opening ◽  
Complex Organization ◽  
Complete Disruption ◽  
In Trans ◽  
Functional States ◽  
Opening And Closing ◽  
Ca2 Channels

In this study, we tested the hypothesis that RyR1 Ca2+ channel closure is sensitive to outward trans-SR membrane Ca2+ gradients established by SERCA1 pumping. To perform these studies we employed stopped-flow rapid-kinetic fluorescence methods to measure and assess how variation in trans-SR membrane Ca2+ distribution affects evolution of RyR1 Ca2+ leaks in RyR1/CASQ1/SERCA1-rich HSR vesicles. Our studies showed that rapid filling of a Mag-Fura-2-sensitive free Ca2+ pool during SERCA1-mediated Ca2+ sequestration appears to be a crucial condition allowing RyR1 Ca2+ channels to close once reloading of luminal Ca2+ stores is complete. Disruption in the filling of this pool caused activation of ruthenium red-inhibitable RyR1 Ca2+ leaks suggesting that SERCA1 pump formation of outward Ca2+ gradients is an important aspect of Ca2+ flux control channel opening and closing. In addition, our observed ryanodine-induced shift in luminal Ca2+ from free to a CTC.Ca+-sensitive, CASQ1-associated bound compartment, underscores the complex organization and regulation of luminal Ca2+. Our study provides, strong evidence that RyR1 functional states directly and indirectly influence the compartmentation of luminal Ca2+. This, in turn, is influenced by the activity of SERCA1 pumps to both fill luminal pools while synchronously reducing Ca2+ levels on the cytosolic face of RyR1 channels.

Fetal lung epithelial cells contain two populations of amiloride-sensitive Na+ channels

1993 ◽  
Vol 264 (4) ◽  
pp. L357-L364 ◽  
Author(s):  
S. Matalon ◽  
M. L. Bauer ◽  
D. J. Benos ◽  
T. R. Kleyman ◽  
C. Lin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Fetal Lung ◽  
Lung Epithelial Cells ◽  
Na Channel ◽  
Na Transport ◽  
Plasma Membrane Vesicles ◽  
Binding Studies ◽  
Channel Protein ◽  
Lung Epithelial

Active Na+ transport by the alveolar epithelium plays a major role in reabsorption of the fetal lung fluid after birth. We characterized the biochemical and physiological characteristics of Na+ conductive pathways in distal fetal lung epithelial (FLE) cells isolated from 20-day-old rat fetuses. We demonstrated that a polyclonal antibody to Na+ channel protein (NaAb) binds to the plasma membranes of FLE cells. In Western blot studies, this NaAb and an anti-idiotypic monoclonal antibody to the amiloride-binding subunit of the Na+ channel protein recognized 150- and 90-kDa polypeptides in plasma membrane vesicles of FLE. 22Na+ flux measurements across plasma membrane vesicles of FLE revealed the existence of electrogenic Na+ transport, which was twice as high as the corresponding adult value. One hundred micromolars of amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride inhibited 30, 40, and 70% of the electrogenic Na+ transport across plasma membrane vesicles of FLE cells, respectively. The half-maximum inhibition of electrogenic Na+ transport by these substances occurred between 0.3 and 1 microM. [3H]benzamil equilibrium binding studies in membrane vesicles of FLE cells revealed the existence of two binding sites that had dissociation constant values of 19 and 1,525 nM, respectively. These data indicate the presence of both high- and low-amiloride affinity Na+ conductive pathways (channels) in FLE cells.

Hepatic Na(+)-dicarboxylate cotransport: identification, characterization, and acinar localization

1992 ◽  
Vol 263 (6) ◽  
pp. G871-G879 ◽  
Author(s):  
R. H. Moseley ◽  
S. Jarose ◽  
P. Permoad
Keyword(s):  
Driving Forces ◽  
Tricarboxylic Acid Cycle ◽  
Liver Perfusion ◽  
Membrane Vesicles ◽  
Inhibitory Effect ◽  
Plasma Membrane Vesicles ◽  
Rat Liver Perfusion ◽  
Perfusion Studies ◽  
Intracellular Binding ◽  
Tricarboxylic Acid Cycle Intermediates

Rat liver perfusion studies suggest that the transport of alpha-ketoglutarate (KG) and related dicarboxylates exhibits acinar heterogeneity, in that the uptake and subsequent metabolism of these organic anions appears to occur predominantly in the perivenous region. However, the isolated perfused liver as an experimental model cannot distinguish intra-acinar differences in either the rate of solute uptake and/or efflux or intracellular binding and/or metabolism. Therefore, the driving forces and acinar localization of KG transport were examined using rat basolateral liver plasma membrane vesicles (blLPMV) isolated from control animals and animals treated 24 h before with selective perivenous and periportal toxins [carbon tetrachloride (CCl4) and allyl alcohol (AA), respectively]. In control blLPMV, [14C]KG uptake into an osmotically sensitive space was markedly stimulated by an inwardly directed Na+ gradient but not by inwardly directed gradients of other monovalent cations. The Na+ ionophore, gramicidin, had a small but significant inhibitory effect on Na(+)-dependent KG uptake, demonstrating that KG uptake was not the result of an intravesicular positive Na+ diffusion potential. The protonophore, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, had no effect on Na+ gradient-driven KG uptake, indicating that KG uptake was not the indirect result of coordinated activities of Na-H and KG-OH exchange. Na+ gradient-driven KG uptake was electrogenic (occurring with the net transfer of positive charge), and cis-inhibited by other tricarboxylic acid cycle intermediates, including succinate, fumarate, and malate and by citrate, but not by the dicarboxylates oxalate and malonate nor by glutamate and taurocholate (TC).(ABSTRACT TRUNCATED AT 250 WORDS)

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