High-affinity pH-dependent passive Ca binding by myometrial plasma membrane vesicles

1983 ◽  
Vol 244 (1) ◽  
pp. C61-C67 ◽  
Author(s):  
A. K. Grover ◽  
C. Y. Kwan ◽  
E. E. Daniel
Keyword(s):  
Plasma Membrane ◽  
Reaction Medium ◽  
Membrane Vesicles ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Ph Dependent ◽  
The Difference ◽  
Ph Buffer

Rat myometrium plasma membrane-(PM) enriched fraction N1 binds calcium passively in a pH-dependent manner at a Ca2+ concentration of 1 microM. The Ca binding increases with increasing pH from 6.27 to 7.47 with a half maximum near 6.8. The difference between binding at 6.27 and 7.07 (the pH-dependent Ca binding) depends on the pH of the reaction medium rather than the pH of the medium in which the membranes had previously been suspended. The pH-dependent Ca binding is not an artifact due to EGTA, the pH buffer used, or soluble protein trapped inside the membrane vesicles. The pH-dependent Ca binding occurs with a dissociation constant value of 0.28 microM and Hill coefficient of 2.37 for Ca2+. The high affinity pH-dependent Ca uptake and the release of Ca2+ from the membranes is virtually complete in 10 s in the presence of 1 microM A23187 but not in its absence. The distribution of the pH-dependent Ca binding in the various rat myometrium subcellular fractions parallels the activity of 5'-nucleotidase in these fractions and not the activities of NADPH-dependent or succinate-dependent cytochrome c reductases. The high affinity and rapid binding and release of Ca at the pH-dependent Ca binding sites in the PM-enriched fraction suggests that the binding and release from these sites may play a key role in excitation-contraction coupling of the smooth muscle.

scholarly journals ATP-dependent transport of glutathione conjugate of 7β,8α-dihydroxy-9α,10α-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles

1998 ◽  
Vol 332 (3) ◽  
pp. 799-805 ◽  
Author(s):  
Sanjay K. SRIVASTAVA ◽  
Xun HU ◽  
Hong XIA ◽  
Richard J. BLEICHER ◽  
Howard A. ZAREN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Dependent Transport ◽  
Detoxification Pathway ◽  
Stimulation Of

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 µM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis–Menten kinetics (Km 46 µM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis–Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 µM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 µM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 µM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

Inhibition of Maize (Zea mays L.) Root Plasma Membrane-Bound Redox Activities by Coumarins

1994 ◽  
Vol 49 (7-8) ◽  
pp. 447-452 ◽  
Author(s):  
Sabine Lüthje ◽  
José A. Gonzaléz-Reyes ◽  
Placido Navas ◽  
Olaf Döring ◽  
Michael Böttger
Keyword(s):  
Zea Mays ◽  
Plasma Membrane ◽  
Zea Mays L ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
Two Phase ◽  
Concentration Dependent Manner ◽  
Oxidoreductase Activity ◽  
Membrane Bound

Modulation of plasma membrane-bound NADH:hexacyanoferrate III oxidoreductase activities by dicumarol and warfarin was investigated with plasma membrane vesicles of Zea mays L. (cv. Sil Anjou 18) roots, prepared by aqueous two phase partitioning. Vesicles were about 65% right-side out orientated as demonstrated by enzyme latency of vanadate sensitive ATPase activity. Dicumarol or warfarin, respectively, inhibited NADH:hexacyanoferrate III oxidoreductase activity in a concentration-dependent manner and inhibition could be reversed partially by addition of quinones

scholarly journals Chemically induced vesiculation as a platform for studying TMEM16F activity

2019 ◽  
Vol 116 (4) ◽  
pp. 1309-1318 ◽  
Author(s):  
Tina W. Han ◽  
Wenlei Ye ◽  
Neville P. Bethel ◽  
Mario Zubia ◽  
Andrew Kim ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Calcium Influx ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
Chemical Induction ◽  
Plasma Membrane Vesicles ◽  
Kinetic Assay ◽  
Membrane Asymmetry ◽  
Phospholipid Scramblase ◽  
Chemically Induced

Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.

scholarly journals Plasma Membrane Vesicles with Engineered Transmembrane Protein Ligands for High-Affinity Cell Targeting

2017 ◽  
Vol 112 (3) ◽  
pp. 592a
Author(s):  
Chi Zhao ◽  
David J. Busch ◽  
Conor P. Vershel ◽  
Hisham A. Ali ◽  
Natalie C. Miroballi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Membrane Vesicles ◽  
Cell Targeting ◽  
Plasma Membrane Vesicles ◽  
Protein Ligands ◽  
High Affinity

scholarly journals A calmodulin-activated (Ca2+-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi

1991 ◽  
Vol 280 (3) ◽  
pp. 715-720 ◽  
Author(s):  
G Benaim ◽  
S Losada ◽  
F R Gadelha ◽  
R Docampo
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Bovine Brain ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
High Affinity

High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.

Ceramide and sphingosine have an antagonistic effect on the plasma-membrane Ca2+-ATPase from human erythrocytes

2002 ◽  
Vol 362 (2) ◽  
pp. 247-251 ◽  
Author(s):  
Claudia COLINA ◽  
Vincenza CERVINO ◽  
Gustavo BENAIM
Keyword(s):  
Plasma Membrane ◽  
Second Messengers ◽  
Membrane Vesicles ◽  
Inhibitory Effect ◽  
Antagonistic Effect ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
Key Enzyme ◽  
Hydroxy Groups ◽  
Dose Dependent

The plasma-membrane Ca2+-ATPase is a key enzyme in the regulation of the intracellular Ca2+ concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca2+. In view of the fact that the Ca2+-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca2+-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca2+ and the Vmax of the enzyme. Furthermore, this second messenger also stimulates Ca2+ transport in inside—out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca2+-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca2+-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca2+ concentration.

scholarly journals Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum

1988 ◽  
Vol 249 (1) ◽  
pp. 223-230 ◽  
Author(s):  
J L Milne ◽  
M B Coukell
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Membrane Vesicles ◽  
High Specificity ◽  
Channel Blockers ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Isolation And Characterization ◽  
Enzyme Markers ◽  
Pumping Activity

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with ‘inside-out’ plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.

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