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

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.

Download Full-text

The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit

Biochemical Journal ◽

10.1042/bj2730429 ◽

1991 ◽

Vol 273 (2) ◽

pp. 429-434 ◽

Cited By ~ 10

Author(s):

A Darnanville ◽

R Bredoux ◽

K J Clemetson ◽

N Kieffer ◽

N Bourdeau ◽

...

Keyword(s):

Plasma Membrane ◽

Time Course ◽

Plasma Membranes ◽

Polyclonal Antibodies ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Glycoprotein Ib ◽

Dependent Protein ◽

Dose Dependent ◽

Fold Stimulation

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3320799 ◽

1998 ◽

Vol 332 (3) ◽

pp. 799-805 ◽

Cited By ~ 11

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.

Download Full-text

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

Zeitschrift für Naturforschung C ◽

10.1515/znc-1994-7-809 ◽

1994 ◽

Vol 49 (7-8) ◽

pp. 447-452 ◽

Cited By ~ 10

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

Download Full-text

3,3′,5′-Triiodothyronine inhibits ontogenetic development of iodothyronine-5′-deiodinase in the liver of the neonatal mouse

Acta Endocrinologica ◽

10.1530/acta.0.1190181 ◽

1988 ◽

Vol 119 (2) ◽

pp. 181-188 ◽

Cited By ~ 3

Author(s):

Doo Chol Han ◽

Kanji Sato ◽

Yuko Fujii ◽

Minoru Ozawa ◽

Hidehito Imamura ◽

...

Keyword(s):

Single Injection ◽

Inhibitory Effect ◽

Antagonistic Effect ◽

Time Dependent ◽

Dependent Manner ◽

Neonatal Mice ◽

Dose Dependent Decrease ◽

Dose Dependent

Abstract. To elucidate the effect of rT3 on iodothyronine-5′-deiodinating activity (I-5′-DA) in the liver of neonatal mice, rT3 was injected sc on the 5–8th day after birth and I-5′-DA in the liver was determined. A single injection of rT3 (0.01–1 μg/g) inhibited the ontogenetically developing I-5′-DA in a dose- and time-dependent manner. The inhibitory effect was reversible and specific for I-5′-DA. Lineweaver-Burk analysis revealed that the time- and dose-dependent decrease in the enzyme activity was due to a decrease in Vmax with no alteration in Km values (5 × 10−8 mol/l). The maximal inhibitory effect was observed at a dose of 1 μg rT3/g, whereas the inhibitory effect was diminished at greater doses (4–10 μg/g), probably owing to a contamination with T4 of the rT3 preparation administered. Furthermore, consistent with our previous in vitro findings, rT3 inhibited the I-5′-DA induced by T3 in the liver of neonatal mice. These findings suggest that rT3 inhibited I-5′-DA in the liver of neonatal mice by decreasing the amount of enzyme available to the substrate and that rT3 also elicited an antagonistic effect against T3 in the induction of I-5′-DA in vivo.

Download Full-text

Chemically induced vesiculation as a platform for studying TMEM16F activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817498116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1309-1318 ◽

Cited By ~ 7

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.

Download Full-text

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

AJP Cell Physiology ◽

10.1152/ajpcell.1983.244.1.c61 ◽

1983 ◽

Vol 244 (1) ◽

pp. C61-C67 ◽

Cited By ~ 18

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.

Download Full-text