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.

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 unconjugated bilirubin by rat liver canalicular plasma membrane vesicles

1998 ◽  
Vol 331 (1) ◽  
pp. 99-103 ◽  
Author(s):  
Lorella PASCOLO ◽  
Enrique J. BAYON ◽  
Felicia CUPELLI ◽  
J. Donald OSTROW ◽  
Claudio TIRIBELLI
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Atp Hydrolysis ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Organic Anion Transporter ◽  
Unconjugated Bilirubin ◽  
Plasma Membrane Vesicles ◽  
Anion Transporter ◽  
Dependent Transport

The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5´-[α,β-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a Km of 26±8 nM and a Vmax of 117±11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59±11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.

Alanine uptake by liver at midpregnancy in rats

1987 ◽  
Vol 252 (3) ◽  
pp. E408-E413 ◽  
Author(s):  
M. Pastor-Anglada ◽  
X. Remesar ◽  
G. Bourdel
Keyword(s):  
Plasma Membrane ◽  
Active Transport ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Pregnant Rats ◽  
Dependent Transport

The participation of the liver to the increase in alanine utilization seen at midpregnancy was studied in 9- and 12-day pregnant rats. Liver fractional extraction of alanine was assessed in vivo from the changes in concentration in afferent and efferent vessels. Hepatic active transport of alanine was determined in vitro using isolated plasma-membrane vesicles. Compared with nonpregnant controls, alanine fractional extraction was significantly increased on day 12 but not on day 9 of pregnancy. Vesicles isolated from 9- and 12-day pregnant animals had a greater capacity for Na+-dependent transport than those from controls. Eadie-Hofstee plotting showed that this increase was due to an increase in Vmax with no change in Km. Both A and ASC systems contributed to the Vmax increase. These results indicate that, although by day 9 the liver has developed an increased capacity for alanine uptake, the actual extraction is seen only by day 12 of pregnancy. At this stage the liver participates actively in the turnover of alanine and the development of hypoalaninemia.

scholarly journals Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium

1988 ◽  
Vol 252 (1) ◽  
pp. 215-220 ◽  
Author(s):  
A Enyedi ◽  
J Minami ◽  
A J Caride ◽  
J T Penniston
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Calcium Uptake ◽  
Limited Proteolysis ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Active Calcium ◽  
Stimulation Of

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.

ATP-Dependent transport for glucuronides in canalicular plasma membrane vesicles

1991 ◽  
Vol 176 (2) ◽  
pp. 622-626 ◽  
Author(s):  
Kazuo Kobayashi ◽  
Satoshi Komatsu ◽  
Tsuyoshi Nishi ◽  
Hisanori Hara ◽  
Koichiro Hayashi
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Dependent Transport

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.

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