scholarly journals Glycosylation is essential for biosynthesis of functional gastric H+, K+-ATPase in insect cells

1997 ◽  
Vol 321 (2) ◽  
pp. 419-424 ◽  
Author(s):  
Corné H. W. KLAASSEN ◽  
Jack A. M. FRANSEN ◽  
Herman G. P. SWARTS ◽  
Jan Joep H. H. M. De PONT
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Functional Properties ◽  
Insect Cells ◽  
Expression Level ◽  
Α Subunit ◽  
Atpase Subunits ◽  
Glycoprotein Processing ◽  
Α Subunits

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and 1-deoxymannojirimycin, inhibitors of glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated α-subunits and not of non-glycosylated α-subunits. Transport of the H+,K+-ATPase α-subunit to the plasma membrane but not its ability to assemble with the α-subunit depended on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

2001 ◽  
Vol 281 (3) ◽  
pp. C982-C992 ◽  
Author(s):  
Craig Gatto ◽  
Scott M. McLoud ◽  
Jack H. Kaplan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Β Subunit ◽  
Α Subunit ◽  
Enzyme Preparations ◽  
High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

Isoproterenol increases Na+-K+-ATPase activity by membrane insertion of α-subunits in lung alveolar cells

1999 ◽  
Vol 276 (1) ◽  
pp. L20-L27 ◽  
Author(s):  
Alejandro M. Bertorello ◽  
Karen M. Ridge ◽  
Alexander V. Chibalin ◽  
Adrian I. Katz ◽  
Jacob I. Sznajder
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Atpase Activity ◽  
Kinase Inhibitor ◽  
Lung Edema ◽  
Alveolar Type ◽  
Type Ii ◽  
Alveolar Cells ◽  
Α Subunit ◽  
Α Subunits

Catecholamines promote lung edema clearance via β-adrenergic-mediated stimulation of active Na+ transport across the alveolar epithelium. Because alveolar epithelial type II cell Na+-K+-ATPase contributes to vectorial Na+ flux, the present study was designed to investigate whether Na+-K+-ATPase undergoes acute changes in its catalytic activity in response to β-adrenergic-receptor stimulation. Na+-K+-ATPase activity increased threefold in cells incubated with 1 μM isoproterenol for 15 min, which also resulted in a fourfold increase in the cellular levels of cAMP. Forskolin (10 μM) also stimulated Na+-K+-ATPase activity as well as ouabain binding. The increase in Na+-K+-ATPase activity was abolished when cells were coincubated with a cAMP-dependent protein kinase inhibitor. This stimulation, however, was not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase α-subunit; rather, it was the result of an increased number of α-subunits recruited from the late endosomes into the plasma membrane. The recruitment of α-subunits to the plasma membrane was prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases Na+-K+-ATPase activity in alveolar type II epithelial cells by recruiting α-subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.

Effects of bafilomycin A1 on cytosolic pH of sheep alveolar and peritoneal macrophages: evaluation of the pH-regulatory role of plasma membrane V-ATPases.

1995 ◽  
Vol 198 (8) ◽  
pp. 1711-1715 ◽  
Author(s):  
T A Heming ◽  
D L Traber ◽  
F Hinder ◽  
A Bidani
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Initial Rate ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Phi Regulation

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (m theta) from sheep. Cytosolic pH was measured using 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir < 6.8) by approximately 35%. Recovery of pHi was abolished by co-treatment of m theta with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal m theta from sheep. Sheep m theta also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.

scholarly journals The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase α-subunit is essential for K+ activation of the enzyme activity

1998 ◽  
Vol 331 (2) ◽  
pp. 465-472 ◽  
Author(s):  
Harm P. H. HERMSEN ◽  
Herman G. P. SWARTS ◽  
Jan B. KOENDERINK ◽  
Jan Joep H. H. M. De PONT
Keyword(s):  
Atpase Activity ◽  
Transmembrane Domain ◽  
The Other ◽  
Lower Sensitivity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Α Subunit ◽  
Activity Maximum ◽  
Activity Measurements ◽  
Atpase Subunits

To investigate the role of Glu820, located in transmembrane domain M6 of the α-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820 → Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40–100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10–20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a] pyridine (SCH 28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH 28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH 28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the α-subunit.

Characterization of an amiloride binding region in the α-subunit of ENaC

2003 ◽  
Vol 285 (6) ◽  
pp. F1279-F1290 ◽  
Author(s):  
Ollie Kelly ◽  
Chaomei Lin ◽  
Mohan Ramkumar ◽  
Nina C. Saxena ◽  
Thomas R. Kleyman ◽  
...  
Keyword(s):  
Single Channel ◽  
Point Mutations ◽  
Binding Affinities ◽  
Extracellular Loop ◽  
Wild Type ◽  
Α Subunit ◽  
Open Time ◽  
The Mean ◽  
Α Subunits

One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the α-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278–283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type α-subunits or α-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities ( Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high- Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type α-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.

Receptor-mediated inhibition of renal Na+-K+-ATPase is associated with endocytosis of its α- and β-subunits

1997 ◽  
Vol 273 (5) ◽  
pp. C1458-C1465 ◽  
Author(s):  
Alexander V. Chibalin ◽  
Adrian I. Katz ◽  
Per-Olof Berggren ◽  
Alejandro M. Bertorello
Keyword(s):  
Atpase Activity ◽  
Gradient Centrifugation ◽  
Coated Vesicles ◽  
Α Subunit ◽  
Kinase C ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Atpase Subunits ◽  
Α And Β Subunits

The mechanisms involved in receptor-mediated inhibition of Na+-K+-ATPase remain poorly understood. In this study, we evaluate whether inhibition of proximal tubule Na+-K+-ATPase activity by dopamine is linked to its removal from the plasma membrane and internalization into defined intracellular compartments. Clathrin-coated vesicles were isolated by sucrose gradient centrifugation and negative lectin selection, and early and late endosomes were separated on a flotation gradient. Inhibition of Na+-K+-ATPase activity by dopamine, in contrast to its inhibition by ouabain, was accompanied by a sequential increase in the abundance of the α-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation between these organelles. A similar pattern was found for the β-subunit. The increased incorporation of both subunits in all compartments was blocked by calphostin C. The results demonstrate that the dopamine-induced decrease in Na+-K+-ATPase activity in proximal tubules is associated with internalization of its α- and β-subunits into early and late endosomes via a clathrin-dependent pathway and that this process is protein kinase C dependent. The presence of Na+-K+-ATPase subunits in endosomes suggests that these compartments may constitute normal traffic reservoirs during pump degradation and/or synthesis.

scholarly journals The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum

PLoS ONE ◽  
2020 ◽  
Vol 15 (8) ◽  
pp. e0238452 ◽  
Author(s):  
Alexander Zhgun ◽  
Mariya Dumina ◽  
Ayrat Valiakhmetov ◽  
Mikhail Eldarov
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Critical Role ◽  
Cephalosporin C ◽  
Acremonium Chrysogenum

scholarly journals ABCB1 and ABCG2 restricts the efficacy of gedatolisib (PF-05212384), a PI3K inhibitor in colorectal cancer cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Changfu Liu ◽  
Wenge Xing ◽  
Haipeng Yu ◽  
Weihao Zhang ◽  
Tongguo Si
Keyword(s):  
Colorectal Cancer ◽  
Atpase Activity ◽  
Cancer Cells ◽  
Cell Line ◽  
Abc Transporters ◽  
Expression Level ◽  
Colorectal Cancer Cell Line ◽  
Colorectal Cancer Cells ◽  
Mtt Assays

Abstract Background Overexpression of ABC transporters is a big challenge on cancer therapy which will lead cancer cells resistance to a series of anticancer drugs. Gedatolisib is a dual PI3K and mTOR inhibitor which is under clinical evaluation for multiple types of malignancies, including colorectal cancer. The growth inhibitory effects of gedatolisib on colorectal cancer cells have been specifically studied. However, the role of ABC transporters on gedatolisib resistance remained unclear. In present study, we illustrated the role of ABC transporters on gedatolisib resistance in colorectal cancer cells. Methods Cell viability investigations of gedatolisib on colorectal cancer cells were determined by MTT assays. The verapamil and Ko143 reversal studies were determined by MTT assays as well. ABCB1 and/or ABCG2 siRNA interference assays were conducted to verify the role of ABCB1- and ABCG2-overexpression on gedatolisib resistance. The accumulation assays of gedatolisib were conducted using tritium-labeled paclitaxel and mitoxantrone. The effects of gedatolisib on ATPase activity of ABCB1 or ABCG2 were conducted using PREDEASY ATPase Kits. The expression level of ABCB1 and ABCG2 after gedatolisib treatment were conducted by Western blotting and immunofluorescence assays. The well-docked position of gedatolisib with crystal structure of ABCB1 and ABCG2 were simulated by Autodock vina software. One-way ANOVA was used for the statistics analysis. Results Gedatolisib competitively increased the accumulation of tritium-labeled substrate-drugs in both ABCB1- and ABCG2-overexpression colorectal cancer cells. Moreover, gedatolisib significantly increased the protein expression level of ABCB1 and ABCG2 in colorectal cancer cells. In addition, gedatolisib remarkably simulated the ATPase activity of both ABCB1 and ABCG2, suggesting that gedatolisib is a substrate drug of both ABCB1 and ABCG2 transporters. Furthermore, a gedatolisib-resistance colorectal cancer cell line, SW620/GEDA, was selected by increasingly treatment with gedatolisib to SW620 cells. The SW620/GEDA cell line was proved to resistant to gedatolisib and a series of chemotherapeutic drugs, except cisplatin. The ABCB1 and ABCG2 were observed overexpression in SW620/GEDA cell line. Conclusions These findings suggest that overexpression of ABCB1 and ABCG2 may restrict the efficacy of gedatolisib in colorectal cancer cells, while co-administration with ABC transporter inhibitors may improve the potency of gedatolisib.

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