ATPase reaction cycle of P4‐ATPases affects their transport from the endoplasmic reticulum

Sarco(endo)plasmic reticulum Ca2+ (SERCA) pumps are important for cell signaling. Three different genes, SERCA1, 2, and 3, encode these pumps. Most tissues, including vascular smooth muscle, express a splice variant of SERCA2 (SERCA2b), whereas SERCA3a is widely distributed in tissues such as vascular endothelium, tracheal epithelium, mast cells, and lymphoid cells. SERCA2b protein is readily inactivated by peroxynitrite that may be formed during cardiac ischemia reperfusion or during immune response after infection. Here, we compared the peroxynitrite sensitivity of SERCA2b and SERCA3a by using microsomes prepared from HEK-293T cells overexpressing the pumps. We incubated the microsomes with different concentrations of peroxynitrite and determined Ca2+ uptake, Ca2+-Mg2+-ATPase, Ca2+-dependent formation of acylphosphate intermediate, and protein mobility in Western blots. Ca2+ uptake, Ca2+-Mg2+-ATPase, and Ca2+-dependent formation of acylphosphate intermediate were inactivated for both SERCA2b and SERCA3a, but the latter was more resistant to the inactivation. Western blots showed that SERCA2b and SERCA3a proteins oligomerized after treatment with peroxynitrite, but each with a slightly different pattern. Compared with monomers, the oligomers may be less efficient in forming the acylphosphate intermediate and in conducting the remainder of the steps in the reaction cycle. We conclude that the resistance of SERCA3a to peroxynitrite may aid the cells expressing them in functioning during exposure to oxidative stress.

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Cdc50p Plays a Vital Role in the ATPase Reaction Cycle of the Putative Aminophospholipid Transporter Drs2p

Journal of Biological Chemistry ◽

10.1074/jbc.m109.013722 ◽

2009 ◽

Vol 284 (27) ◽

pp. 17956-17967 ◽

Cited By ~ 90

Author(s):

Guillaume Lenoir ◽

Patrick Williamson ◽

Catheleyne F. Puts ◽

Joost C. M. Holthuis

Keyword(s):

Vital Role ◽

Reaction Cycle ◽

Atpase Reaction

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The Work Performed By Biological Motors

10.1101/580241 ◽

2019 ◽

Author(s):

Josh E. Baker

Keyword(s):

Free Energy ◽

Molecular Motors ◽

Molecular Motor ◽

Free Energy Change ◽

Energy Change ◽

Constant Force ◽

Reaction Cycle ◽

Atpase Reaction ◽

Definition Of ◽

Mechanochemical Model

AbstractMolecular motors are enzymes that perform work (F · x) when they move along a track a distance x against a constant force F. This work is performed through intermediate chemical steps in a motor’s ATPase reaction cycle, each step having a free energy change associated with it that is a sum of chemical, Δµchem, and mechanical, Δµext, potentials. Defining Δµext is fundamental to our understanding of how molecular motors work, yet after decades of study the definition of Δµext remains disputed. Some postulate that Δµext is a function of both F and x, while others assume that Δµext is a function of neither F nor x, and still others argue that Δµext is a function of F but not x. Here we evaluate these models and conclude that only the latter – a mechanochemical model proposed by A.V. Hill in the 1930’s – describes molecular motor mechanochemistry.

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The basal Mg2+-dependent ATPase activity is not part of the (H++K+)-transporting ATPase reaction cycle

Biochemical Journal ◽

10.1042/bj2670565 ◽

1990 ◽

Vol 267 (3) ◽

pp. 565-572 ◽

Cited By ~ 4

Author(s):

H T W M Van der Hijden ◽

S Kramer-Schmitt ◽

E Grell ◽

J J H H M de Pont

Keyword(s):

Gastric Mucosa ◽

Atpase Activity ◽

Hydrolytic Activity ◽

Reaction Cycle ◽

Dependent Atpase ◽

Atpase Reaction ◽

Phosphorylation Reaction ◽

Rate Of Hydrolysis ◽

Hydrolysis Of

Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the ‘stretched-out’ version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.

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Structural Characterization of the ATPase Reaction Cycle of Endosomal AAA Protein Vps4

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.09.067 ◽

2007 ◽

Vol 374 (3) ◽

pp. 655-670 ◽

Cited By ~ 43

Author(s):

Junyu Xiao ◽

Hengchuan Xia ◽

Kae Yoshino-Koh ◽

Jiahai Zhou ◽

Zhaohui Xu

Keyword(s):

Structural Characterization ◽

Reaction Cycle ◽

Atpase Reaction ◽

Aaa Protein

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Kinetic characterization of tetrapropylammonium inhibition reveals how ATP and Pi alter access to the Na+-K+-ATPase transport site

AJP Cell Physiology ◽

10.1152/ajpcell.00043.2005 ◽

2005 ◽

Vol 289 (2) ◽

pp. C302-C311 ◽

Cited By ~ 15

Author(s):

Craig Gatto ◽

Jeff B. Helms ◽

Megan C. Prasse ◽

Krista L. Arnett ◽

Mark A. Milanick

Keyword(s):

Conformational Changes ◽

Inorganic Phosphate ◽

Cation Transport ◽

Cation Binding ◽

Atp Binding ◽

Cation Site ◽

Reaction Cycle ◽

Atpase Reaction ◽

Transport Site

Current models of the Na+-K+-ATPase reaction cycle have ATP binding with low affinity to the K+-occluded form and accelerating K+ deocclusion, presumably by opening the inside gate. Implicit in this situation is that ATP binds after closing the extracellular gate and thus predicts that ATP binding and extracellular cation binding to be mutually exclusive. We tested this hypothesis. Accordingly, we needed a cation that binds outside and not inside, and we determined that tetrapropylammonium (TPA) behaves as such. TPA competed with K+ (and not Na+) for ATPase, TPA was unable to prevent phosphoenzyme (EP) formation even at low Na+, and TPA decreased the rate of EP hydrolysis in a K+-competitive manner. Having established that TPA binding is a measurement of extracellular access, we next determined that TPA and inorganic phosphate (Pi) were not mutually exclusive inhibitors of para-nitrophenylphosphatase (pNPPase) activity, implying that when Pi is bound, the transport site has extracellular access. Surprisingly, we found that ATP and TPA also were not mutually exclusive inhibitors of pNPPase activity, implying that when the cation transport site has extracellular access, ATP can still bind. This is consistent with a model in which ATP speeds up the conformational changes that lead to intracellular or extracellular access, but that ATP binding is not, by itself, the trigger that causes opening of the cation site to the cytoplasm.

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