Topic
Vanadate
About: Vanadate is a research topic. Over the lifetime, 4497 publications have been published within this topic receiving 120109 citations. The topic is also known as: vanadate.
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TL;DR: This evaluation of CFTR chloride channel activity under conditions known to inhibit the activity of ATPases shows that CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics.
Abstract: CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mm). Higher concentrations of vanadate (10 mm) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN−) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for this transition state analogue is considerably different than that of other ABC transporters.
50 citations
TL;DR: The results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na, k-ATpase and the gastric H, K- ATPase enzymes.
50 citations
TL;DR: A 72 kDa protein was phosphorylated when membranes were incubated with Mg 2+ and [ 32 P]orthophosphate, and phosphorylation was inhibited by both vanadate and delta endotoxin, suggesting that destabilization of this phosphoprotein was responsible for phosphatase inhibition.
50 citations
TL;DR: The primary or secondary energized transport of Ca2+, Mg2+ and H+ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically and it was suggested that this ATP-dependent high-affinity Ca2+ transport mechanism is the only functioning Ca2- transporter of the tonopine under in-vivo conditions i.e. under the low cytosolic Ca2+.
Abstract: The primary or secondary energized transport of Ca2+, Mg2+ and H+ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca2+ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca2+ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca2+-pumping and tonoplast H+-pyrophosphatase (PPiase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca2+-pumping activity could be detected. The ATP-dependent Ca2+ uptake into tonoplast vesicles (using Ca2+ concentrations from 0.8–1 μM) was completely inhibited by the Ca2+ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H+ accumulation had no effect. Vanadate (IC50 = 43 μM) and diethylstilbesterol (IC50 = 5.2 μM) were potent inhibitors of this type of Ca2+ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K
m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca2+ transport mechanism is the only functioning Ca2+ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca2+ concentration. In contrast, the secondary energized Ca2+-transport mechanism of the tonoplast, the low-affinity Ca2+/H+-antiporter, which was reported to allow the uptake of Ca2+ in exchange for H+, functions chiefly as an Mg2+ transporter under physiological conditions because cytosolic Mg2+ is several orders of magnitude higher than the Ca2+ concentration. This conclusion was deduced from experiments showing that Mg2+ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H+ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H+-ATPase and H+-PPiase was found to be influenced by Ca2+ differently from and independently of the Mg2+ concentration. Calcium was a strong inhibitor for the H+-PPiase (IC50 = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H+-ATPase (IC50 = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca2+-and Mg2+-regulated H+-PPiase, (ii) the newly described high-affinity Ca2+-AT-Pase, (iii) the low-affinity Mg2+(Ca2+)/H+-antiporter and (iv) the H2+-ATPase.
50 citations
TL;DR: The resolution-reconstitution approach has been employed in order to gain information as to the subunit composition of the Neurospora plasma membrane H+-ATPase, which indicates that this enzyme has no subunits even vaguely resembling any that have been found to be associated with other transport ATPases and that if this enzymes has any subunits at all other than the 105,000-dalton hydrolytic moiety, they must be very small.
50 citations