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.

Fast axonal transport in permeabilized lobster giant axons is inhibited by vanadate

Abstract: We have developed a method for permeabilizing axons and reactivating the fast transport of microscopically visible organelles. Saltatory movements of organelles in motor axons isolated from lobster walking legs were observed using Nomarski optics and time-lapse video microscopy. In the center of the axon most of the particles and mitochondria moved in the retrograde direction, but immediately below the axolemma the majority moved in the anterograde direction. When axons were permeabilized with 0.02% saponin in an adenosine 5′- triphosphate (ATP)-free “internal” medium, all organelle movement ceased. Saltatory movements resembling those in intact axons immediately reappeared upon the addition of MgATP. Very slight movement could be detected with ATP concentrations as low as 10 microM, and movement appeared to be maximal with 1 to 5 mM ATP. Vanadate, which does not affect axonal transport in intact axons, inhibited the reactivated organelle movements in permeabilized axons. Movement was rapidly and reversibly inhibited by 50 to 100 microM sodium orthovanadate. The effects of vanadate, including the time course of inhibition, its reversibility, and its concentration dependence, are consistent with the hypothesis that a dyneinlike like molecule may play a role in the mechanism of fast axonal transport.

Tungsten Transport Protein A (WtpA) in Pyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

Abstract: Molybdenum and tungsten have similar ionic radii and chemical properties. Tungsten is the heaviest atom and the only third-row transition element that exhibits biological activity in enzymes. Molybdenum is the only second-row transition metal that exhibits biological activity when it is present in a cofactor of a metalloenzyme. Both metals are present mainly in enzymes that catalyze oxygen atom transfer reactions. In these enzymes they are coordinated by the two dithiolene sulfur atoms of a pterin molecule, called a molybdopterin cofactor (25). In the case of tungsten, the metal is always coordinated by two pterin moieties, forming a so called bis-pterin cofactor (9, 13). Molybdenum is an essential trace metal for many forms of life whereas tungsten is found mostly in archaea and in some bacteria. The molybdenum cofactor-containing enzymes can be divided into three families depending on the coordination chemistry of the Mo ligand: the sulfite oxidases; the xanthine oxidoreductases, also including the aldehyde oxidases; and the dimethyl sulfoxide reductases, which are found only in prokaryotes (18, 35). The tungsten cofactor-containing enzymes consist only of the aldehyde oxidoreductases (AORs), formate dehydrogenases (FDHs), and an acetylene hydratase (13). Based on sequence comparison the FDHs and acetylene hydratase are part of the molybdenum-containing dimethyl sulfoxide reductase family. The transport of molybdate has been well characterized in particular for Escherichia coli, which expresses a high-affinity ABC transporter for molybdate encoded by the modABC genes (27). The periplasmic molybdate binding protein ModA binds specifically molybdate and tungstate and not sulfate or other anions (27). Crystal structures of the E. coli and the Azotobacter vinelandii ModA indicate that the specificity for molybdate and tungstate is mostly determined by the size of the binding pocket. The Cambridge Structural Database (2) gives 1.75 ± 0.04 A and 1.76 ± 0.02 A for molybdate and tungstate, respectively, and 1.47 ± 0.02 A for sulfate. The ModA proteins cannot discriminate between molybdate and tungstate. The first tungsten-specific ABC transporter was identified in Eubacterium acidaminophilum (17). The periplasmic tungsten uptake protein (TupA) was cloned and expressed in E. coli and was shown to bind only tungstate with a high affinity. A crystal structure of TupA is not yet available, and it is not clear what the structural basis is of the specificity for tungstate over molybdate. Recently, a high-affinity vanadate transporter, which was highly selective for vanadate compared to tungstate, was identified in the cyanobacterium Anabaena variabilis ATCC 29413 based on the sequence similarity with the TupA protein from E. acidaminophilum (58% sequence similarity) (24). A. variabilis ATCC 29413 expresses an alternative V-dependent nitrogenase for the fixation of nitrogen, and therefore it requires vanadate (24). The specificity of this transporter for vanadate indicates that high sequence similarities are not conclusive for the selectivity of the transporter. Our goal was to study tungstate transport in an organism that is strictly dependent on tungstate, the hyperthermophilic archaeon Pyrococcus furiosus. This organism grows optimally at 100°C under strict anaerobic conditions (7). In the last decade five tungsten-containing aldehyde oxidoreductase enzymes were purified and characterized from P. furiosus. AOR (21), formaldehyde oxidoreductase (31), and tungsten-containing oxidoreductase 5 (WOR5) (5) all have a broad substrate specificity for aldehydes varying from shorter chains and C4 to C6 semialdehydes (FOR) to longer, aromatic, and aliphatic backbones (AOR and WOR5). These broad substrate specificities do not immediately imply a clear physiological function for these proteins; microarray experiments indicate that they might play a role in peptide fermentation or in stress response (34, 38). In contrast, glyceraldehyde-3-phosphate (GAP) oxidoreductase is known only to convert the substrate GAP (22). It is the only W-containing aldehyde oxidoreductase with an assigned function, namely, in the Embden-Meyerhof type of glycolysis, where it converts GAP to 3-phosphoglycerate, replacing glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. Tungsten oxidoreductase 4 (WOR4) was purified from P. furiosus grown in the presence of elemental sulfur (S0) (30). No substrate has been identified yet for WOR4. Besides these five tungsten-containing enzymes of the AOR family, the genome of P. furiosus also includes two genes for putative tungsten- or molybdenum-containing FDHs (29). Cultivation experiments indicated that P. furiosus has a highly specific tungstate uptake mechanism. When molybdate was added to the growth medium in a 1,000-fold excess, the cells were able to selectively scavenge the traces of tungstate from the medium and use it for the incorporation in the cofactor of the AOR enzymes (23). The genome of P. furiosus does not carry a tupA homologue; however, a putative sulfate/thiosulfate/molybdate transporter is present that has 30% sequence similarity with the ModA protein from E. coli (PF0080/PF0081/PF0082) (18% sequence identity). The other components of this putative transporter, WtpB and WtpC, have a high sequence similarity to ModB/TupB (53% and 50% similarity) and ModC/TupC (51% and 56% similarity), respectively. Besides this putative sulfate/thiosulfate/molybdate transporter, the only ABC transporter encoded in the genome with some similarity (28%) to ModA is annotated as a putative phosphate transporter (PF1003/PF1006/PF1007/PF1008). However, the sequence identity is much lower, only 11%. Since no molybdenum enzymes have been identified yet from P. furiosus, we hypothesized that the operon that contains the PF0080, PF0081, and PF0082 genes codes for a tungstate-selective ABC transporter. An mRNA fragment carrying the PF0080 gene has previously been detected in microarray experiments (38), indicating that the protein is expressed in vivo. In this paper we describe the cloning, expression, and binding characteristics of this new tungstate transport protein (WtpA).