Molecular cloning and sequencing of the cDNA for vacuolar H+-pyrophosphatase from Chara corallina

Nakanishi, Yoichi; Matsuda, Nao; Aizawa, Kenji; Kashiyama, Taku; Yamamoto, Keiichi; Mimura, Tetsuro; Ikeda, Mikiko; Maeshima, Masayoshi

doi:10.1016/s0005-2736(99)00037-1

Cited by 23 publications

(7 citation statements)

References 20 publications

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“…This was supported by immunochemical study with an antibody specific to this sequence (DVGADLVGKVE) (12). This sequence is common among VPPases not only from land plants but also from Chara corallina (10), Acetabularia acetabulum (13), R. rubrum (5), Thermotoga maritima (GenBank™ accession number AE001702), and P. aerophilum (6). Studies using substrate analogs, such as aminomethylenebisphosphonate, have also provided information on the catalytic domain (14 -16).…”

Section: Vacuolar Hmentioning

confidence: 94%

“…Thus, V-PPase should have a specific binding site for its substrate (Mg-PP i ), Mg 2ϩ , K ϩ , and Ca 2ϩ in addition to a H ϩ transport channel. Multiple amino acid sequence alignment of V-PPases of various organisms revealed highly conserved regions (2,3,10). There is a putative substrate-binding motif of DXXXXXXXKXE in the cytoplasmic loop (1,11).…”

Section: Vacuolar Hmentioning

confidence: 99%

“…We prepared a line of constructs, in which charged residues in a putative substrate-binding site were replaced, expressed in Saccharomyces cerevisiae, and then examined for enzymatic properties. V-PPase has been proposed to have three conserved regions (3,10). In addition to a putative PP i -binding site in the first conserved region, we investigated the two acidic motifs in the first and third conserved regions.…”

Section: Vacuolar Hmentioning

confidence: 99%

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Mutagenic Analysis of Functional Residues in Putative Substrate-binding Site and Acidic Domains of Vacuolar H+-Pyrophosphatase

Nakanishi

Saijo

Wada

et al. 2001

Journal of Biological Chemistry

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. Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases. Vacuolar Hϩ -pyrophosphatase (V-PPase) 1 belongs to the fourth class of electrogenic proton pump in addition to the P-, F-, and V-type ATPases. The proton pumping reaction couples with the hydrolysis of PP i . V-PPase acidifies vacuoles together with vacuolar H ϩ -ATPase in the plant cell and actively exports protons from the cytosol in the bacterial plasma membrane (1-3). V-PPase has the simplest structure among the proton pumps except for bacteriorhodopsin, a light-driven proton pump. The molecular mass calculated from the cDNA sequences range from 80 to 81 kDa for V-PPases of land plants and algae (for a review, see Refs. 3 and 4), while V-PPases in photosynthetic bacteria Rhodospirillum rubrum (5) and archaebacteria Pyrobaculum aerophilum (6) are relatively small. The simplicity of the enzyme structure and its substrate is an advantage to analyze the structure-function relationship. The enzyme activity is stimulated by K ϩ at relatively high concentrations. Also, Mg 2ϩ is essential to form a Mg-PP i complex and to keep the active conformation of V-PPase (1, 7, 8). Ca There is a putative substrate-binding motif of DXXXXXXXKXE in the cytoplasmic loop (1, 11). This was supported by immunochemical study with an antibody specific to this sequence (DVGADLVGKVE) (12). This sequence is common among VPPases not only from land plants but also from Chara corallina (10), Acetabularia acetabulum (13), R. rubrum (5), Thermotoga maritima (GenBank™ accession number AE001702), and P. aerophilum (6). Studies using substrate analogs, such as aminomethylenebisphosphonate, have also provided information on the catalytic domain (14 -16). Furthermore, the N-ethylmaleimide-binding cysteine residue (Cys 634 ) (17) and the N,NЈ-dicyclohexylcarbodiimde-binding residues (Glu 305 and Asp 504 ) (18) have been identified by a combination of site-directed mutagenesis of Arabidopsis V-PPase and heterologous expression in yeast.The aim of this study is to clarify the substrate-binding site of V-PPase by the method of site-directed and random mutagenesis. We prepared a line of constructs, in which charged residues in a putative substrate-binding site were replaced, expressed in Saccharomyces cerevisiae, and then examined for enzymatic properties. V-PPase has been proposed to have three conserved regions (3, 10). In addition to a putative PP i -binding site in the first conserved region, we investigated the two acidic motifs in the first and third conserved regions. Each aspartic acid residue in the two acidic regions was substituted and examined for enzymatic properties. Here, the functional roles of these residues on the substrate hydrolysis, binding of free Mg 2ϩ , and a coupling reaction between PP i hydrolysis and proton transport were examined. The similarity of the conserved functional motifs of V-PPase with the P-type ATPase is also discussed.

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Section: Vacuolar Hmentioning

confidence: 94%

Section: Vacuolar Hmentioning

confidence: 99%

Section: Vacuolar Hmentioning

confidence: 99%

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Mutagenic Analysis of Functional Residues in Putative Substrate-binding Site and Acidic Domains of Vacuolar H+-Pyrophosphatase

Nakanishi

Saijo

Wada

et al. 2001

Journal of Biological Chemistry

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“…This experimental system clearly needs revisiting. Nakanishi et al (1999) found the cDNA sequence of the C. corallina PPase 71% identical to that of land plants and 46% identical to that of chlorophyte Acetabularia and phototropic bacterium Rhodospirillum rubrum .…”

Section: One Plant – Many Experimental Systemsmentioning

confidence: 94%

Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism

Beilby

2016

Front. Plant Sci.

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The morphology of characean algae could be mistaken for a higher plant: stem-like axes with leaf-like branchlets anchored in the soil by root-like rhizoids. However, all of these structures are made up of giant multinucleate cells separated by multicellular nodal complexes. The excised internodal cells survive long enough for the nodes to give rise to new thallus. The size of the internodes and their thick cytoplasmic layer minimize impalement injury and allow specific micro-electrode placement. The cell structure can be manipulated by centrifugation, perfusion of cell contents or creation of cytoplasmic droplets, allowing access to both vacuolar and cytoplasmic compartments and both sides of the cell membranes. Thousands of electrical measurements on intact or altered cells and cytoplasmic droplets laid down basis to modern plant electrophysiology. Furthermore, the giant internodal cells and whole thalli facilitate research into many other plant properties. As nutrients have to be transported from rhizoids to growing parts of the thallus and hormonal signals need to pass from cell to cell, Characeae possess very fast cytoplasmic streaming. The mechanism was resolved in the characean model. Plasmodesmata between the internodal cells and nodal complexes facilitate transport of ions, nutrients and photosynthates across the nodes. The internal structure was found to be similar to those of higher plants. Recent experiments suggest a strong circadian influence on metabolic pathways producing indole-3-acetic acid (IAA) and serotonin/melatonin. The review will discuss the impact of the characean models arising from fragments of cells, single cells, cell-to-cell transport or whole thalli on understanding of plant evolution and physiology.

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“…33 The protein topologies were predicted using MEMSAT-SVM prediction, 34 TMHMM prediction 35 and TMpred server. 36 The MEMSAT algorithm employed empirically derived topological data, from available membrane proteins. All residues were assigned to one of five states (inner helix end, middle helix, outer helix end, intracellular, and extracellular).…”

Section: Molecular Pharmaceuticsmentioning

confidence: 99%

Mechanistic Insights into PEPT1-Mediated Transport of a Novel Antiepileptic, NP-647

Khomane

Nandekar²,

Wahlang³

et al. 2012

Mol. Pharmaceutics

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The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 μM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

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Molecular cloning and sequencing of the cDNA for vacuolar H+-pyrophosphatase from Chara corallina

Cited by 23 publications

References 20 publications

Mutagenic Analysis of Functional Residues in Putative Substrate-binding Site and Acidic Domains of Vacuolar H+-Pyrophosphatase

Mutagenic Analysis of Functional Residues in Putative Substrate-binding Site and Acidic Domains of Vacuolar H+-Pyrophosphatase

Multi-Scale Characean Experimental System: From Electrophysiology of Membrane Transporters to Cell-to-Cell Connectivity, Cytoplasmic Streaming and Auxin Metabolism

Mechanistic Insights into PEPT1-Mediated Transport of a Novel Antiepileptic, NP-647

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