Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Subunit interaction of vacuolar H+-pyrophosphatase as determined by high hydrostatic pressure.

ABSTRACT: Vacuolar H+-pyrophosphatase (H+-PPase) from etiolated hypocotyls of mung bean (Vigna radiata L.) is a homodimer with a molecular mass of 145 kDa. The vacuolar H+-PPase was subjected to high hydrostatic pressure to investigate its structure and function. The inhibition of H+-PPase activity by high hydrostatic pressure has a pressure-, time- and protein-concentration-dependent manner. The Vmax value of vacuolar H+-PPase was dramatically decreased by pressurization from 293.9 to 70.2 micromol of PPi (pyrophosphate) consumed/h per mg of protein, while the Km value decreased from 0.35 to 0.08 mM, implying that the pressure treatment increased the affinity of PPi to vacuolar H+-PPase but decreased its hydrolysis. The physiological substrate and its analogues enhance high pressure inhibition of vacuolar H+-PPase. The HPLC profile reveals high pressure treatment of H+-PPase provokes the subunit dissociation from an active into inactive form. High hydrostatic pressure also induces the conformational change of vacuolar H+-PPase as determined by spectroscopic techniques. Our results indicate the importance of protein-protein interaction for this novel proton-translocating enzyme. Working models are proposed to interpret the pressure inactivation of vacuolar H+-PPase. We also suggest that association of identical subunits of vacuolar H+-PPase is not random but proceeds in a specific manner.

SUBMITTER: Yang SJ

PROVIDER: S-EPMC1219367 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation.

Project description:Vacuolar proton-pyrophosphatase (H(+)-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H(+)-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5 +/- 10.8 and 158.4 +/- 19.5 kDa for PP1 hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H(+)-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H(+)-PPase was 71.1 +/- 6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H(+)-PPase was decreased to approx. 63.4 +/- 6.3 kDa. A working model was proposed to elucidate the structure of native H(+)-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.

| S-EPMC1217314 | biostudies-other

Substrate-induced changes in domain interaction of vacuolar H⁺-pyrophosphatase.

Project description:Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H(+)-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H(+)-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H(+)-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H(+)-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H(+)-pyrophosphatase in the presence and absence of substrate and its analog.

| S-EPMC4294485 | biostudies-literature

Response to high hydrostatic pressure

Project description:S. cerevisiae Y440 Mat a leu2 was grown in YEPD at 28 degrees C with aeration to exponential growth phase and was subjected to a hydrostatic pressure of 50 and 200 MPa for 30 minutes at room temperature. Total RNA was extracted using phenol/chloroform and further precipitated with 3 M sodium acetate / absolute ethanol. Extracted RNA samples were treated for 10 min with 0.5 U of RNAse-free DNAse I / ]g RNA at 37 oC to remove any residual genomic DNA. RNA pellets were washed in 70 % ethanol and resuspended in DEPC treated water. Purified mRNA from pressurized and unpressurized cells was reversed-transcribed, labeled with fluorescent-tagged nucleotides, and hybridized against a common refernce pool of mRNA for 18 h at 65 0C on cDNA microarray. After several washes, arrays were scanned using a commercially available scanning laser microscope (GenePix 4000) from Axon Instruments (Foster City, CA), the data obtained was normalized (mean value) applying a linear regression method. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract Keywords: Computed

2005-12-30 | GSE3935 | GEO

Thermococcus eurythermalis A501, high hydrostatic pressure

Project description:In this study, we performed a global quantitative proteomic analysis under extreme temperatures, pH, hydrostatic pressure (HP) and salinity on an archaeal strain, Thermococcus eurythermalis A501. Here is the result of pressure adaptation: HP (40 MPa) tested under 85°C and 95°C, and the optimal culture condition (85°C, pH 7, 2.3% NaCl, 10 MPa) was used as the control.

2020-09-09 | PXD019007 | Pride

Vacuolar Proton Pyrophosphatase Is Required for High Magnesium Tolerance in Arabidopsis.

Project description:Magnesium (Mg2+) is an essential nutrient in all organisms. However, high levels of Mg2+ in the environment are toxic to plants. In this study, we identified the vacuolar-type H⁺-pyrophosphatase, AVP1, as a critical enzyme for optimal plant growth under high-Mg conditions. The Arabidopsisavp1 mutants displayed severe growth retardation, as compared to the wild-type plants upon excessive Mg2+. Unexpectedly, the avp1 mutant plants retained similar Mg content to wild-type plants under either normal or high Mg conditions, suggesting that AVP1 may not directly contribute to Mg2+ homeostasis in plant cells. Further analyses confirmed that the avp1 mutant plants contained a higher pyrophosphate (PPi) content than wild type, coupled with impaired vacuolar H⁺-pyrophosphatase activity. Interestingly, expression of the Saccharomyces cerevisiae cytosolic inorganic pyrophosphatase1 gene IPP1, which facilitates PPi hydrolysis but not proton translocation into vacuole, rescued the growth defects of avp1 mutants under high-Mg conditions. These results provide evidence that high-Mg sensitivity in avp1 mutants possibly resulted from elevated level of cytosolic PPi. Moreover, genetic analysis indicated that mutation of AVP1 was additive to the defects in mgt6 and cbl2 cbl3 mutants that are previously known to be impaired in Mg2+ homeostasis. Taken together, our results suggest AVP1 is required for cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells.

| S-EPMC6274811 | biostudies-literature

Transcription profiling of yeast at high hydrostatic pressure

Project description:S. cerevisiae Y440 Mat a leu2 was grown in YEPD at 28 0C with aeration to exponential growth phase and was subjected to a hydrostatic pressure of 50 and 200 MPa for 30 minutes at room temperature. Total RNA was extracted using phenol/chloroform and further precipitated with 3 M sodium acetate / absolute ethanol. Extracted RNA samples were treated for 10 min with 0.5 U of RNAse-free DNAse I /g RNA at 37 oC to remove any residual genomic DNA. RNA pellets were washed in 70 % ethanol and resuspended in DEPC treated water. Purified mRNA from pressurized and unpressurized cells was reversed-transcribed, labeled with fluorescent-tagged nucleotides, and hybridized against a common refernce pool of mRNA for 18 h at 65 0C on cDNA microarray. After several washes, arrays were scanned using a commercially available scanning laser microscope (GenePix 4000) from Axon Instruments (Foster City, CA), the data obtained was normalized (mean value) applying a linear regression method.

2005-10-27 | E-SMDB-2406 | biostudies-arrayexpress

Mouse oocytes: High hydrostatic pressure (HP) treated vs. Control

2012-01-06 | GSE28410 | GEO

Mouse oocytes: High hydrostatic pressure (HP) treated vs. Control

Project description:Transcription profiling of mouse oocytes treated with 20 MPa hydrostatic pressure for 60 minutes at 37 °C comparing control oocytes kept under identical conditions as pressure treated ones, except HHP treatment. One-condition experiment, HP treated oocytes vs. Control oocytes. Biological replicates: 4 HP treated replicates, 4 control replicates.

2012-01-06 | E-GEOD-28410 | biostudies-arrayexpress

Shewanella piezotolerans WP3 wild-type strain under high hydrostatic pressure

Project description:This SuperSeries is composed of the SubSeries listed below.

2016-09-10 | GSE82267 | GEO

Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure.

Project description:The majority of the Earth's microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth's crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth's surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.

| S-EPMC7264842 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data