Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds

Abrecht, Helge; Wattiez, Ruddy; Ruysschaert, Jean‐Marie; Homblé, Fabrice

doi:10.1104/pp.124.3.1181

Cited by 31 publications

(30 citation statements)

References 44 publications

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“…VDAC Purification-As a modification to the previously reported protocol (22), purification of the two VDAC isoforms was achieved in a single purification step. Mitochondria were isolated from the cotyledons of 24-h imbibed kidney beans (P. vulgaris) seeds and solubilized with 2% (v/v) Genapol X-080 and 4 M urea.…”

Section: Resultsmentioning

confidence: 99%

“…1, lanes 2 and 4). The staining intensities reflect the abundance of each isoform in the seed mitochondria (22).…”

Section: Resultsmentioning

confidence: 99%

“…Two VDAC isoforms were purified from kidney bean cotyledons in a single purification step using chromatofocusing chromatography as previously reported (22). Highly purified proteins were reconstituted in a mixture of asolectin and stigmasterol, a major sterol of plant membranes.…”

Section: Discussionmentioning

confidence: 99%

“…The two VDAC proteins were purified and separated in a single chromatofocusing chromatography step, i.e. the HTP batch described in the previous protocol (22) was omitted. Briefly, seeds from P. vulgaris were soaked in tap water for 24 h and mitochondria were isolated from the cotyledons by differential centrifugation steps and further purified on a 28% Percoll gradient (23).…”

Section: Methodsmentioning

confidence: 99%

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Structure and Orientation of Two Voltage-dependent Anion-selective Channel Isoforms

Abrecht

Goormaghtigh²,

Ruysschaert³

et al. 2000

Journal of Biological Chemistry

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Two VDAC (voltage-dependent anion-selective channel) isoforms were purified from seed cotyledons of Phaseolus vulgaris by chromatofocusing chromatography. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to study the structural properties of the two isoforms reconstituted in a mixture of asolectin and 5% stigmasterol. The IR spectra of the two VDAC isoforms were highly similar indicating 50 to 53% anti-parallel ␤-sheet. The orientation of the ␤-strands relative to the barrel axis was calculated from the experimentally obtained dichroic ratios of the amide I ␤-sheet component and the amide II band. Comparing the IR spectra of the reconstituted VDAC isoforms with the IR spectra of the bacterial porin OmpF, for which a high resolution structure is available, provided evidence for a general structural organization of the VDAC isoforms similar to that of bacterial porins. Hydrogen-deuterium exchange measurements indicated that the exchange of the amide protons occurs to a higher extent in the two VDAC isoforms than in the OmpF porin.Mitochondria are surrounded by two distinct membranes. The mitochondrial outer membrane is freely permeable for small hydrophilic solutes up to approximately 6 kDa. This property has been attributed to the presence of a pore forming protein, the voltage-dependent anion-selective channel (VDAC) 1 or mitochondrial porin (1). Biophysical properties of VDAC channels inserted in planar lipid bilayers have been studied in a large variety of organisms. VDAC channels switch from a high conducting fully open state (3.6 to 4.5 nS in 1 M KCl) at low voltages to several low-conducting substates upon application of voltages higher than Ϯ20 mV. VDAC is slightly anion selective in the fully open state but switches to cation selectivity in the low-conducting substates (2). In these substates, the flow of negatively charged metabolites such as succinate, citrate, phosphate (3), and adenine nucleotides (4, 5) through the channel is strongly reduced compared with the fully open state. Therefore, VDAC is thought to regulate the mitochondrial metabolism by regulating the flux of metabolites across the mitochondrial outer membrane.VDAC channels are involved in different cellular events like the induced release of cytochrome c, which constitutes an early step in apoptosis (6). VDAC is the binding site for cytoplasmic enzymes like glycerol kinase and hexokinase (7) and for the cytoskeleton (8). The different VDAC isoforms that exist in yeast, plants, and mammals could be involved in specific functions. Two human VDAC isoforms have different binding affinities for hexokinase (9). Plant VDAC isoforms have slightly different electrophysiological properties (10), and mitochondria isolated from yeast vdac minus mutants show differences in their outer membrane permeability to NADH depending on which of the three mouse VDAC isoforms was expressed (11). Therefore, it seems reasonable to assume that the multitude of functional properties that are attributed to VDAC could arise from ...

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Section: Resultsmentioning

confidence: 99%

“…1, lanes 2 and 4). The staining intensities reflect the abundance of each isoform in the seed mitochondria (22).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Structure and Orientation of Two Voltage-dependent Anion-selective Channel Isoforms

Abrecht

Goormaghtigh²,

Ruysschaert³

et al. 2000

Journal of Biological Chemistry

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“…Since then, VDAC proteins have been discovered in most eukaryotic organisms ranging from yeast (2) to animals (3,4) to plants (5,6). Functional characterization of these proteins has been mainly achieved by purified protein reconstitution into planar bilayer membranes (for a review see Ref.…”

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confidence: 99%

Voltage-dependent Anion-selective Channels VDAC2 and VDAC3 Are Abundant Proteins in Bovine Outer Dense Fibers, a Cytoskeletal Component of the Sperm Flagellum

Hinsch

Pinto

Aires

et al. 2004

Journal of Biological Chemistry

107

110

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Outer dense fibers (ODF) are specific subcellular components of the sperm flagellum. The functions of ODF have not yet been clearly elucidated. We have investigated the protein composition of purified ODF from bovine spermatozoa and found that one of the most abundant proteins is a 30 -32-kDa polypeptide. This protein was analyzed by sequencing peptides derived following limited proteolysis. Peptide sequences were found to match VDAC2 and VDAC3. VDACs (voltage-dependent, anion-selective channels) or eukaryotic porins are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. In mammals, three VDAC isoforms (VDAC1, -2, -3) have been identified by cDNA cloning and sequencing. Antibodies against synthetic peptides specific for the three mammal VDAC isoforms were generated in rabbits. Their specificity was demonstrated by immunoblotting using recombinant VDAC1, -2, and -3. In protein extracts of bovine spermatozoa, VDAC1, -2, and -3 were detected by specific antibodies, while only VDAC2 and -3 were found as solubilized proteins derived from purified bovine ODFs. Immunofluorescence microscopy of spermatozoa revealed that anti-VDAC2 and anti-VDAC3 antibodies clearly bound to the sperm flagellum, in particular to the ODF. Transmission electron immunomicroscopy supported the finding that VDAC2 protein is abundant in the ODF. Since the ODF does not have any known membranous structure, it is tempting to speculate that VDAC2 and VDAC3 might have an alternative structural organization and different functions in ODF than in mitochondria.

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Mitochondrial Porins, Eukaryotic

Benz

2006

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds

Cited by 31 publications

References 44 publications

Structure and Orientation of Two Voltage-dependent Anion-selective Channel Isoforms

Structure and Orientation of Two Voltage-dependent Anion-selective Channel Isoforms

Voltage-dependent Anion-selective Channels VDAC2 and VDAC3 Are Abundant Proteins in Bovine Outer Dense Fibers, a Cytoskeletal Component of the Sperm Flagellum

Mitochondrial Porins, Eukaryotic

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