Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

2009 ◽  
Vol 29 (6) ◽  
pp. 351-362 ◽  
Author(s):  
Viviana A. Menzel ◽  
M. Carolina Cassará ◽  
Roland Benz ◽  
Vito De Pinto ◽  
Angela Messina ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Functional Characterization ◽  
Membrane Conductance ◽  
Peptide Sequencing ◽  
Blue Staining ◽  
Voltage Dependent Anion Channel ◽  
Bovine Spermatozoa

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30–35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

scholarly journals Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

2018 ◽  
Vol 150 (4) ◽  
pp. 637-646 ◽  
Author(s):  
Laura-Marie Winterstein ◽  
Kerri Kukovetz ◽  
Oliver Rauh ◽  
Daniel L. Turman ◽  
Christian Braun ◽  
...  
Keyword(s):  
Ion Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Functional Characterization ◽  
In Vitro Translation ◽  
Target Membrane ◽  
Electrophysiological Characterization ◽  
Channel Properties

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F− channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels.

CFTR in K562 human leukemic cells

2003 ◽  
Vol 285 (2) ◽  
pp. C480-C488 ◽  
Author(s):  
Yanina A. Assef ◽  
Alicia E. Damiano ◽  
Elsa Zotta ◽  
Cristina Ibarra ◽  
Basilio A. Kotsias
Keyword(s):  
Single Channel ◽  
Functional Characterization ◽  
Leukemic Cells ◽  
Cftr Gene ◽  
Human Leukemia ◽  
Selectivity Ratio ◽  
Open Probability ◽  
Selective Channel ◽  
Human Leukemic Cells

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 μM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.

Prokaryotic Expression and Functional Characterization of the 19 kDa Protein in Balanus albicostatus Cement

2013 ◽  
Vol 461 ◽  
pp. 445-450
Author(s):  
Chao Liang ◽  
Yun Qiu Li ◽  
Bi Ru Hu ◽  
Wen Jian Wu
Keyword(s):  
Amino Acid ◽  
Molecular Design ◽  
Functional Characterization ◽  
Blue Staining ◽  
Amino Acid Residues ◽  
Adhesive Ability ◽  
Solid Liquid ◽  
Dental Applications ◽  
The Relationship

Barnacle is a unique sessile crustacean, which produces a multi-protein complex historically called barnacle cement to attach to diverse immersed materials permanently. The proteinaceous cement exhibits powerful adhesive property and special waterproof capability to cure at solid-liquid boundaries, which makes it ideal biomaterial for technical, medical and dental applications. It has been proved that a 19 kDa protein component, termed cp-19k in the cement plays a key role in surface coupling during underwater attachment. To verify whether the bacterial recombinant 19 kDa protein retains the adhesive ability, we cloned and sequenced the Bacp-19k gene in Balanus albicostatus. It encodes 173 amino acid residues, with seven biased ones, Thr, Lys, Gly, Ala, Val, Ser and Leu, comprising about 80% of the total. Two amino acid substitutions (F69L, I106L) were discovered in Bacp-19k due to the polymorphisms in barnacle cp-19ks, compared with the submitted one (GenBank: AB242295.1). Recombinant Bacp-19k was highly expressed in host strain Escherichia coli BL21 (DE3) and purified by affinity chromatography. Adsorption of recombinant Bacp-19k to glass substrata was examined by Coomassie brilliant blue staining. Future study will reveal the relationship between specific structures and functions for molecular design of novel biomimetic underwater adhesives.

scholarly journals Functional characterization of a voltage-gated anion channel from teleost fish intestinal epithelium

1988 ◽  
Vol 136 (1) ◽  
pp. 383-403 ◽  
Author(s):  
C. A. Loretz ◽  
C. R. Fourtner
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Anion Channel ◽  
Intestinal Epithelial ◽  
Channel Conductance ◽  
Patch Clamp Technique ◽  
Voltage Gated

An anion channel was isolated, using patch-clamp technique, from the basolateral membrane of goby intestinal epithelial cells. Single-channel conductance varied over a range from 20 to 90 pS. The channel was voltage-gated over the physiological range of cell membrane potential with depolarization increasing the proportion of time in the open state. There was no Ca2+ sensitivity. The selectivity sequence was SO4(2-) greater than Cl- greater than Mes-. The channel may function in vivo as one of several avenues of basolateral membrane Cl- exit with the voltage-gating property serving to match basolateral Cl- exit to apical entry.

scholarly journals Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

2020 ◽  
Author(s):  
Fabian C. Herbert ◽  
Sameera Abeyrathna ◽  
Nisansala Abeyrathna ◽  
Yalini Wijesundara ◽  
Olivia Brohlin ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Elevated Temperatures ◽  
Functional Characterization ◽  
Transmembrane Proteins ◽  
Protein Assemblies ◽  
Metal Transporters ◽  
Structure And Activity ◽  
Detergent Micelles

<div> <div> <div> <p>Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamel- lar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these com- plexes remains challenging because of their chemical composition, the hydrophobicity and structural instabil- ity of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid su- pramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework- 8 (ZIF-8) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF-8 bio- composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embed- ded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization. </p> </div> </div> </div>

Solid-supported lipid bilayers – A versatile tool for the structural and functional characterization of membrane proteins

Methods ◽  
2020 ◽  
Vol 180 ◽  
pp. 56-68 ◽  
Author(s):  
Jakob Andersson ◽  
Pierluigi Bilotto ◽  
Laura L.E. Mears ◽  
Stefan Fossati ◽  
Ulrich Ramach ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Functional Characterization ◽  
Supported Lipid Bilayers ◽  
Versatile Tool

Functional characterization of the low-molecular-mass phosphotyrosine-protein phosphatase of Acinetobacter johnsonii 1 1Edited by M. Yaniv

1998 ◽  
Vol 278 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Christophe Grangeasse ◽  
Patricia Doublet ◽  
Carole Vincent ◽  
Elisabeth Vaganay ◽  
Mylène Riberty ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Protein Phosphatase ◽  
Functional Characterization ◽  
Acinetobacter Johnsonii ◽  
Phosphotyrosine Protein Phosphatase

scholarly journals Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers

Langmuir ◽  
2017 ◽  
Vol 33 (38) ◽  
pp. 9988-9996 ◽  
Author(s):  
Marco Maccarini ◽  
Landry Gayet ◽  
Jean-Pierre Alcaraz ◽  
Lavinia Liguori ◽  
Barry Stidder ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid Bilayers ◽  
Functional Characterization
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