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.
Skip Nav Destination
Article navigation
Research Article|
July 22 2009
Molecular and functional characterization of VDAC2 purified from mammal spermatozoa
Viviana A. Menzel;
Viviana A. Menzel
*Center of Dermatology and Andrology, Justus Liebig University of Giessen, Gaffkystrasse 14, DE-35392, Germany
Search for other works by this author on:
M. Carolina Cassará;
M. Carolina Cassará
*Center of Dermatology and Andrology, Justus Liebig University of Giessen, Gaffkystrasse 14, DE-35392, Germany
Search for other works by this author on:
Roland Benz;
Roland Benz
†Lehrstuhl für Biotechnologie, Universität Würzburg, DE-97074 Würzburg, Germany
Search for other works by this author on:
Vito De Pinto;
Vito De Pinto
1
‡Department of Chemistry, Laboratory of Biochemistry and Molecular Biology, University of Catania, IT-95125 Catania, Italy
§Institute for Biomembranes and Biosystems, Rome, Italy
1To whom correspondence should be addressed, at Department of Chemical Sciences, University of Catania, v.le A. Doria, 6, IT-95125 Catania, Italia (email vdpbiofa@unict.it).
Search for other works by this author on:
Angela Messina;
Angela Messina
‡Department of Chemistry, Laboratory of Biochemistry and Molecular Biology, University of Catania, IT-95125 Catania, Italy
§Institute for Biomembranes and Biosystems, Rome, Italy
Search for other works by this author on:
Vincenzo Cunsolo;
Vincenzo Cunsolo
‡Department of Chemistry, Laboratory of Biochemistry and Molecular Biology, University of Catania, IT-95125 Catania, Italy
Search for other works by this author on:
Rosaria Saletti;
Rosaria Saletti
‡Department of Chemistry, Laboratory of Biochemistry and Molecular Biology, University of Catania, IT-95125 Catania, Italy
Search for other works by this author on:
Klaus-Dieter Hinsch;
Klaus-Dieter Hinsch
*Center of Dermatology and Andrology, Justus Liebig University of Giessen, Gaffkystrasse 14, DE-35392, Germany
∥Borkum Riff Rehabilitation Clinic of the Federal Insurance for Salaried Employees' Institution (DRV-Bund), DE-26757 Borkum, Germany
Search for other works by this author on:
Elvira Hinsch
Elvira Hinsch
*Center of Dermatology and Andrology, Justus Liebig University of Giessen, Gaffkystrasse 14, DE-35392, Germany
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
August 29 2008
Revision Received:
October 17 2008
Accepted:
October 31 2008
Accepted Manuscript online:
October 31 2008
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biosci Rep (2009) 29 (6): 351–362.
Article history
Received:
August 29 2008
Revision Received:
October 17 2008
Accepted:
October 31 2008
Accepted Manuscript online:
October 31 2008
Citation
Viviana A. Menzel, M. Carolina Cassará, Roland Benz, Vito De Pinto, Angela Messina, Vincenzo Cunsolo, Rosaria Saletti, Klaus-Dieter Hinsch, Elvira Hinsch; Molecular and functional characterization of VDAC2 purified from mammal spermatozoa. Biosci Rep 1 December 2009; 29 (6): 351–362. doi: https://doi.org/10.1042/BSR20080123
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.