A simple and rapid method for the purification of the mitochondrial porin from mammalian tissues.

A new, simple and rapid procedure for the purification of high amounts of mitochondrial porins from different tissues of mammalia is described. The method consists in a single step hydroxyapatite/celite chromatography of Triton X-100 solubilized mitochondrial membranes. For optimal purification several factors are critical such as the absence of salts, a low protein/detergent ratio and an exact hydroxyapatite/celite ratio of 2:1.

Porin pores of mitochondrial outer membranes from high and low eukaryotic cells: biochemical and biophysical characterization.

The mitochondrial porins from mammalian tissues and from low eukaryotic cells were purified with a high yield, and their biochemical and functional properties were investigated. When analyzed by SDS gel electrophoresis, all mammalian porins show a very similar apparent molecular mass (35-35.5 kDa). In contrast yeast and Paramecium porins have a molecular mass of 30 and 37 kDa, respectively. The peptide maps of mammalian porins are very similar although small differences are apparent between porins of different tissues of the same organism and also between those of the same tissue of different organisms. The peptide patterns of porins from yeast and Paramecium are completely different from those of mammalian porins. Antibodies raised against the rat liver porin cross-react with all the other mammalian porins but not with that of yeast. The incorporation of porins into artificial lipid bilayer membranes showed that they are able to form pores with approximately the same specific activity. The single-channel conductance is for all porins, except for that of Paramecium, about 4 nS in 1 M KCl, corresponding to an effective pore diameter of 1.7 nm. They are voltage-dependent and switch to substates at transmembrane potentials higher than 10 mV. The number of gating charges varies, however, for pores from different tissues, indicating a different sensitivity to the potential as a result of a possible different function.

Pore formation by the mitochondrial porin of rat brain in lipid bilayer membranes.

The porin of the outer membrane of rat-brain mitochondria was isolated and purified. The protein showed a single band of apparent Mr 35,500 on dodecyl sulfate-containing polyacrylamide gels. The incorporation of rat-brain porin into artificial lipid bilayer membranes showed that it is able to form pores with an average single-channel conductance of 400 pS in 0.1 M KCI. The pores were found to be voltage-dependent and switched to substrates at higher transmembrane potentials. The voltage-dependence of the rat brain pore was considerably smaller than that of the other known eukaryotic porins. The possible role of the rat-brain porin in the regulation of transport process across the outer mitochondrial membrane is discussed.

Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla. Two porins in mitochondria?

A fast purification procedure for the isolation and purification of eukaryotic porin (De Pinto et al., (1987) Biochim. Biophys. Acta 905, 499-502) was applied to liver mitochondria of the fish Anguilla anguilla. A protein preparation was obtained which formed slightly anionically selective pores in reconstitution experiments with lipid bilayer membranes. The distribution of single-channel conductances had two maxima of 2.4 nS and 4.0 nS in 1 M KCl. Sodium dodecylsulfate electrophoretograms of the protein preparation showed the presence of two bands of very similar electrophoretic mobility (32 and 32.5 kDa). Both bands cross-reacted with antibodies raised against purified bovine heart porin and with antibodies raised against the 19 amino acids N-terminal end of human porin. No cross-reactivity was observed with antibodies against yeast porin. The peptide maps of the two bands showed slight differences. The possibility of the presence of two different porins in liver mitochondria of Anguilla anguilla is discussed. An extensive immunological comparison of different mitochondrial porins is presented.

Characterization of the mitochondrial porin from Drosophila melanogaster.

Mitochondrial porin was isolated from the fruit fly Drosophila melanogaster at different developmental stages, starting from whole mitochondria. The porin from adults' mitochondria was fully characterized. The protein had a molecular mass of 31 kDa as judged from sodium dodecylsulfate electrophoretograms. It was very resistive against digestion with V8 proteinase of Staphylococcus aureus and a larger number of fragments were only obtained after digestion with papain. Drosophila porin showed little interaction with antibodies raised against mitochondrial porins from mammalia and Neurospora crassa, but a strong reactivity with antibodies raised against yeast porin. Reconstitution experiments with planar lipid bilayer membranes showed that the protein was able to form ion-permeable pores with a single-channel conductance of 0.41 nS in 0.1 M KCl. At low transmembrane voltages Drosophila porin had the properties of a general diffusion pore with an estimated effective diameter of about 1.7 nm and a small selectivity for anions over cations. Voltages larger than 20 to 30 mV resulted in a closure of the pore. The closed states of the pore were found to be cation-selective. The addition of a synthetic polyanion to the aqueous phase on one side of the membrane resulted in an asymmetric shift of the voltage dependence and the pore became already closed at very small voltages negative at the cis-side (the side of the addition of the polyanion).

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin.

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the Mr region of 30 000-35 000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent Mr 35 000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.

Purification and Characterization of Porin from Corn (Zea mays L.) Mitochondria.

Mitochondrial porin from corn (Zea mays L. B 73) shoots was solubilized with lauryl(dimethyl)-amine oxide and purified by chromatography on a hydroxyapatite:celite column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein had an apparent molecular mass of 35 kD. When reconstituted in planar lipid bilayer membranes the porin formed ion-permeable channels with single-channel conductance of 2.0 and 4.0 nanosiemens in 1 M KCl. At low transmembrane voltages corn porin had the properties of a general diffusion pore with an estimated effective diameter of 1.6 nm and a small selectivity for anions over cations. The primary structure of corn porin seems to be quite different from that of other mitochondrial porins, because it did not cross-react with monoclonal antibodies against human porin and with polyclonal antibodies against yeast porin. Furthermore, the peptide maps of corn and bovine heart porins were very different. A sequence of 21 amino acids obtained by Edman degradation of peptides generated by porin proteolysis with Staphylococcus aureus V8 protease did not show any significant homology with known sequences of mitochondrial porins. Results of our investigation suggest that corn porin possesses functional properties similar to those of other mitochondrial porins, despite major structural differences.

Prediction of the transmembrane regions of beta-barrel membrane proteins with a neural network-based predictor.

A method based on neural networks is trained and tested on a nonredundant set of beta-barrel membrane proteins known at atomic resolution with a jackknife procedure. The method predicts the topography of transmembrane beta strands with residue accuracy as high as 78% when evolutionary information is used as input to the network. Of the transmembrane beta-strands included in the training set, 93% are correctly assigned. The predictor includes an algorithm of model optimization, based on dynamic programming, that correctly models eight out of the 11 proteins present in the training/testing set. In addition, protein topology is assigned on the basis of the location of the longest loops in the models. We propose this as a general method to fill the gap of the prediction of beta-barrel membrane proteins.

The mitochondrial permeability transition pore may comprise VDAC molecules. II. The electrophysiological properties of VDAC are compatible with those of the mitochondrial megachannel.

The electrophysiological properties of isolated mitochondrial porin (VDAC), reconstituted in planar bilayers or proteoliposomes, resemble those of the mitochondrial megachannel believed to be the permeability transition pore. In particular, a correspondence was found with regard to the voltage dependence: VDAC was driven to closed states by potentials of either sign, but the effect was not symmetrical; voltages negative in the compartment to which VDAC was added were more effective. The results are consistent with the hypothesis that the PTP may consist of two cooperating VDAC channels, plus presumably an adenine nucleotide carrier dimer and a third component known to be part of the mitochondrial benzodiazepine receptor.

Positive residues involved in the voltage-gating of the mitochondrial porin-channel are localized in the external moiety of the pore.

The role of positive charges located on the hydrophilic surface of the mitochondrial outer membrane channel was investigated by studying the interaction between LDAO-solubilized porin and a cation-exchanger column. The binding of porin to the column material was inhibited when the elution buffer had a pH of 9 or when 2 mM dextran sulfate was added to the buffer at neutral pH. Interestingly, the addition of a synthetic copolymer of methacrylate, maleate and styrene known as a potent modulator of the voltage-dependence, did not influence the interaction between column material and porin. Incubation of porin with fluorescein isothiocyanate (FITC) resulted in the isolation of a porin fraction in which on average two lysines located on the surface of the pore-forming complex per 35 kDa polypeptide were modified. The voltage-dependence of the fluorescein isothiocyanate modified porin was strongly decreased as compared with the unmodified porin. The experiments presented here give the first biochemical evidence that positively charged lysine residues located on the surface of the channel-forming complex are responsible for the gating of the mitochondrial porin-channel.

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse.

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9-13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3' untranslated sequence (3'-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5'-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5'-UTR sequence, demonstrated to be transcribed by 5'-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2-1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.

Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster.

We have raised polyclonal antibodies against purified the Drosphila melanogaster mitochondrial porin. They showed high titre and specificity and were thus used as a tool for screening an expression library. The isolated clone 1T1 showed 74% sequence identity in the last 19 residues at the C-terminus of human porin. A subclone of 1T1, containing the porin-like sequence, was thus used as a probe for re-screening a cDNA library and several positive clones were plaque-purified. We present here the sequence of a 1363 bp cDNA encoding a protein of 279 amino acids. Its identity with porin was also confirmed by N-terminal Edman degradation of the purified protein. The D. melanogaster porin shows an overall 51.8% identity with human porin isoform 1 (porin 31HL or HVDAC1) and an overall 55.7% identity with human porin isoform 2 (HVDAC2). Hydrophobicity plots and secondary structure predictions showed a very high similarity with data obtained from known porin sequences. The D. melanogaster porin cDNA was used as a probe for in situ hybridization to polytenic salivar gland chromosomes. It hybridizes with different intensities in two sites, in chromosome 2L, at region 31E and in chromosome 3L at region 79D. Thus, also in Drosophila melanogaster porin polypeptide(s) belong(s) to a multigene family.

Characterization of channel-forming activity in muscle biopsy from a porin-deficient human patient.

A bioptic specimen from the muscles of a patient suffering from severe myopathy was inspected for the presence of human porin 31HL. Western blotting suggested that the specimen was free of the most abundant eukaryotic porin 31HL (HVDAC1). The specimen was treated with detergent and the soluble protein fraction was passed through a dry hydroxyapatite column. The passthrough of this column was inspected for channel formation in artificial lipid-bilayer membranes. The channel observed under these conditions had a single-channel conductance of about 2.5 nS in 1 M KCl, was cation selective, and was found to be virtually voltage independent. Experiments with a control specimen from a healthy human being, without any indication for muscle myopathy, revealed the presence of the voltage-dependent porin 31HL in the sample. It is discussed whether the patient's bioptic specimen contained another human porin, which has not been studied to date in its natural environment.

Presence of a voltage-dependent anion channel 1 in the rat postsynaptic density fraction.

Little is known about the molecular organization and functions of the postsynaptic density (PSD), a cytoskeletal specialization on the postsynaptic membrane. In an attempt to elucidate the protein composition of PSD, we have sequenced a 35 kDa protein of the rat forebrain PSD fraction. Amino acid sequence information of the tryptic peptides and immunoblot analyses revealed that the protein is a voltage-dependent anion channel 1 (VDAC1). VDAC1 was enriched in the PSD fraction and was partially soluble in 1% n-octyl glucoside (NOG) or Triton X-100. Our data indicate that VDAC1, which is originally found in the outer mitochondrial membrane, is also present in the central nervous system (CNS) synapses in association with the PSD 'core'.

Bilateral hip disarticulation in paraplegics with decubitus ulcers.

A small percentage of paraplegic patients develop chronic decubitus ulcers that are unresponsive to the usual plastic surgical maneuvers. We used anatomic and nonanatomic (filleting) approaches to hip disarticulation in three patients with severe chronic cavernous decubitus ulcers. All patients were rehabilitated to wheelchair ambulation, with subsequent healing of the operative sites. This type of therapy might be considered in paraplegics with less compelling reasons for amputation, because of the associated rehabilitation potential.

VDAC1 selectively transfers apoptotic Ca2+ signals to mitochondria.

Voltage-dependent anion channels (VDACs) are expressed in three isoforms, with common channeling properties and different roles in cell survival. We show that VDAC1 silencing potentiates apoptotic challenges, whereas VDAC2 has the opposite effect. Although all three VDAC isoforms are equivalent in allowing mitochondrial Ca(2+) loading upon agonist stimulation, VDAC1 silencing selectively impairs the transfer of the low-amplitude apoptotic Ca(2+) signals. Co-immunoprecipitation experiments show that VDAC1, but not VDAC2 and VDAC3, forms complexes with IP(3) receptors, an interaction that is further strengthened by apoptotic stimuli. These data highlight a non-redundant molecular route for transferring Ca(2+) signals to mitochondria in apoptosis.

Filters for the reduction of baseline wander and muscle artifact in the ECG.

Contamination of the electrocardiographic (ECG) signal by extraneous electrical potentials is a significant problem in exercise electrocardiography. Baseline wander and muscle artifact are particularly troublesome sources of interference. This paper describes two digital filters that were constructed and found effective in reducing these two types of signal contamination. The baseline wander filter presented here is a linear phase high-pass filter having a cutoff frequency lower than the heart rate. The low computational overhead makes this filter practical for multiple channel implementation on a low-cost digital signal processor. Because the muscle artifact signal, generated by skeletal muscle activity, occupies the same spectrum space as the heart-generated signal, spectral filtering is not very effective in eliminating it. Described here is a time-varying filter that employs a combination of linear and nonlinear filtering techniques.

Purification and properties of the voltage-dependent anion channel of the outer mitochondrial membrane.

The methods for the purification of functionally active mitochondrial porin or voltage-dependent anion channel of the outer mitochondrial membrane are critically evaluated. Two rapid and efficient methods are now available. Both make use of a hydroxyapatite/celite column as a single chromatographic step. However, in one method with long polar head-group detergents, porin passes through the column, whereas in the other method, with shorter polar head-group detergents, porin is first bound to the column and then eluted by the addition of salts. On the basis of these results, a model for the arrangement of porin in the detergent-protein micelles is proposed.

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC).

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided beta-strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 A resolution. In mitochondrial porins the N-terminal region forms an amphipathic alpha-helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.