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At least 19 recordsLinked to original sources
PubMed1989

[Identification of human porins. II. Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL)].

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.

Amino Acid Sequence↗
PubMed1989

[Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell].

We describe for the first time a porin (Porin 31HL) on the plasmalemm of an eukaryontic cell line, where porins have been found only on the outer mitochondrial membranes. The expression of the porin on the plasmalemm of transformed human B-lymphocytes is demonstrated by cytotoxicity- and indirect immunofluorescence techniques with living and fixed cells. The rabbit xenoantisera used were directed against purified Porin 31HL and free or acetylated synthetic peptides of its nineteen N-terminal amino acids. The three-step purification procedure for Porin 31HL started from a total membrane fraction of the B-cell line, followed by ion-exchange chromatography on CM- and DEAE-cellulose and a final gel filtration in SDS on Sephacryl S-300.

Amino Acids↗
PubMed1992

Studies on human porin. VII. The channel properties of the human B-lymphocyte membrane-derived "Porin 31HL" are similar to those of mitochondrial porins.

Porin 31HL was isolated and purified from total membrane preparations of a human B-lymphocyte cell line. The protein showed a single band of apparent molecular mass 31 kDa on SDS-PAGE. Reconstitution of the protein into artificial lipid bilayer membranes defines its function as a channel-forming protein. The distribution of single-channel conductances had two maxima of 2.4 and 4.3 nS in 1M KCl. The channel formed by Porin 31HL of human B-lymphocytes was found to be voltage-dependent and switched to ion-permeable substates at membrane voltage larger than 20mV. In the open state the pore exhibited the characteristics of a general diffusion pore because the mobility sequence of the ions inside the pore was similar to that in the bulk aqueous phase. The effective diameter was estimated to be about 1.7 nm. The properties of the low conductance state of the channel were studied in detail. In this state the pore favored the passage of cations, in contrast to the open state which favored anions slightly. Monoclonal antibodies against the N-terminal end of Porin 31HL blocked its reconstitution but had otherwise no influence on the channel properties. This result suggested that the amphipathic alpha-helical structure at the N-terminal end is probably not involved in channel gating. The channel-forming properties of Porin 31HL were compared to those of porins isolated from mitochondrial outer membranes and to those of the "maxi chloride channel" observed in the cytoplasmic membrane of several eukaryotic cells.

Animals↗
PubMed1994

Studies on human porin. XII. Eight monoclonal mouse anti-"porin 31HL" antibodies discriminate type 1 and type 2 mammalian porin channels/VDACs in western blotting and enzyme-linked immunosorbent assays.

Eight mouse monoclonal antibodies directed against the acetylated N-terminal part of the type 1 human VDAC Porin 31HL clearly discriminate type 1 and type 2 mammalian porin channels. This is shown by comparing synthetic N-terminal peptides of either channel type in Western dot blots or by ELISA. The data support the specificity of the anti-Porin 31HL antibodies and thus give further support to our recent observations on extramitochondrial expression of VDAC. In the plasmalemma of different mammalian cells VDAC forms part of an ubiquitous chloride channel complex, which in patch clamp measurements may figure as the outwardly rectifying depolarization-induced chloride channel that is affected in cystic fibrosis.

Amino Acid Sequence↗
PubMed1991

Studies on human porin. IV. The primary structures of "Porin 31HM" purified from human skeletal muscle membranes and of "Porin 31HL" derived from human B lymphocyte membranes are identical.

We report on the purification of "Porin 31HM" from the crude plasma membrane fraction of human skeletal muscle. Furthermore, all tryptic peptides of the molecule were purified and characterized by different methods. The alignment of the peptides with the complete primary structure of the human B lymphocyte plasma membrane-derived "Porin 31HL", published by us recently (Kayser, H. et al. (1989) this Journal 370, 1265-1278), proved both structures to be completely identical. Our data demonstrate that porin fractions from crude plasma membranes of different human cell types do not show any variation on the primary structure level.

Amino Acid Sequence↗
PubMed1994

Porins from plants. Molecular cloning and functional characterization of two new members of the porin family.

Porins are voltage-gated diffusion pores found in all eukaryotic kingdoms. Here we describe, for the first time, the identification and characterization of two cDNAs encoding porins from plants. Peptide sequences obtained from a 30-kDa protein of envelope membranes from pea root plastids allowed the isolation of two cDNA clones from pea and maize. On the protein level, both proteins are homologous by 58%. Sequence comparison against the Swiss-Prot sequence data base revealed a homology of about 25% to mitochondrial porins from fungi and human. Computer-aided predictions of the secondary structure of the plant porins revealed the presence of 16 antiparallel beta-strands that are also found in mitochondrial porins. Porins from non-green plastids and from the outer mitochondrial membrane were reconstituted into planar lipid bilayers. The proteins showed high pore-forming activities and similar single-channel conductances. In vitro translated porin was preferentially imported only into non-green plastids but not into chloroplasts. To our knowledge, this is the first example of selective import of a plastid protein into different types of plastids. This finding is in line with the observation that an immunoreactive 30-kDa band was only found in non-green plastids and mitochondria but not in chloroplasts. We conclude that mitochondria and non-green plastids possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin.

Amino Acid Sequence↗
PubMed1993

A mitochondrial porin cDNA predicts the existence of multiple human porins.

Pores formed in the outer membrane of mitochondria by mitochondrial porin make it permeable to water-soluble metabolites smaller than approximately 5 kDa. We have isolated a full-length cDNA encoding a human porin. This probe detects a single approximately 1.5-kilobase mRNA species on Northern blots, but multiple hybridizing bands on genomic Southern blots. The open reading frame predicts a 38.1-kDa protein with a pI of 6.59 that is homologous but not identical to a previously reported protein sequence of a 31-kDa porin isolated from human lymphocytes (porin 31HL). The most striking difference between the two porins is that the sequence predicted by the cDNA is longer than the 31HL porin by 27 amino acids at the amino terminus and 38 amino acids at the carboxyl terminus. The porin cDNA directs the in vitro translation of two protein species of approximately 32 and approximately 36 kDa, which appear to result from alternate usage of AUG initiation codons. The 32-kDa protein is the predominant species imported into both rat and yeast mitochondria in vitro. Taken together, these results suggest that multiple porin proteins can be expressed in humans. Additionally, a porin consensus protein sequence has been identified that is conserved in eukaryotic organisms as diverse as yeast and man.

Amino Acid Sequence↗
PubMed1991

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Götz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.

Animals↗
PubMed1993

Studies on human porin. IX. Immunolocalization of porin and CFTR channels in human surface respiratory epithelium.

The expression of the voltage-dependent anion channel (VDAC) "Porin 31 HL" and its cellular and subcellular immunocytochemical localization in the human respiratory epithelium were studied with monoclonal and polyclonal antibodies using immunofluorescence and immunogold labelling with light (LM) and transmission electron microscopy (TEM), respectively. Porin was identified in the apical domain of the ciliated cells and in the basal cells of the respiratory epithelium. Immunogold labelling was present in the apical plasma membrane and subapical vesicles of the ciliated cells. In pre-embedded freshly dissociated surface epithelial cells, porin could also be identified with TEM at the outer part of the plasma membrane of basal cells. By LM double immunolabelling, both porin and cystic fibrosis transmembrane conductance regulator (CFTR) were identified in the apical domain of ciliated cells but not in basal cells where CFTR was never identified. On Western blots of solubilized total membrane protein preparations from the same frozen surface epithelial respiratory cells, the antibodies recognized a group of 3 proteins of 31, 60 and 130-140 kDa with a strong reactivity for a 31 kDa protein, corresponding to the porin and a protein of 170 kDa which is consistent with mature CFTR. These results suggest that porin might be part of a multi-component chloride channel complex which could interact with CFTR.

Antibodies↗
PubMed1994

Refined structure of the porin from Rhodopseudomonas blastica. Comparison with the porin from Rhodobacter capsulatus.

The structure of the membrane channel porin from the phototrophic bacteria Rhodopseudomonas blastica has been refined at 1.96 A resolution yielding an R-factor of 17.6%. The final model consists of all 289 amino acid residues, 247 water molecules and three detergent molecules modelled as n-octyltetraoxyethylene. One of these detergent molecules binds together with its two symmetry-related molecules tightly in a pocket at the molecular 3-fold axis. This pocket may bind three alkyl chains of a lipopolysaccharide which in turn would stabilize the trimer and could possibly play a role in membrane insertion. The overall shape of this porin resembles OmpF of Escherichia coli more than the only known sequence-related porin from Rhodobacter capsulatus. The membrane contacting surface is similar in all structurally known porins; it shows exceptional frequencies of amino acid residues and side-chain rotamers. The 46-residue loop beta 5-beta 6 of the porin is shown to be tightly fastened to the beta-barrel, excluding an in vivo loop movement that closes the pore. The trimer interface region has the structure of a water-soluble protein with an extensive non-polar core and numerous hydrogen bonds at the surface. The loops at the external end of the barrel are long and rigid whereas those at the periplasmic barrel end are short and mobile. The crystal packing is discussed.

Amino Acid Sequence↗
PubMed1991

Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.

Amino Acid Sequence↗
PubMed1992

Studies on human porin. VIII. Expression of "Porin 31HL" channels in the plasmalemma of the acute-lymphoblastic-leukemia cell line KM3 as revealed by light- and electron-microscopy.

First electron microscopy data on the expression of the VDAC "Porin 31HL" in the plasmalemma of a eucaryotic cell are presented. In a light and electron microscopic study we demonstrate the expression of the porin channel in the outer cell membrane of the pre-B lymphocyte type acute-lymphoblastic-leukemia cell line KM3. Monoclonal mouse anti-"Porin 31HL" antibodies were applied in indirect immunofluorescence or immunogold labelling experiments. The results confirm our early topological data on the expression of porin channels in the cytoplasmic membrane of different human cell types as revealed on the light microscopy level. Interestingly, only a pre-embedding immunoreaction approach was successful for gold particle labelling of the plasmalemma. This again is in agreement with our recent data on the accessibility of the acetylated N-terminal part of "Porin 31HL" molecules on the outer cell surface.

Antibodies, Monoclonal↗
PubMed1987

Polyclonal activation of human peripheral blood lymphocytes by bacterial porins and defined porin fragments.

Bacterial porins were isolated from Escherichia coli B and Salmonella typhimurium S 1135. The proteins were cleaved either by cyanogen bromide treatment or by enzymatic digestion into a variety of small fragments, and the compounds were characterized by SDS-polyacrylamide gel electrophoresis. Both the porins and the porin fragments constituted potent mitogens for human peripheral blood lymphocytes, comparable to the human B-lymphocyte activator pokeweed mitogen. In the cultures, B-lymphocytes were stimulated into immunoglobulin production, as measured by ELISA. In all experiments, the activity of the mitogens extracted from S. typhimurium was superior to that of the compound isolated from E. coli B. The well-defined porins constitute valuable tools for investigating the molecular mechanism of human lymphocyte activation.

Bacterial Outer Membrane Proteins↗
PubMed1991

Prediction of the general structure of OmpF and PhoE from the sequence and structure of porin from Rhodobacter capsulatus. Orientation of porin in the membrane.

By comparing the hydrophilicity profiles and sequences of porin from Rhodobacter capsulatus with those of OmpF and PhoE from Escherichia coli, a set of insertions and deletions for alignment of the sequences has been deduced. With this alignment a similar folding of OmpF and PhoE has been predicted as found by X-ray structure analysis of porin from Rhodobacter capsulates. Furthermore, the orientation of the porin trimer in the outer membrane was inferred from topological data on PhoE. According to this result a single channel of approx. 30 A diameter starts at the outer surface. Near the middle of the outer membrane bilayer this channel branches out into three separate channels, each running within a single porin monomer to the periplasmic surface.

Amino Acid Sequence↗
PubMed1990

Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis?

"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle.

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL. In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC "Porin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin. Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines. VDAC in the plasmalemma is discussed as the molecular basis of the Blatz and Magleby channel.

Animals↗
PubMed1994

On the structure of mitochondrial porins and its homologies with bacterial porins.

By use of computer modelling, we have predicted a model of 16 transmembrane beta-strands for mitochondrial porins structure from human, Saccharomyces cerevisiae, Neurospora crassa and Dictyostelium discoideum. The proposed model takes into account biochemical and immunological data reported in the literature, as well as electrophysiological results obtained with yeast mitochondrial porins with mutations at selected amino acids. The predicted structure is very similar to that of some bacterial porins, as apparent from the homology of their hydropathic profiles.

Amino Acid Sequence↗
PubMed1995

Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: construction of mutants with deletions in the multiple porin genes.

We inserted deletions in the chromosomal genes of Pseudomonas aeruginosa coded for the outer membrane porins, proteins C, D2, or E1, and all possible combinations of these proteins by the gene replacement technique and selecting for imipenem-resistance. Determination of the minimum inhibitory concentrations of beta-lactams, fluoroquinolones, chloramphenicol and gentamicin in these mutants revealed that most mutants showed equal susceptibility to the porin-sufficient strain. The only exception was that imipenem and meropenem showed increased minimum inhibitory concentrations in all of the mutants lacking protein D2. These results firmly established that the P. aeruginosa porins identified so far form the pores do not accommodate the passage of most antipseudomonal antibiotics, with the exception of carbapenems.

Bacterial Outer Membrane Proteins↗