Junko Ishii

Determination of ion permeability through the channels made of porins from the outer membrane ofSalmonella typhimurium in lipid bilayer membranes

SummaryThe three types of porin (matrix-proteins) fromSalmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10−12m), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1m KCl. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins fromSalmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris+, glucosamine+, Hepes−_ a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1m KCl (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single condutance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.

Lipopolysaccharide promoted opening of the porin channel

We show here that the imipenem (a carbapenem, β‐lactam antibiotic)‐permeable porin channels (protein D2 or OprD2) of Pseudomonas aeruginosa were closed mostly in the lipopolysaccharide (LPS)‐free membrane and were openable by adding LPS to the membrane as assayed by ion conductivity measurements using planar lipid bilayers. Open and closed states of the OprD2 channels exhibited conductivities of about 400 and 30 pS, respectively, in 1 M NaCl. The OprD2 channel in the LPS‐containing membrane showed very rapid opening and closing events in a second order and the duration of closure became longer at low membrane potentials.

Characterization of lamB protein from the outer membrane of Escherichia coli that forms diffusion pores selective for maltose-maltodextrins

The outer membrane of Escherichia coli harbors 2 kinds of transport proteins that facilitate the diffusion of maltose-maltodextrins (Glc,), namely porins forming general diffusion pores for up to Glc3 [ 1,2], and h-receptor proteins or 1amB proteins for up to Glc,j [2,3]. The role of ZamB-protein oligomers in the formation of Glc,-selective pores was demonstrated in vitro using liposomes containing ZamB proteins [2,4]. To understand the molecular mechanism of solute selectivity by ZamB protein, it is essential to analyze the higher structure of ZamB-protein oligomers. This communication reports the subunit structure of pore-forming ZamB proteins; the relative molecular masses (M,) of oligomers and monomers, and some physicochemical properties of ZamB proteins. was determined from the dry weight of proteins and their UV-absorption profile in a solution of 0.3 M NaCl, O&25% SDS, and 10 mM Tris-HCl (pH 7.0). Purified ZamB proteins (16 mg) were dialyzed exhaustively against a large excess of 3 mM NaNs, lyophilized and dried over PZ05 for a week. The amount of SDS bound to the dialyzed proteins was <2% (w/w) of the weight of protein. Sedimentation equilibrium measurements and circular dichroism spectroscopy were described in [6]. C12Es, DzO, SDS and guanidine-HCl were purchased from Nikko Chemicals, Merck, BioRad and Nakarai Chemicals, respectively. TSK-gel G3000SW was obtained from Tokyo Soda.

Specific interaction of the protein-D2 porin of Pseudomonas aeruginosa with antibiotics

The protein-D2 porin of Pseudomonas aeruginosa is lacking in carbapenem or fluoroquinolone-resistant strains and hence was thought to facilitate the diffusion of these antibiotics. We examined the effect of several antibiotics on the single channel conductivity of protein-D2 in planar lipid bilayers and found that fluoroquinolones and carbapenems at concentrations of around 1 mM caused closure of the protein-D2 channel. Tetracycline, ampicillin, piperacillin, and latamoxef did not exert any detectable effect on the protein-D2 channel activity.

Modification of the porin function by the membrane components used for the reconstitution of model membranes

Diffusion rate of the cationic solute through the porin pore was five times higher in the vesicles made of acidic phospholipids than in that of zwitterionic phospholipids. In contrast, diffusion of the anionic solute through these vesicles was reciprocally reversed. Diffusion ofpara-nitrophenyl phosphate was enhanced in the presence of lipopolysaccharide and the phospholipids extracted from bacterial cells as compared with the diffusion through porin in phosphatidyl choline.

A simple method for determining the outer membrane permeability of gram-negative bacteria in intact cells

Abstract The permeability of the intact outer membrane of gram-negative bacteria was determined by weighing the centrifuged pellets of cells treated with hypertonic solutes [10]. The present study refined the existing procedures by focusing on the factors that may affect the results. The study revealed the following findinds: ( i ) The weight of cells treated with outer membrane-impermeable hypertonic saccharide was significantly reduced and the reduction was proportional to the solute osmolarity. On the other hand, the weight of cells treated with outer membrane-permeable hypertonic saccharide was unchanges. ( ii ) The centrifugal force up to 165,000 × g for 60 s or NaCl up to 150 mM produced no detectable influence on the assay. ( iii ) It was possible to measure the outer membrane permeability even in the hypotonic test solutes using plasmolyzed cells. ( iv ) This method proved equally applicable for measuring the outer membrane permeabilities of both smooth and rough strains. ( v ) Application of this technique to several species of gram-negative bacteria confirmed the previously reported exclusion limits. All these data show that the weighing of centrifuged pellets of hypertonic solute-treated cells is a useful method for determining the outer membrane permeability of gram-negative bacteria.