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Biomedical subjects

T Nakae

Publications and source records attributed to T Nakae.

At least 127 records · Page 7Linked to original sources
PubMed1983

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.

Bacterial Outer Membrane Proteins↗
PubMed1982

Estimation of molecular weights of membrane proteins in the presence of SDS by low-angle laser light scattering combined with high-performance porous silica gel chromatography. Confirmation of the trimer structure of porin of the E. coli outer membrane.

An assessment study was carried out to evaluate the performance of the low-angle laser light-scattering technique combined with high-performance porous silica gel chromatography in the presence of sodium dodecyl sulfate and precision differential refractometry. It was found that the combined technique is highly promising as a reliable method for determining the molecular weight of a membrane protein solubilized by the surfactant. As a test, molecular weights of porin forming the permeability channel of the outer membrane of E. coli B in an oligomeric form were measured before and after heat treatment, which is known to cause dissociation. The results obtained indicate that the porin oligomer is a trimer with stoichiometric composition.

Chromatography, High Pressure Liquid↗
PubMed1982

Diffusion of aminoglycoside antibiotics across the outer membrane of Escherichia coli.

The diffusion of aminoglycoside antibiotics (gentamicin, kanamycin, streptomycin, fradiomycin, lividomycin, and mannosylparomomycin) through porin pores was examined in vitro by the liposome swelling technique, using vesicle membranes reconstituted from phospholipids and purified porin trimers. Results showed that aminoglycoside antibiotics diffuse through porin-pores very efficiently, as rapidly as hexoses and disaccharides, despite the fact that the molecular weights of the aminoglycosides used were higher than or close to the exclusion limit of porin pores. The susceptibility to aminoglycoside antibiotics of mutant strains producing 3 to 4% of porin was not significantly different from that of a strain producing a wild-type quantity of porin. These results were interpreted to mean that aminoglycoside antibiotics diffuse through porin-pores very efficiently. Therefore, the diffusion of these drugs through the mutant outer membranes producing 3 to 4% of porin is not a rate-limiting step for aminoglycoside diffusion and its action.

Aminoglycosides↗
PubMed1982

Diffusion of beta-lactam antibiotics through liposome membranes containing purified porins.

A method to determine the diffusion of cephalosporins through porin pores in vitro was developed, using liposomes reconstituted from phospholipids, lipopolysaccharides, and purified porin trimers. With this method, the roles of several species of porin pores from Escherichia coli and Salmonella typhimurium in the diffusion of cephalexin, cephaloridine, and cephalothin were examined. Results clearly showed that porins from E. coli B and 39,000-molecular-weight porins from S. typhimurium formed the most efficient pores. Thus, these were considered to represent a single functional group. OmpF and OmpE porins of E. coli K-12 and 38,000-molecular-weight porins of S. typhimurium formed moderately efficient pores. OmpC porins of E. coli K-12 and 40,000-molecular-weight porins of S. typhimurium were the least efficient pore formers. The present method can be used to distinguish the role of individual porin pores in the diffusion of cephalosporins.

Anti-Bacterial Agents↗
PubMed1982

The solute selectivity of porin pores of Escherichia coli and Salmonella typhimurium.

The solute selectivity of porin pores of Escherichia coli and Salmonella typhimurium was examined using vesicle membranes reconstituted from phospholipids and purified porin trimers. OmpE- and 34K-porin-pores allowed a preferential diffusion of phosphorylated and/or negatively charged compounds. They allowed the diffusion of positively charged solutes reasonably well. OmpF- and 35K-porin-pores favored the diffusion of positively charged solutes a little more than negatively charged solutes. OmpC- and 36K-porin-pores showed poor diffusibility to all solute tested. B-porins from E. coli B formed highly efficient pores for the diffusion of both positively and negatively charged solutes. On the basis of these observations, possible mechanisms of solute selectivity were discussed.

Cell Membrane↗
PubMed1982

Molecular weights and subunit structure of LamB proteins.

Phage lambda-receptor proteins of Escherichia coli, LamB proteins, form oligomeric aggregates to build transmembrane diffusion pores selective for maltose and maltodextrins. The molecular weights (MW) of functional oligomers as well as dissociated monomers were determined by sedimentation equilibrium analysis in homogeneous non-ionic surfactant and deuterium oxide and in 6 M guanidine-HCl, respectively. The MW of oligomers and monomers appeared as 135 600 and 45 900, respectively. Thus, functional Lamb proteins consisted of three identical subunits.

Bacterial Outer Membrane Proteins↗
PubMed1982

Effects of cations on the outer membrane permeability of Escherichia coli.

The outer membrane permeability to beta-lactam antibiotics was determined with intact Escherichia coli cells treated with various concentrations of NaCl. It was found that the treatment of cells with moderate concentrations of NaCl caused dramatic increase in the diffusion rate of beta-lactam across the outer membrane. This increased rate of beta-lactam diffusion was not due to the high osmolarity exerted by NaCl, since the elevated osmolarity of the assay medium by sucrose had no significant effect. This increased rate of beta-lactam diffusion was restored by addition of a low concentration of MgSO4 or CaCl2 into the assay medium. These results were interpreted as that the treatment of intact E. coli cells with moderate concentration of monovalent cations squeezed out divalent cations, which presumably destabilized the outer membranes, causing electrostatic repulsion of negatively charged lipopolysaccharide molecules in site.

Ampicillin↗
PubMed1982

The use of the laser light scattering for the molecular weight determination of membrane proteins in surfactants.

A method is described for the determination of the molecular weight of the protein moiety of protein surfactant complexes using of low angle laser light (633 nm) scattering in combination with high performance porous silica gel chromatography, precision differential refractometry and differential UV absorption was described. The calibration curves in sodium dodecyl sulfate (SDS) and in a non-ionic surfactant, octaethyleneglycol dodecyl ether, using several reference proteins yielded linear lines. Molecular weight of porin oligomers and monomers, an intrinsic membrance protein that forms the permeability channel in the outer membrane of Escherichia coli, were calculated and were found to be 109,000 and 36,300, respectively in SDS and that of oligomers in octaethy leneglycol dodecyl ether was 114,200. Similarly, the molecular weights of maltoporin oligomers and monomers, which form maltose-maltodextrin specific channels, appeared to be 148,500 and 48,200, respectively, in SDS and that of oligomers in octaethyleneglycol dodeclyl ether was 149,000.

Chromatography, Gel↗
PubMed1981

Permeability properties of chemically modified porin trimers from Escherichia coli B.

The pore-forming protein of the outer membrane of Escherichia coli, porin, was chemically modified with acetic anhydride, succinic anhydride, and glycinamide. Extensive modification of amino groups of the functional porin trimers caused reduced diffusion rates of the negatively charged solutes such as p-nitrophenyl phosphate and AMP, but did not reduce significantly the diffusion of positively charged molecules carbobenzoxy-glycyl-prolyl-arginine-p-nitranilide and tosyl-glycyl-prolyl-arginine-p-nitranilide. Modification of carboxyl groups of trimers caused decreased diffusion rates of the positively charged solutes more significantly than the diffusion rates of negatively charged solutes. The results suggest that the ionic interactions play an important role for the diffusion of charged solutes through the porin pore. The diffusion of p-nitrophenyl alpha-D-glucoside, an uncharged solute, ws not influenced significantly by modification of either amino or carboxyl groups. This observation suggests that modifications only occurred in areas outside of the narrowest portion of the pore or, alternatively, that amino and carboxyl groups are exclusively located at noncylindrical area of the pore. The structural integrity of the acetylated and the succinylated trimers seemed well preserved. On the other hand, modification of carboxyl groups decreased the thermal stability of trimers and extensive modifications caused the dissociation of trimers into monomers at 37 degrees C.

Acetic Anhydrides↗
PubMed1980

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

The three types of porin (matrix-proteins) from Salmonella 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)(-12)M), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1 M KC1. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39 K 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 from Salmonella 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 1 M KC1 (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 conductance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.

Bacterial Proteins↗
PubMed1980

Subunit constituent of the porin trimers that form the permeability channels in the outer membrane of Salmonella typhimurium.

The polypeptide composition of the functional porin trimers that produced the permeability channels in the outer membrane of Salmonella typhimurium was examined on two-dimensional slab gels. The results suggested that the majority of porin trimers from strains producing mixed species of porin polypeptides consisted of homologous subunit polypeptides. The present results do not exclude the possibility that a small fraction of porin trimer is constructed from heterologous subunit polypeptides.

Bacterial Proteins↗
PubMed1980

Permeability properties of Escherichia coli outer membrane containing, pore-forming proteins: comparison between lambda receptor protein and porin for saccharide permeation.

Outer membrane permeability conferred by lambda receptor protein and porins to maltose-maltodextrins and other oligosaccharides was studied in vitro with reconstituted vesicle membranes and in vivo with mutant strains lacking either one of these proteins. The vesicle membranes reconstituted from phospholipids, lipopolysaccharide, and purified lambda receptor allowed rapid diffusion of maltose and maltose-maltodextrins of up to six glucose residues, but the membranes acted essentially as a molecular sieve for sucrose, raffinose, stachyose, and inulins of molecular weights 800, 920, and 1,380. The vesicle membranes containing porins allowed rapid diffusion of maltose but not of maltose-maltodextrins larger than maltose. The apparent transport Km values for maltose-maltodextrins of up to six glucose residues from the strain carrying lamB+ ompB (lambda receptor+, porin-) were similar (about 5 X 10(-6) M), whereas the transport Km values for maltose- and maltotriose of the strain carrying lamB ompB+ (lambda receptor-, porin+) alleles appeared to be 300 and about 20,000 X 10(-6) M. These results suggest that lambda receptor protein forms permeability pores that facilitate the diffusion of maltose-maltodextrins and function as a molecular sieve for other saccharides.

Bacterial Proteins↗
PubMed1979

Subunit structure of functional porin oligomers that form permeability channels in the other membrane of Escherichia coli.

Oligomers of a protein, porin, form permeability channels in the outer membrane of Escherichia coli B. A functional porin oligomer was identified and was purified to homogeneity by gel filtration in the presence of salts and sodium dodecyl sulfate. Molecular weights of purified porin oligomer and heat-dissociated monomer appeared to be 102,900 and 32,600, respectively, when determined by sedimentation equilibrium in the presence of sodium dodecyl sulfate. We concluded that the porin oligomer thus consists of three identical subunits. These data and results from other laboratories suggest porin trimers exist also in the outer membrane of intact cells, and participate in the formation of permeability channels. It was found that porin trimer bound less sodium dodecyl sulfate than the porin monomer.

Bacterial Proteins↗