Alkali-treated Membranes Research Articles

Investigation of Effective Modification Treatments for Titanium Membranes

Titanium membranes are used for guided bone regeneration in implant therapy. However, as a bioinert material, titanium does not have the ability to accelerate bone formation. Various titanium surface treatments to confer bioactivity have been demonstrated; however, there are concerns about the influence of chemical treatments for thin titanium membranes. This study investigated the influence of surface modifications on the structure of titanium membranes. Titanium membranes of 20 μm thickness were treated with acid or alkali solutions, and we evaluated their surface structure, wettability, thickness, and mechanical strength compared to non-treated membranes. Alkali-treated titanium membranes displayed the formation of nanoscale pore structures on their surfaces, enhanced hydrophilicity, and almost same thickness compared with acid-treated membranes. Furthermore, the tensile strength of alkali-treated membranes was comparable to non-treated membranes. These results suggest that alkali treatment is an appropriate surface modification method for titanium membranes.

Interactions between Hemoglobin and Cod Muscle Constituents following Treatment at Extreme pH Values

Acid and/or alkaline solubilization is a recent method developed to separate proteins from muscle foods with good functional properties. However, exposure of the muscle and its components to low pH values has been shown to promote lipid oxidation, limiting therefore the applications of this novel method. This research aimed primarily to study the physicochemical changes of the fish membranes brought about during acid or alkali solubilization processes. The effect on lipid oxidation and the possible role of the water soluble fraction of the muscle (press juice) as a potent antioxidant were also investigated. Model systems comprising minced cod muscle or cod microsomal suspensions were used. Results showed that acid or alkaline treatment (pH < 3.5 or pH > 10.5) of cod membranes significantly delayed lipid oxidation. Added triacylglycerols to washed cod system treated at low pH did not enhance hemoglobin-mediated lipid oxidation. Decreased precipitation of hemoglobin was observed with the alkali-treated membranes at all protein concentrations compared to the acid-treated and the untreated membranes. Finally, the addition of press juice to washed cod muscle tissue or to the membrane model system, significantly delayed hemoglobin lipid oxidation. The results of this study can be used to improve pH-shifting technologies to avoid or decrease lipid oxidation problems. Also, the use of press-juice from cod muscle as means of protecting the muscle against lipid oxidation is suggested.

Changes in the electrochemical characteristics of a bipolar membrane immersed in high concentration of alkaline solutions

The chemical stability of a commercial bipolar membrane (Neosepta BP-1, Tokuyama Co.) was examined after immersing it in concentrated NaOH solution at different temperatures, for various periods of time. The physical and electrochemical changes in the alkali-treated membranes were measured and the effects on membrane performance were investigated thoroughly by analyzing FTIR spectra, water-splitting capabilities, and current–voltage curves. The FTIR spectra showed that the quaternary ammonium groups in the anion-exchange layer (AEL) of the BP-1 membrane decomposed via the Hofmann reaction when the membranes were immersed in 5.0 M NaOH at over 40 °C, and in 2.5 M NaOH at 50 °C. In addition, the water-splitting capability of the membranes that had undergone structural change decreased. This was attributed to decreased Donnan exclusion resulting from the decomposition of functional groups in the AEL of the BP-1 membrane. The results of the current–voltage experiments were similar to those of FTIR and water-splitting capability studies. The electric resistance for water dissociation increased when the structure of the AEL changed in concentrated alkaline solution, and at high temperature. We concluded that a more stable bipolar membrane anion-exchange layer is required for use in concentrated alkaline solutions.

Characterization and application of radiation grafted membranes in waste treatment

Ionic membranes were prepared by radiation-induced grafting of acrylic acid onto low density polyethylene films. To elucidate the possibility of practical use, a study has been made for the characterization of the grafted and chemically treated membranes. The selectivity of such prepared membranes towards the chelation or complexation of different alkali metals was investigated, to find that the higher affinity is observed for K+, Na+ and Li+ ions compared to other alkali metals used. The metal uptake percent was determined using different techniques: flame photometer and X-ray fluorescence (XRF). The uptake of metal from its feed solution by the grafted membrane increased as the degree of grafting increased, i.e., it is directly proportional to the content of functional carboxylic acid groups in the graft copolymer. As a consequence, the electrical conductivity of metal feed solution decreased during such process of metal chelation by the membrane. The higher the grafting degree of membrane, the lower the electrical conductivity of metal feed solutions observed. The changes in thermal properties of the prepared membranes were investigated and characterized using differential scanning calorimeter (DSC), and thermal gravimetric analysis (TGA). The thermal stability of these membranes increased with a degree of grafting due to the formation of crosslinked network structure via hydrogen bonding. Furthermore, such stability is enhanced for the alkali-treated membranes even at high elevated temperatures. The prepared membranes showed a great promise for possible use in the recovery of uranium from zirconium in their wastes.

Characterization and application of radiation grafted membranes in treatment of intermediate active waste

Graft copolymers that possess ionic characters were prepared by radiation grafting of acrylic acid onto polyethylene and fluorinated polymers followed by chemical treatments. Thermal stability, mechanical and ion exchange properties of the grafted and alkali-treated membranes were studied. The selectivity of the prepared membranes towards the adsorption of different metals from a simulated waste solution has been studied. The feasibility of zirconium separation from solution containing uranium was also investigated.

Proteolytic cleavage sites of band 3 protein in alkali-treated membranes: fidelity of hydropathy prediction for band 3 protein.

To assess the fidelity of hydropathy prediction for band 3 protein, we determined the cleavage sites of the protein and the portions of the protein tightly bound to the membrane lipid bilayer by means of in situ proteolytic digestion. For the removal of all anticipated hydrophilic connector loops from membranes, we had to denature the band 3 protein molecule in situ by alkali treatment. When the alkali-treated membranes were digested with trypsin, chymotrypsin, and pepsin, the majority of the anticipated transmembrane portions remained in the membrane fraction. However, five anticipated transmembrane portions were released into the supernatant fraction. Thus, the first, second, third, sixth and tenth anticipated transmembrane portions, in accordance with the hydropathy prediction, were released into the supernatant with the proteolytic digestion method. This indicates that these anticipated transmembrane portions are not bound with the boundary lipids although the hydrophobicity of these portions is comparable to that of the portions experimentally remaining in the membrane fraction. It is conceivable that the membrane peptide portions of band 3 protein could be classified into at least two categories, i.e. one bound to the boundary lipids and the other free from the boundary lipids. Approximately 90% of the transmembrane domain of the band 3 protein are recovered in either the supernatant fraction or the membrane fraction. The fidelity of hydropathy prediction for polytopic membrane proteins and the nature of the membrane embedded peptide portions are discussed.

Reconstitution of high-affinity opioid agonist binding in brain membranes.

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.

Transition of affinity states for leukotriene B4 receptors in sheep lung membranes

Leukotriene B4 (LTB4) is a pro-inflammatory arachidonate metabolite. We have characterized the LTB4 receptors in sheep lung membranes and have assessed the contribution of the guanine-nucleotide-binding (G) protein in the regulation of receptor affinity states. Saturation isotherms have demonstrated a single class of LTB4 receptor with a Kd of 0.18 +/- 0.03 nM and a density (Bmax.) of 410 +/- 84 fmol/mg of protein in sheep lung membranes. The effect of the G-protein on receptor affinity was assessed in the presence of non-hydrolysable GTP analogues (e.g. GTP[S]) and in membranes following alkali treatment (pH 12.1) to remove the G-protein. Saturation isotherms produced either in the presence of GTP[S] (Kd.GTP[S] = 0.51 +/- 0.02 nM) or with alkali-treated membranes (Kd.alk. = 0.52 +/- 0.02 nM) demonstrated a 3-fold shift in receptor affinity for [3H]LTB4 binding. In competition experiments, the rank order of affinity of LTB4 analogues was LTB4 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than 20-COOH-LTB4, using either untreated or alkali-treated membranes, both in the presence and absence of GTP[S]. These findings demonstrate that, in sheep lung membranes, there is only one class of LTB4 receptor. Removal of the G-protein or uncoupling of the receptor from the G-protein shifted the agonist-binding affinity of the receptor by 3-4-fold, without affecting the specificity of the LTB4 receptor in either the high- or the low-affinity state.

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61–63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37°C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4–8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two separate spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

Isolation of reconstitutively active succinate dehydrogenase in highly purified state.

Existing procedures for the isolation of mammalian succinate dehydrogenase yield preparations of high purity or retain reconstitution activity, but not both. A new procedure is described for the isolation in good yield of virtually homogeneous preparations with full reconstitution activity, and retaining iron-sulfur center 3 and the "low Km" reaction site of ferricyanide. On reincorporation of the soluble enzyme into alkali-treated membranes, the same high turnover number (approximately 21,000/min at 38 degrees) is obtained in catalytic assays as with intact inner membrane preparations.

Interrelations of reconstitution activity, reactions with electron acceptors, and iron-sulfur centers in succinate dehydrogenase

Reconstitutively active, soluble preparations of succinate dehydrogenase have been examined for the stoichiometric relationship of the covalently bound flavin to the electron paramagnetic resonance (epr) signals of the iron-sulfur centers known to be present in membrane-bound forms of the enzyme. The Fe-S center ( g = 2.01), which is paramagnetic in the oxidized state (the high-potential Fe-S cluster of the enzyme, HiPIP) and which was identified in earlier studies as a component of succinate-ubiquinone reductase (Beinert et al., 1975, Eur. J. Biochem. 54, 185), can be demonstrated in reconstitutively active, soluble preparations (0.20 to 0.35 equiv/mol of enzyme) at a significantly higher level than in enzyme preparations extracted without succinate. Studies of the kinetics of the reduction of the epr-detectable Fe-S centers revealed no difference between reconstitutively active and inactive preparations in the behavior of centers 1 and 2 and, of the oxidized Fe-S center detected in reconstitutively active preparations, only a part or none at all was reduced by succinate at catalytically significant rates. The failure to demonstrate the HiPIP center in stoichiometric amounts even when the enzyme is extracted and purified under anaerobic conditions in the presence of succinate may be due to the experimental conditions needed to convert this center to the paramagnetic state for observation, i.e., the obligatory use of artificial electron acceptors, such as ferricyanide. A procedure devised to circumvent this problem by incorporating the soluble enzyme into alkali-treated membranes resulted in a reconstituted preparation which was more stable to oxidizing agents. After incorporation into the membrane, between 50 and 100% of the HiPIP center was then detectable, suggesting a relationship between this Fe-S component and reconstitution activity. It has also been noted that, under aerobic conditions, the rates of decay of this epr signal, the reconstitution activity, and the catalytic activity of soluble preparations with low concentrations of ferricyanide as electron acceptor go parallel and may, indeed, be functions of the integrity of the same property of the protein. During this time, about half of the phenazine methosulfate reductase activity also declines, and the process is accompanied by a major increase in K m for phenazine methosulfate. The content of the two ferredoxin-type Fe-S cehters, one of which is reduced by the substrate (center 1) and the other by dithionite but not by succinate (center 2), in reconstitutively active preparations did not differ significantly from that of reconstitutively inactive, soluble preparations. On the basis of these data, the notion that the ferredoxin-type Fe-S centers or signals of reconstitutively active enzyme have properties very different from those of other preparations is considered lacking in experimental support.