Phosphatidylinositol 3,4,5-trisphosphate (PIP_3: 1) is a polyphosphoinositide produced from phosphatidylinositol 4,5-bisphosphate (PI4, 5P_2) by phosphatidylinositol-3 kinase (PI-3 kinase) in response to a wide range of growth factor stimulation. It is still unclear what a important role of PIP_3 is. To investigate how PIP_3 interact with proteins in the intracellular signal transduction system, we have developed two affinity gels with novel PIP_3 analogs. IP_3-APBgel is a affinity gel bearing 1-O-acyl type ligand (IP_3-APB: 6) and is expected to associate with proteins which specifically recognize head group of PIP_3. Using this IP_3-APBgel, we have isolated a novel protein with a molecular mass of 43kD from bovine brain extract and was termed PIP_3 binding protein. The protein bound PIP_3 with a higher affinity than it did inositol 1,3,4,5-tetrakisphosphate (IP_4), PI4, 5P_2, phosphatidylinositol 3,4-bisphosphate (PI3, 4P_2) and phosphatidylinositol 3-phosphate (PI3P), suggesting that the binding to PIP_3 was specific. It contained one zinc finger motif and two pleckstrin homology (PH) domains and the entire sequence was 83% similar to centaurin α, another PIP_3 binding protein. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each PH domains and the introduction of both mutations caused loss of the binding activity. These suggest PIP_3BP binds PIP_3 throuth two PH domains present in the molecule. To detect PIP_3 binding proteins in a more exact manner, we designed PIP_3 analog containing a glycerol group as well as fatty acid groups (PIP_3-APB: 12). One of the peptides identified from the lysates of calf thymus had a molecular mass of 70kD and was identified to be the Tec protein-tyrosine kinase. This interaction between the PIP_3 analog beads and Tec could be efficiently inhibited by free PIP_3. Significantly higher concentrations of PI4, 5P_2, PI4P and IP_4 were required to compete this interaction. Studies with the various deletion mutants of Tec protein revealed that the binding site of PIP_3 was localized within the PH domain of Tec. Now we have demonstrated that our affinity gels are very useful biochemical tools to detect PIP_3 associated proteins. Moreover, we isolated a novel PIP_3 binding protein, Tee and Aktγ (which was also isolated by PIP_3-APBgel). These proteins have PH domains which turned out to be essential to interact with PIP_3. This PIP_3-PH interaction is likely to have a significance in the PI-3 kinase signal transdution pathway.

抄録全体を表示

形を問わず適用できるという放射線グラフト重合法の利点を生かして, 多孔性中空糸膜状のイオン交換材料を作成した。多孔性膜の孔表面にイオン交換基をもつ高分子鎖を膜厚方向に一様に取り付けて, そこヘイオンを拡散移動抵抗なく供給するという仕組みと, 高分子鎖上のイオン交換基同志の静電反発によって高分子鎖が伸長してイオン交換場ができるという仕組みをつくって, 従来のイオン交換材料に比べて優れた性能を示すことができた。

抄録全体を表示

Membrane technology introduced entirely new concepts into the food industry such as concentration of liquid food without heating and complete removal of microorganisms from food system at room temperature. The present situation of the application of membrane technology in food industry is overviewed along with its brief history. Recent topics suggest that the following subjects are to be explored more for the further promotion of the present technology in food industry and biotechnology : (1) development of highly selective membrane technology; (2) application of membrane technology to nonaqueous systems; (3) breakthrough in the concentration limitation of membrane technology; (4) membrane bioreactors; (5) membrane technology as an integration system between reaction and separation; (6) analysis and control of the fouling phenomena of membranes.

抄録全体を表示

ある特定の血球細胞を血液中から分離・除去する試みは, 近年, 成分輸血, 治療あるいは多くの基礎研究において重要な課題となっている。特に, 目的細胞に対して特異的な親和牲を有する吸着材を用いた分離・除去法は, 簡便, 迅速かつ効率的な処理を可能にするものであると考えられる。本研究では, リンパ球の亜集団であるBリンパ球に対する非生物学的なアフィニドを用いて, 単核球分画あるいは全血からのBリンパ球の分離あるいは除去を試みた。その結果, イムノグロブリン(Ig)に対してアフィニティを有するスルファチアゾール(ST)を固定化した吸着材は, 膜表面にIgを有するBリンパ球に対しても高い親和性を有することが観察された。また, 担体―リガンド間へのスペーサーの導入は, Tリンパ球の非特異接着を抑制し, 分離材の選択性を高めるのに効果的であることも明かとなった。このような分離材は, リガンドの多様化によって, 幅広い応用を実現できるものと思われる。

抄録全体を表示

An epoxy-group-containing monomer, glycidyl methacrylate (GMA), was grafted onto a porous hollow-fiber membrane made of polyethylene, by radiation-induced graft polymerization. Four kinds of alcohol, i.e., methanol, ethanol, 1-propanol, and 1-butanol, were employed as the solvents for GMA. Grafting rate of GMA was decreased with increasing carbon number of alcohols. The epoxy group of the GMA-grafted membrane with a degree of grafting of 90 or 120% was converted to a sulfonic acid group at a molar conversion of 10 to 12%. Hen-egg lysozyme (HEL) dissolved in carbonate buffer solution (pH 9.0) was forced to permeate through the pores of the sulfonic-acid-group-containing porous hollow-fiber membranes prepared with various alcohol solvents. The binding capacity of HEL onto the membrane in equilibrium with the feed concentration of 0.5 g-HEL/L increased with the increase in the carbon number of alcohols. For example, the binding capacities of the membranes prepared with methanol and 1-butanol were 0.18 and 0.38 g-HEL/g-membrane, respectively. In contrast, the flux of the protein solution was decreased with increasing carbon number of alcohols. This demonstrates that longer polymer brushes are formed on the pore surface of the membrane prepared with a higher carbon number of alcohol employed as the solvent for GMA.

抄録全体を表示

We proposed a method for purifying proteins based on an interaction between dually-immobilized affinity ligands and dually-tagged proteins. Streptavidin (SA) and nickel (Ni) ions as two kinds of ligands were immobilized to the polymer brush grafted onto a porous hollow-fiber membrane. Whereas, green fluorescent protein (GFP) containing both streptavidin binding peptide (SBP) and poly histidine tag (His-tag) as two kinds of tags was genetically produced. The resultant dually-tagged protein solution was permeated through the pores of the dual-affinity-ligands- immobilized porous hollow-fiber membrane. The dual-affinity ligands and dually-tagged proteins were found to fit on the polymer chain grafted onto the pore surface at a binding efficiency 2%.

抄録全体を表示

AOTとタウロデオキシコール酸 (TDCA) を用いて, 逆相ミセルによるlipase (Chromobacterium viscosum lipase) の正・逆抽出を行った.AOTとTDCAは, イソオクタン中で安定な混合ミセルを形成した.混合ミセルは, AOTミセルより優れた選択性・活性収率を示した.ミセルサイズは, 50mMAOTではTDCAの濃度 (0-4mM) によらず, AOTミセルとほぼ等しい.ミセル界面でTDCA 1分子の占める面積は, AOT6.2分子に相当する.混合ミセルは, α-chymotrypsinやpapainとは対照的に, lipaseに対して選択的に分配係数を8倍程度まで増加させ, 微量注入法を用いなくても, 二相抽出法や液膜抽出法によって活性収率を改善できる.

抄録全体を表示

An acid extractant, di (2, 4, 4-trimethylpentyl) phosphinic acid (Cyanex 272), was impregnated to the hydrophobic　ligand of a polymer chain grafted onto the pore surface of a porous membrane of a hollow-fiber form. The hydrophobic　ligand was introduced by a reaction of an epoxy group of the poly-glycidyl methacrylate, which was appended to　the porous membrane by radiation-induced graft polymerization, with n-alkylamine (CnH2n+1NH2) or n-octadecanethiol　(C18H37SH). The carbon number of the n-alkylamino group ranged from 0 to 18. The amount of Cyanex 272　impregnated onto the porous membrane increased with an increasing density of the n-octadecylamino group, whereas　the amount of impregnated Cyanex 272 was constant irrespective of the density of the n-octadecanethiol group.　No leakage of Cyanex 272 impregnated to the n-dodecylamino or n-octadecylamino group of the graft chain was　observed. The capturing of zinc ions by the Cyanex 272-impregnated porous hollow-fiber membrane was achieved　during the permeation of a zinc chloride solution through the pores of the porous membrane. The equilibrium binding　capacity of the Cyanex 272-impregnated porous hollow-fiber membrane for zinc ions was 0.37 mol/kg of the　GMA-grafted membrane.

抄録全体を表示

PEG (polyethylene glycol) /Dex (dextran) およびPEG/PK (リン酸カリウム) 系による水性二相抽出法をパパイヤラテックスからのパパインの分離・精製に用いた.分配係数はパパインと分配系との間に作用する静電的, 立体的, 疎水的などの各種の相互作用に依存するが, PEG/PK系では, PEGとパパインとの疎水的相互作用の寄与が大きく, 分配係数と選択性はPEG/Dex系より高い.また, パパイヤラテックス粉末を水性二相系に直接添加し, 浸出と分離を同時に行わせる方式はパパイヤラテックスの浸出液を分配させる方式よりも有効である.
PEGのOHをパパインと特異的アフィニティを持つ各種のリガンドで置換し, それらの修飾PEGを水性二相抽出に用いた.その結果, 分配係数と選択性は大きく向上するが, その結果はPB (Procion-Blue) -PEGを用いたPEG/PK系で顕著である.

抄録全体を表示

Membrane-based affinity chromatography has advantages over conventional bead-based chromatography for the following two reasons : (1) the module charged with hollow fibers requires a much lower operating pressure than a bead-packed bed ; and (2) as the biomolecule can be transported by convection to the ligand immobilized on the inner surface of the microporous membrane, faster adsorption onto the affinity membrane can be attained. Hollow-fiber affinity membranes containing phenylalanine and tryptophan as ligands were prepared by the radiation-induced grafting of glycidyl methacrylate onto a porous polyethylene hollow fiber, followed by coupling of the epoxide group produced with Phe and Trp. The remaining epoxide group is quantitatively converted into a diol group. The diol group renders the polymer surface hydrophilic and prevents nonselective adsorption. To evaluate the adsorption behavior of the membrane, the gamma-globulin-containing buffer solution was permeated from the inside to the outside of the Trp-containing hollow-fiber affinity membrane. The breakthrough curves as a function of effluent volume coincided irrespective of the flow-rate, i. e., the residence time of the solution across the membrane, as a result of negligible diffusional mass transfer resistance.

抄録全体を表示

市販の多孔性中空糸膜(膜厚0.6 mm，孔径0.4 µm，空孔率70%)に接ぎ木したグリシジルメタクリレート(GMA)高分子鎖にさまざまな官能基，たとえば，キレート基，イオン交換基を導入した．膜間に圧力差を与えて金属やタンパク質溶液を透過させると，金属イオンやタンパク質の孔内から官能基までの拡散物質移動抵抗を最小化できる．GMAのグラフト率およびエポキシ基から官能基へのモル転化率を調整して，多孔性中空糸膜の官能基密度を従来の吸着材のそれに比べて高くできた．まず，分離対象として金属イオンやタンパク質を，それぞれグラフト鎖のキレート基やイオン交換基へ膜孔内の透過流に乗せて輸送した．対象溶液の透過速度を速くすればするほど，多孔性中空糸膜への対象イオンの総括吸着速度はそれだけ速くなった．そのうえ，グラフト鎖のうち，孔表面から孔内部に向けて存在するポリマーブラシは，イオン交換基どうしの静電反発によって伸長するので，タンパク質を多層に吸着した．つぎに，約1 mmの膜厚のキレートおよびイオン交換多孔性中空糸膜を，それぞれ二成分の金属イオンおよびタンパク質溶液が透過する間に置換吸着が観察された．さらに，アルブミンが多層に吸着した多孔性中空糸膜を使ってDLトリプトファンのキラル分離を実証した．

抄録全体を表示

Protein binding amounts of the modules consisting of anion-exchange porous membranes were determined as a function of flow rate of the protein solution. Two kinds of anion-exchange porous membranes of a hollow-fiber form were compared : EA membrane containing a 2-hydroxylethylamino group and DEA-EA membrane containing a diethylamino group along with the 2-hydroxylethylamino group. The module has an inner diameter of 1.8 cm and effective length of 8 cm, respectively, into which nine or eight numbers of the anion-exchange porous hollow-fiber membrane (ID=2.8 mm, OD=4.4 mm) were incorporated. A 2 kg/m³ bovine serum albumin (BSA) in a Tris-HCl buffer (pH 8.0) was fed to the module from the lumen side to the shell side at various flow rates. The dynamic binding amount of BSA calculated from breakthrough curves was constant irrespective of flow rate ranging from 0.17×10⁶ m³/s to 1.7×10⁶ m³/s because of a negligible diffusional mass-transfer resistance assisted by convective flow through the pores. The DEA-EA module exihibited about three-fold equilibrium binding amount of BSA compared to the EA module. This can be explained by multilayer binding of BSA into the polymer chains grafted onto the pore surface.

抄録全体を表示

Radioisotopes were released by Fukushima Daiichi nuclear power plant, which was seriously damaged by the magnitude 9.0 earthquake and the tsunami that followed. To achieve the rapid capture of cesium ions and easy processing of radioactive water, a novel potassium-cobalt-hexacyanoferrate (KCo-HCFe)-impregnated fiber was prepared by radiation-induced graft polymerization and subsequent chemical modifications. Hexacyanoferrate ions were bound to vinyl benzyl trimethylammonium chloride-grafted 6-nylon fiber. Subsequently, the bound hexacyanoferrate ions were reacted with cobalt ions to form insoluble KCo-HCFe. The resultant KCo-HCFe-impregnated fiber exhibited a high affinity to cesium ions in seawater. Such fibers can be fabricated into various fiber modules suitable for the removal of cesium at sites.

抄録全体を表示