生物物理化学
Online ISSN : 1349-9785
Print ISSN : 0031-9082
ISSN-L : 0031-9082
44 巻, 1 号
選択された号の論文の8件中1~8を表示しています
  • 水摩 仁美, 山口 奈緒, 真鍋 敬
    2000 年 44 巻 1 号 p. 1-7
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    未変性条件のミクロ2次元電気泳動で分離されたヒト血漿蛋白質を, 市販画像処理ソフトウェアとパーソナルコンピュータを組み合わせた画像処理システムで定量する方法について検討した. 画像上の各蛋白質の同定は, 電気泳動転写後の免疫化学的染色によってすでに作成済みの「ヒト血漿蛋白質同定地図」を参照して行った. 試料添加体積を変化させた2次元ゲルについて比較すると, ほとんどの血漿蛋白質スポットについて, 試料添加体積と積算濃度とは比例していた. しかし, アルブミンとIgGについては, 積算濃度の増加が頭打ちになる現象が観察された. アルブミン精製品によるタンパク量-積算濃度標準曲線を用いて, 2次元ゲル上のスポットのアルブミン換算量 (IgGについては精製IgGへの換算量) を求めたところ, すでに報告されている各タンパク質の定量値とよい一致が見られた.
  • 井本 真由美, 篠原 兵庫, 櫻林 郁之介, 山本 和彦, 秋山 利行, 山住 俊晃, 尾鼻 康朗, 古田 格
    2000 年 44 巻 1 号 p. 9-14
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    We encountered a case of IgD-type multiple myeloma in which we could not identify the type of light (L) chains of the IgD M-protein in the patient's serum by conventional immunoelectrophoresis or immunofixation electrophoresis. To clarify the cause of this failure, the immunochemical and structural properties of IgD in the patient's serum were characterized. The immunochemical reactivity of IgD M-protein against anti-λ anti- bodies was extremely low and the IgD-IgG complex was present in the patient's serum. The complex was dissociated in the presence of SDS, indicating that the association was formed via non-covalent binding. In a reassociation test, IgG prepared from normal subjects did not form a complex with the patient's IgD pretreated with a reducing agent, but formed a complex with IgD pretreated with papain. These results suggest that IgG formed a complex with the Fab of the patient's IgD.
  • 橋本 修一, 戸円 智幸
    2000 年 44 巻 1 号 p. 15-19
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    グリシンを用いた非還元型PAGE後のゲル内ALP活性を高感度に検出するために, グリシンおよび亜鉛の影響を活性染色で調べた. 192mMグリシンを用いた電気泳動後のゲル内ALP活性は, ホウ酸を用いた時の半分に減少した. これに対して, グリシン緩衝液にZn2+を事前添加すると, 電気泳動後の酵素活性は亜鉛の濃度に依存して増加し, 1mMで最大となり, ホウ酸を用いた場合と同等かそれ以上になった. しかし, 泳動試料中あるいは酵素活性染色液中にZn2+を添加しても再活性化は起こらなかった. また蛋白質に結合した65Znはグリシンを用いた電気泳動で遊離し, ゲル内を陽極に移動した. グリシンの錯体形成能力は, 200mMグリシンに対し0.1mM以上のZn2+で消失することが, ECBTを用いた実験で明らかになった.
  • 島崎 洋次, 藤原 麻衣, 真鍋 敬
    2000 年 44 巻 1 号 p. 21-25
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    We have reported that the third component of complement (C3) is observed in the pattern of microscale non-denaturing two-dimensional electrophoresis (2-DE) after removal of IgG and IgA using protein A agarose and antibodies.1) For the investigation of constitutive polypeptides of the C3, the constituent polypeptides of human plasma proteins were analyzed using non-denaturing isoelectric focusing (IEF) in the first dimension and treated with mercaptoethanol/SDS, and then subjected to the second dimensional SDS electrophoresis (non-denaturing IEF/reduced-SDS PAGE). The α chain of C3 spot was detected in the pattern of non-denaturing IEF/reduced-SDS PAGE, but, the β chain of C3 spot was hidden by albumin spot. After albumin was removed using polyethylene glycol, both C3α and C3β spots were observed in the pattern of the non-denaturing IEF/reduced-SDS PAGE. The combining analysis of C3 in the non-denaturing 2-DE and the constituent polypeptides in the non-denaturing IEF/reduced-SDS PAGE can be useful for the analysis of C3 and its fragments during the activation of complements.
  • 孝口 裕一, 相根 義昌, 渡部 俊弘, 大山 徹
    2000 年 44 巻 1 号 p. 27-34
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    Clostridium botulinum type C strain Stockholm (C-St) produced two types of progenitor toxins (M and L). The purified L toxin consisted of neurotoxin (NT), nontoxic-nonhemagglutinin (NTNHA) and hemagglutinin (HA) components which could be separated into four subcomponents, HA-33, HA-17, HA-22-23 and HA-55. On the other hand, purified M toxin lacked HA components. We isolated all components from the progenitor L toxin by following chromatographic methods. The L toxin was separated into an NT and NTNHA/HAs complex via Mono Q anion exchange column chromatography. Using gel filtration with a TSK-gel HW-55S column in the presence of 6M guanidine hydrochloride, the NTNHA/HAs complex derived from the L toxin could be separated into three fractions, NTNHA single component, HA-55/33 mixture and HA-22-23/17 mixture. Then, the HA-55/33 mixture was separated into each component using Mono S cation exchange column chromatography in the presence of 8M urea. The HA-22-23/17 mixture was also separated from each other with Superose 12 gel filtration column chromatography in the presence of 8M urea. The separated NT, NTNHA and HA subcomponents were homogenous on SDS-PAGE, and were identified to be C-St progenitor toxin gene products based on their N-terminal amino acid sequence analysis. This study presents for the first time an attempt to isolate components from the progenitor toxin. Although reassociation of the isolated components was also attempted via mixing of NTNHA, HA-55, HA-33, HA-22-23 and HA-17, HA activity of the mixture was lower than that of parent NTNHA/HAs complex.
  • 井本 真由美, 篠原 兵庫, 秋山 利行, 古田 格
    2000 年 44 巻 1 号 p. 35-37
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    Sensitivity and specificity of commercial anti-human free light (L) chains antibodies (DAKO Co. and Binding Site Co.) for detection of Bence Jones protein (BJP) were compared with those of conventional anti-human L chains antibodies (DAKO Co.), and the usefulness and problems of these antibodies were investigated. When BJP positive serum and urine were used, sensitivity of anti-human free L chains antibodies against BJP was lower than that of conventional anti-human L chains antibodies in many cases. The anti-human free L chains antibodies also reacted with not only free L chains but also with intact Igs, i.e., L chains linked to H chains. Therefore, care must be taken when the antibodies are used in a specific sensitive method such as immunoblotting.
  • 飯島 史朗, 志村 佐恵子, 土田 敦子, 木村 都, 芝 紀代子, 平塚 信夫, 井上 潤子, 山口 信隆
    2000 年 44 巻 1 号 p. 39-42
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    SEPARAX-SP membrane is used generally as supporting medium for serum protein fractionation in many clinical laboratories, but it has not been used for isoelectric focusing (IEF). Although the exclusive cellulose acetate membrane for IEF is a SEPARAX-EF membrane, we studied usefulness of SEPARAX-SP membrane as supporting medium for cellulose acetate membrane IEF as comparison with SEPARAX-EF membrane. Serum sample was applied and electrophoresed in according to our method. After electrophoresis, all cellulose acetate membranes were stained with Coomassie brilliant blue G-250. The both membranes showed clear and similar isoelectrophoretic patterns of serum protein. Furthermore, we confirmed that isoelectric focusing can be carried out on six layers of SEPARAX-SP membranes as same as SEPARAX-EF membrane. All six SEPARAX-SP membranes were showed clear isoelectrophoretic patterns of serum protein, but detected serum protein concentration was decreased in lower layers of SEPARAX-SP membrane. Therefore, we examined the distribution of protein among the six layers of SEPARAX-SP membrane and compared to those of SEPARAX-EF membrane. The ratio of protein distribution between top membrane and bottom membrane was 2:1 on SEPARAX-SP membrane against to the ratio of 30:1 on SEPARAX-EF membrane. Furthermore, protein distribution of six layers on SEPARAX-SP membrane was more uniform than that on SEPARAX-EF membrane. These results indicated that SEPARAX-SP membrane was useful supporting medium for cellulose acetate membrane IEF.
  • 栗原 由利子, 芝 紀代子, 横山 和則, 亀井 幸子
    2000 年 44 巻 1 号 p. 43-46
    発行日: 2000/03/15
    公開日: 2009/03/31
    ジャーナル フリー
    We measured total protein (TP) and immunoglobulin levels in cyst fluid taken from 19 patients who had postoperative maxillary cysts (POMC). In the 19 cases, TP, IgG, IgA, and IgM levels were 7990±3558 (mean±1 SD), 1890±1085, 648±1085, and 664±1489mg/dl, respectively. Immunoglobulin correlations for IgG and IgA, IgG and IgM, and IgA and IgM were 0.706, 0.654, and 0.951, respectively. Using cellulose acetate membrane electrophoresis, we also examined the glycosaminoglycan fractions in POMC fluid. Four (21.1%) of the 19 samples showed a separation-impossible fraction of glycosaminoglycans, while 15 samples (78.9%) showed a separation-possible fraction. By digestion with pronase, most samples could be separated into 3 bands: an unknown band, a hyaluronic acid band, and a chondroitin sulfate A band (CSA). The unknown bands of samples having a separation-impossible fraction and of those having a separation-possible fraction were 32.4±3.5% (mean±1 SD) and 58.4±9.6%. The hyaluronic acid bands in the two fractions were 56.0±12.9% and 30.7±8.1%, and the CSA bands were 5.7±8.0% and 8.7±4.3%. On the unknown and CSA bands, samples having a separation-possible fraction were higher than those having a separation-impossible fraction. On the hyaluronic acid band, samples having a separation-impossible fraction were higher than those having a separation-possible fraction, but there were no significant differences.
feedback
Top