JP2011063541A - Purification method of l-arginine-binding factor and screening method of l-arginine-like compound - Google Patents
Purification method of l-arginine-binding factor and screening method of l-arginine-like compound Download PDFInfo
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- JP2011063541A JP2011063541A JP2009215216A JP2009215216A JP2011063541A JP 2011063541 A JP2011063541 A JP 2011063541A JP 2009215216 A JP2009215216 A JP 2009215216A JP 2009215216 A JP2009215216 A JP 2009215216A JP 2011063541 A JP2011063541 A JP 2011063541A
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- arginine
- binding factor
- methyl ester
- binding
- derivative
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Abstract
【課題】 細胞抽出液などの試料中に含まれるアルギニン結合因子を効率的に精製する手段を提供する。
【解決手段】 担体に固定されたL-アルギニンメチルエステル等に試料を接触させることにより、試料中のL-アルギニン結合因子とL-アルギニンメチルエステル等を結合させる工程、及びL-アルギニンメチルエステル等と結合したアルギニン結合因子を回収する工程を含むL-アルギニン結合因子の精製方法、及びL-アルギニン結合因子とL-アルギニン等とからなる結合体に、被験物質を接触させ、L-アルギニン等からL-アルギニン結合因子を解離させる被験物質を選択することを特徴とするL-アルギニン様化合物のスクリーニング方法。
【選択図】 なしPROBLEM TO BE SOLVED: To provide a means for efficiently purifying an arginine binding factor contained in a sample such as a cell extract.
SOLUTION: A step of bringing a sample into contact with L-arginine methyl ester or the like immobilized on a carrier to bind L-arginine binding factor and L-arginine methyl ester or the like in the sample, and L-arginine methyl ester or the like A method for purifying L-arginine-binding factor, including a step of recovering arginine-binding factor bound to lignin, and contacting a test substance with a conjugate comprising L-arginine-binding factor and L-arginine, etc. A method for screening an L-arginine-like compound, comprising selecting a test substance that dissociates an L-arginine-binding factor.
[Selection figure] None
Description
本発明は、L-アルギニンと結合する物質（L-アルギニン結合因子）を効率的に精製する方法、及びその方法によって精製されたL-アルギニン結合因子等を利用して、L-アルギニンと同様の生理活性を持つ化合物をスクリーニングする方法に関する。 The present invention uses a method for efficiently purifying a substance that binds to L-arginine (L-arginine binding factor), and an L-arginine binding factor purified by the method, and is similar to L-arginine. The present invention relates to a method for screening a compound having physiological activity.
L-アルギニンは成長期や病気・怪我のような期間において充分に合成されず、不足しやすいため、準必須アミノ酸として分類される(Morrisら2004)。体内のL-アルギニンの源は食事のタンパク質からや体内タンパク質のターンオーバーや体内での合成による。L-アルギニンは細胞質内酵素であるarginosuccinate synthase及びarginosucciinate lyaseの連続反応によりcitrullineより合成される。一方、L-アルギニンは４種類の酵素により細胞内にて代謝される。NO synthase (NOS)、arginase、arginine:glycine amidinotransferase、arginine decarboxylaseがそれら酵素であり、NO、creatine phophate、agmatine、polyamines、ornithine、citrullineのような生理活性化合物に変換される(Morrisら2006)。また、L-アルギニンは免疫(Angele ら2002)、創傷治癒(Stechmillerら2005)、血管緊張(Koizumiら1994)、血管内皮機能(Schlaichら2004)、インスリン分泌(Weinhausら1997)等の機能が存在する。このようにL-アルギニンは生理活性を有する食物中の化合物であり、直接ないしは代謝されて多くの機能が存在する。 L-Arginine is classified as a quasi-essential amino acid because it is not sufficiently synthesized during growth, illness, or injury and is prone to deficiency (Morris et al. 2004). The source of L-arginine in the body is derived from dietary proteins, by turnover of body proteins, or by synthesis in the body. L-arginine is synthesized from citrulline by the continuous reaction of cytoplasmic enzymes arginosuccinate synthase and arginosucciinate lyase. On the other hand, L-arginine is metabolized intracellularly by four types of enzymes. NO synthase (NOS), arginase, arginine: glycine amidinotransferase, and arginine decarboxylase are these enzymes and are converted into bioactive compounds such as NO, creatine phophate, agmatine, polyamines, ornithine, citrulline (Morris et al. 2006). L-arginine also has functions such as immunity (Angele et al. 2002), wound healing (Stechmiller et al. 2005), vascular tone (Koizumi et al. 1994), vascular endothelial function (Schlaich et al. 2004), insulin secretion (Weinhaus et al. 1997) To do. Thus, L-arginine is a physiologically active food compound that has many functions either directly or metabolized.
L-アルギニンを基質として代謝する酵素群はL-アルギニン結合因子として知られているが、L-アルギニンが直接生理活性を有することに関してはほとんど知られていない。タンパク質に結合し、反応するようなシグナル分子のほとんどは細胞表面に存在するため、アルギニンシグナルの解明のためにはL-アルギニン結合因子の同定は不可欠である。 Enzymes that metabolize L-arginine as a substrate are known as L-arginine-binding factors, but little is known about L-arginine having direct physiological activity. Since most signal molecules that bind to and react with proteins are present on the cell surface, identification of L-arginine-binding factors is essential for the elucidation of arginine signals.
本発明者らは既にグリシジルメタクリレート（GMA）-スチレン共重合体をコアとし、GMAで覆ったナノビーズを作製した(SGビーズ、200nm直径、Kawaguchiら1989)。このナノビーズは従来のアフィニティ精製担体に比べていくつかの利点がある。１）ポア（穴）がないため、洗浄時に残存タンパク質が効果的に除け、また、目的タンパク質が固定化したリガンドにアクセスしやすい。２）直径が小さいため（200nm）大きな表面積が得られる（１gのビーズで２０m2の表面積があり、固定化するリガンドの容量も大きい。）、３）化学的・物理的な安定であるため、種々の有機溶媒中にてリガンドのビーズへの固定化反応が可能である。このような利点から、SGビーズは、迅速で効率的なリガンド結合タンパク質の精製を可能にする(転写因子精製：非特許文献１、非特許文献２、薬剤受容体精製：非特許文献３、非特許文献４、)。SGビーズをもとにして最近新しい磁性ナノビーズ(FGビーズ、200nm直径、磁性粒子をGMA-スチレン共重合体で覆い、さらにGMAポリマーで覆った)が開発された(Nishioら2008)。このFGビーズは市販されている他のビーズ〔adembeads(200nm, ADENTECH)、nanomag-D(130nm, micromod)、Dyabeads(2800nm, Invitrogen)。〕に比べて標的タンパク質に対する高い精製効率を示した(Nishioら2008)。 The present inventors have already produced nanobeads having a glycidyl methacrylate (GMA) -styrene copolymer as a core and covered with GMA (SG beads, 200 nm diameter, Kawaguchi et al. 1989). This nanobead has several advantages over conventional affinity purification supports. 1) Since there is no pore (hole), the remaining protein is effectively removed at the time of washing, and the ligand to which the target protein is immobilized is easily accessible. 2) Because of its small diameter (200 nm), a large surface area can be obtained (1 g of beads has a surface area of 20 m 2 and the capacity of the ligand to be immobilized is large). 3) Because it is chemically and physically stable, The ligand can be immobilized on beads in various organic solvents. Because of these advantages, SG beads enable rapid and efficient purification of ligand-binding proteins (transcription factor purification: non-patent document 1, non-patent document 2, drug receptor purification: non-patent document 3, non-patent document Patent Document 4,). Recently, new magnetic nanobeads (FG beads, 200 nm diameter, magnetic particles covered with GMA-styrene copolymer and further covered with GMA polymer) were developed based on SG beads (Nishio et al. 2008). These FG beads are other commercially available beads (adembeads (200 nm, ADENTECH), nanomag-D (130 nm, micromod), Dyabeads (2800 nm, Invitrogen). ] Higher purification efficiency for the target protein (Nishio et al. 2008).
アルギニンには、NO産生、尿素産生、インスリン分泌促進など多様な機能を持つが、それらの機能については必ずしも十分に解明されているわけではなく、特にインスリン分泌促進についてはほとんどわかっていない。このような機能の解明には、アルギニンがどのような物質と結合しているのかを明らかにすることが重要であるが、現在のところそのような物質（アルギニン結合因子）を効率的に精製する手段が開発されていない。 Arginine has various functions such as NO production, urea production, and insulin secretion promotion, but these functions are not always fully elucidated, and in particular, little is known about insulin secretion promotion. To elucidate such functions, it is important to clarify what kind of substance arginine is bound to, but at present, such substance (arginine binding factor) is efficiently purified. Means have not been developed.
本発明は、このような技術的背景の下になされたものであり、細胞抽出液などの試料中のアルギニン結合因子を効率的に精製する手段を提供することを目的とする。 The present invention has been made under such a technical background, and an object thereof is to provide a means for efficiently purifying an arginine binding factor in a sample such as a cell extract.
本発明者は、上記課題を解決するため鋭意検討を重ねた結果、アフィニティ精製によりアルギニン結合因子を精製する際、アルギニンそのものではなく、アルギニンメチルエステルを用いることにより、効率的にアルギニン結合因子を精製できることを見出した。その理由は、以下の通りである。 As a result of intensive studies to solve the above problems, the present inventor has efficiently purified arginine binding factor by using arginine methyl ester instead of arginine itself when purifying arginine binding factor by affinity purification. I found out that I can do it. The reason is as follows.
アルギニンは、分子中にカルボキシル基とアミノ基を持つため、自己縮合して多量体を形成してしまう。このため、アルギニンを用いたのではアルギニン結合因子を効率的に精製することができない。これに対し、アルギニンメチルエステルはカルボキシル基を持たないのでアルギニンのような自己縮合の問題が生じない。一方、自己縮合を回避できたとしても、メチルエステル化により、アルギニン本来の機能が失われてしまえば、本来アルギニンと結合する物質と結合できなくなってしまい、アルギニン結合因子の効率的な精製は不可能になる。しかし、アルギニンメチルエステルは、アルギニン同様にインスリン分泌活性とNOSアンタゴニスト活性持つことから、アルギニンの持つ機能は失われておらず、アルギニンと結合する物質は、アルギニンメチルエステルにも結合すると推測される。 Arginine has a carboxyl group and an amino group in the molecule, and thus self-condenses to form a multimer. For this reason, the use of arginine cannot efficiently purify the arginine binding factor. On the other hand, since arginine methyl ester does not have a carboxyl group, the problem of self-condensation like arginine does not occur. On the other hand, even if self-condensation can be avoided, if the original function of arginine is lost due to methyl esterification, it cannot bind to the substance that originally binds arginine, and efficient purification of the arginine binding factor is not possible. It becomes possible. However, since arginine methyl ester has insulin secretion activity and NOS antagonist activity like arginine, the function of arginine is not lost, and it is assumed that the substance that binds to arginine also binds to arginine methyl ester.
更に本発明者は、精製されたアルギニン結合因子を利用して、L-アルギニン様化合物をスクリーニングできることを見出した。 Furthermore, the present inventor has found that L-arginine-like compounds can be screened using a purified arginine-binding factor.
本発明は、以上の知見に基づき完成されたものである。 The present invention has been completed based on the above findings.
即ち、本発明は、以下の（１）〜（８）を提供するものである。
（１）担体に固定されたL-アルギニン誘導体に試料を接触させることにより、試料中のL-アルギニン結合因子とアルギニンン誘導体を結合させる工程、及びL-アルギニンン誘導体と結合したアルギニン結合因子を回収する工程を含むL-アルギニン結合因子の精製方法であって、L-アルギニンン誘導体が、アルギニン分子中のカルボキシル基とアミノ基による自己縮合を起こさない化学構造をとり、かつインスリン分泌活性とNOSアンタゴニスト活性を有するものであることを特徴とするL-アルギニン結合因子の精製方法。
（２）L-アルギニン誘導体が、L-アルギニンメチルエステルであることを特徴とする（１）に記載のL-アルギニン結合因子の精製方法。
（３）担体が、磁性を持つ担体であることを特徴とする（１）又は（２）に記載のL-アルギニン結合因子の精製方法。
（４）L-アルギニン結合因子とL-アルギニン又はL-アルギニン誘導体とからなる結合体に、被験物質を接触させ、L-アルギニン又はL-アルギニン誘導体からL-アルギニン結合因子を解離させる被験物質を選択することを特徴とするL-アルギニン様化合物のスクリーニング方法。
（５）L-アルギニン結合因子が、（１）乃至（３）のいずれかに記載の精製方法によって精製されたL-アルギニン結合因子であることを特徴とする（４）に記載のL-アルギニン様化合物のスクリーニング方法。
（６）L-アルギニン結合因子が、ホスホフラクトキナーゼ、RuvB-like 2、又はRuvB-like 1であることを特徴とする（４）に記載のL-アルギニン様化合物のスクリーニング方法。
（７）L-アルギニン誘導体が、アルギニン分子中のカルボキシル基とアミノ基による自己縮合を起こさない化学構造をとり、かつインスリン分泌活性とNOSアンタゴニスト活性を有するL-アルギニンン誘導体であることを特徴とする（４）乃至（６）のいずれかに記載のL-アルギニン様化合物のスクリーニング方法。
（８）L-アルギニンン誘導体が、L-アルギニンメチルエステルであることを特徴とする（４）乃至（６）のいずれかに記載のL-アルギニン様化合物のスクリーニング方法。
That is, the present invention provides the following (1) to (8).
(1) The step of bringing the sample into contact with the L-arginine derivative immobilized on the carrier to bind the L-arginine binding factor and the arginine derivative in the sample, and the arginine binding factor bound to the L-arginine derivative A method for purifying an L-arginine-binding factor comprising a step of recovering, wherein the L-arginine derivative has a chemical structure that does not cause self-condensation due to a carboxyl group and an amino group in the arginine molecule, and has an insulin secretion activity and NOS A method for purifying an L-arginine binding factor, characterized by having an antagonist activity.
(2) The method for purifying an L-arginine binding factor according to (1), wherein the L-arginine derivative is L-arginine methyl ester.
(3) The method for purifying an L-arginine binding factor according to (1) or (2), wherein the carrier is a magnetic carrier.
(4) A test substance for bringing a test substance into contact with a conjugate comprising an L-arginine binding factor and L-arginine or an L-arginine derivative and dissociating the L-arginine binding factor from the L-arginine or L-arginine derivative. A screening method for L-arginine-like compounds, which comprises selecting.
(5) The L-arginine binding factor according to (4), wherein the L-arginine binding factor is an L-arginine binding factor purified by the purification method according to any one of (1) to (3) Screening method for like compounds.
(6) The method for screening an L-arginine-like compound according to (4), wherein the L-arginine-binding factor is phosphofructokinase, RuvB-like 2, or RuvB-like 1.
(7) The L-arginine derivative is a L-arginine derivative having a chemical structure that does not cause self-condensation due to a carboxyl group and an amino group in the arginine molecule, and has insulin secretion activity and NOS antagonist activity. A method for screening an L-arginine-like compound according to any one of (4) to (6).
(8) The method for screening an L-arginine-like compound according to any one of (4) to (6), wherein the L-arginine derivative is L-arginine methyl ester.
本発明の方法により、アルギニン結合因子の効率的な精製が可能になる。また、この方法によって精製されたアルギニン結合因子は、L-アルギニン様化合物のスクリーニングに利用できる。スクリーニングによって取得されたL-アルギニン様化合物は、L-アルギニンと同様にインスリン分泌促進作用を持つ可能性があるので、糖尿病の治療薬などの候補になる。 The method of the present invention enables efficient purification of arginine binding factor. The arginine-binding factor purified by this method can be used for screening for L-arginine-like compounds. Since L-arginine-like compounds obtained by screening may have an insulin secretion-promoting action similar to L-arginine, they are candidates for therapeutic agents for diabetes.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
（Ａ）L-アルギニン結合因子の精製方法
本発明のL-アルギニン結合因子の精製方法は、担体に固定されたL-アルギニン誘導体に試料を接触させることにより、試料中のL-アルギニン結合因子とアルギニンン誘導体を結合させる工程、及びL-アルギニンン誘導体と結合したアルギニン結合因子を回収する工程を含むものである。
(A) Method for purifying L-arginine binding factor The method for purifying L-arginine binding factor according to the present invention comprises contacting a sample with an L-arginine derivative immobilized on a carrier, The method includes a step of binding an arginine derivative and a step of recovering an arginine binding factor bound to the L-arginine derivative.
使用するL-アルギニン誘導体は、アルギニン分子中のカルボキシル基とアミノ基による自己縮合を起こさない化学構造をとり、かつインスリン分泌活性とNOSアンタゴニスト活性を有するものであれば特に限定されない。このようなL-アルギニン誘導体としては、L-アルギニンメチルエステルを例示できるが、これ以外にも、L-NAME、L-アルギニンエチルエステルなどを挙げることができる。 The L-arginine derivative to be used is not particularly limited as long as it has a chemical structure that does not cause self-condensation by a carboxyl group and an amino group in the arginine molecule and has insulin secretion activity and NOS antagonist activity. Examples of such L-arginine derivatives include L-arginine methyl ester, but other examples include L-NAME and L-arginine ethyl ester.
担体は、L-アルギニン誘導体を固定できるものであれば特に限定されないが、粒子状のものが好ましく、また、磁性を有するものが好ましい。好ましい担体の例としては、有機ポリマー被膜を有する磁性ナノビーズを挙げることができる。有機ポリマー被膜を有する磁性ナノビーズの粒径は特に限定されないが、1-500nmが好ましく、20-300nmがより好ましい。有機ポリマーとしては、GMA、GMAとスチレンの共重合体、（ポリ）メタクリル酸、（ポリ）アクリル酸などを挙げることができる。有機ポリマー被膜を有する磁性ナノビーズの具体例としては、SGビーズ（Kawaguchiら1989）、FGビーズ（Nishioら2008）、Dynabeads, Adembeads,nanomagなどを挙げることができる。 The carrier is not particularly limited as long as it can fix the L-arginine derivative, but is preferably in the form of particles, and preferably has a magnetic property. Examples of preferred carriers include magnetic nanobeads having an organic polymer coating. The particle size of the magnetic nanobead having an organic polymer coating is not particularly limited, but is preferably 1-500 nm, and more preferably 20-300 nm. Examples of the organic polymer include GMA, a copolymer of GMA and styrene, (poly) methacrylic acid, and (poly) acrylic acid. Specific examples of magnetic nanobeads having an organic polymer coating include SG beads (Kawaguchi et al. 1989), FG beads (Nishio et al. 2008), Dynabeads, Adembeads, nanomag, and the like.
試料は、L-アルギニン結合因子を含む可能性のあるものであればどのようなものでもよく、例えば、細胞抽出液を用いることができるが、他にも血漿、血清、臓器・組織抽出液などを用いることもできる。細胞抽出液はどのような細胞から得られたものでもよく、例えば、HeLa細胞、膵臓β細胞、繊維芽細胞、骨芽細胞などを挙げることができる。細胞抽出液はどのような生物由来のものでもよく、例えば、ヒト、マウス、ラット、魚、昆虫、酵母など由来の細胞抽出液を用いることができる。 The sample may be any sample that may contain an L-arginine binding factor. For example, a cell extract can be used, but plasma, serum, organ / tissue extract, etc. can also be used. Can also be used. The cell extract may be obtained from any cell, and examples thereof include HeLa cells, pancreatic β cells, fibroblasts, and osteoblasts. The cell extract may be derived from any organism, and for example, a cell extract derived from human, mouse, rat, fish, insect, yeast or the like can be used.
L-アルギニン誘導体に試料を接触させる方法は、試料中のL-アルギニン結合因子とアルギニンン誘導体が結合できるような方法であれば特に限定されない。具体的な方法としては、担体に固定されたL-アルギニン誘導体を試料と混合する方法を挙げることができるが、これ以外にも、担体に固定されたL-アルギニン誘導体をカラムに充填し、そのカラムに試料を通液する方法、バッチ法、競合阻害法などを挙げることができる。担体に固定されたL-アルギニン誘導体を試料と混合する方法は、例えば、L-アルギニン誘導体と試料を適当な緩衝液中で混合し、一定時間この混合状態を維持することにより行うことができる（この際、攪拌しながら混合状態を維持してもよい。）。混合状態を維持する時間は特に限定されないが、0.5-12時間程度が好適である。また、このときの温度も特に限定されないが、4〜37℃程度が好適である。 The method of bringing the sample into contact with the L-arginine derivative is not particularly limited as long as the L-arginine binding factor and the arginine derivative in the sample can be bound. As a specific method, a method of mixing an L-arginine derivative immobilized on a carrier with a sample can be mentioned, but in addition to this, an L-arginine derivative immobilized on a carrier is packed in a column, Examples include a method of passing a sample through a column, a batch method, and a competitive inhibition method. The method of mixing the L-arginine derivative immobilized on the carrier with the sample can be performed, for example, by mixing the L-arginine derivative and the sample in an appropriate buffer and maintaining this mixed state for a certain period of time ( At this time, the mixed state may be maintained while stirring.) The time for maintaining the mixed state is not particularly limited, but is preferably about 0.5 to 12 hours. Further, the temperature at this time is not particularly limited, but is preferably about 4 to 37 ° C.
L-アルギニンン誘導体と結合したアルギニン結合因子を回収する方法は特に限定されない。例えば、担体に固定されたL-アルギニンン誘導体と結合したアルギニン結合因子を担体ごと回収し、そこから、アルギニン結合因子を解離させればよい。アルギニン結合因子を担体ごと回収する方法は特に限定されず、例えば、担体が磁性を持つものであれば、磁力により担体を回収できる。L-アルギニンン誘導体からアルギニン結合因子を解離させる方法は特に限定されず、例えば、煮沸する方法や高濃度のL-アルギニン溶液と接触させる方法などを挙げることができる。煮沸する方法における煮沸時間は特に限定されないが、２〜１０分が好ましい。高濃度のL-アルギニン溶液と接触させる方法におけるL-アルギニン溶液の濃度は特に限定されないが、５０〜２００mMが好ましい。 The method for recovering the arginine binding factor bound to the L-arginine derivative is not particularly limited. For example, the arginine binding factor bound to the L-arginine derivative immobilized on the carrier may be recovered together with the carrier, and the arginine binding factor may be dissociated therefrom. The method for recovering the arginine binding factor together with the carrier is not particularly limited. For example, if the carrier has magnetism, the carrier can be recovered by magnetic force. The method for dissociating the arginine binding factor from the L-arginine derivative is not particularly limited, and examples thereof include a boiling method and a method of contacting with a high concentration L-arginine solution. Although the boiling time in the boiling method is not particularly limited, it is preferably 2 to 10 minutes. The concentration of the L-arginine solution in the method of contacting with a high concentration L-arginine solution is not particularly limited, but is preferably 50 to 200 mM.
（Ｂ）L-アルギニン様化合物のスクリーニング方法
本発明のL-アルギニン様化合物のスクリーニング方法は、L-アルギニン結合因子とL-アルギニン又はL-アルギニン誘導体とからなる結合体に、被験物質を接触させ、L-アルギニン又はL-アルギニン誘導体からL-アルギニン結合因子を解離させる被験物質を選択することを特徴とするものである。
(B) Screening method for L-arginine-like compound The screening method for an L-arginine-like compound of the present invention comprises contacting a test substance with a conjugate comprising an L-arginine binding factor and L-arginine or an L-arginine derivative. A test substance that dissociates the L-arginine-binding factor from L-arginine or an L-arginine derivative is selected.
本発明において「L-アルギニン様化合物」とは、L-アルギニンと同様の生理活性（例えば、インスリン分泌活性、NOSアンタゴニスト活性など）を持つ化合物をいう。 In the present invention, “L-arginine-like compound” refers to a compound having the same physiological activity as L-arginine (eg, insulin secretion activity, NOS antagonist activity, etc.).
L-アルギニン結合因子としては、例えば、上述したL-アルギニン結合因子の精製方法によって試料中から精製されたものを使用することができる。その具体例としては、ホスホフラクトキナーゼ（PFK）、RuvB-like 2、RuvB-like 1などを挙げることができる。 As the L-arginine binding factor, for example, one purified from the sample by the above-described purification method of L-arginine binding factor can be used. Specific examples thereof include phosphofructokinase (PFK), RuvB-like 2, RuvB-like 1 and the like.
L-アルギニン誘導体としては、アルギニン結合因子と結合できるものであれば特に限定されないが、上述したL-アルギニン結合因子の精製方法において使用されるアルギニン分子中のカルボキシル基とアミノ基による自己縮合を起こさない化学構造をとり、かつインスリン分泌活性とNOSアンタゴニスト活性を有するL-アルギニン誘導体を使用することができ、より好適には、L-アルギニンメチルエステル等を使用することができる。 The L-arginine derivative is not particularly limited as long as it can bind to an arginine binding factor, but self-condensation is caused by a carboxyl group and an amino group in the arginine molecule used in the above-described method for purifying L-arginine binding factor. L-arginine derivatives having a non-chemical structure and having insulin secretion activity and NOS antagonist activity can be used, and L-arginine methyl ester and the like can be more preferably used.
被験物質はどのようなものでもよく、精製されているものが好ましいが、未精製の細胞抽出液のようなものであってもよい。 The test substance may be any substance, preferably purified, but may be an unpurified cell extract.
L-アルギニン結合因子とL-アルギニン又はL-アルギニン誘導体とからなる結合体に、被験物質を接触させる方法は特に限定されず、例えば、担体（担体は、上述したL-アルギニン結合因子の精製方法に用いたものを使用できる。）にL-アルギニン又はL-アルギニン誘導体を固定し、固定されたL-アルギニン又はL-アルギニン誘導体にL-アルギニン結合因子結合させて担体固定化結合体を調製し、この担体固定化結合体を被験物質と混合する方法、担体固定化結合体をカラムに充填し、そのカラムに試料を通液する方法、バッチ法、競合阻害法などを挙げることができる。担体固定化結合体を被験物質と混合する方法は、例えば、担体固定化結合体を被験物質と適当な緩衝液中で混合し、一定時間この混合状態を維持することにより行うことができる（この際、攪拌・振盪しながら混合状態を維持してもよい。）。混合状態を維持する時間は特に限定されないが、１分〜１０時間程度が好適である。また、このときの温度も特に限定されないが、４〜３７℃程度が好適である。 A method for bringing a test substance into contact with a conjugate comprising an L-arginine binding factor and L-arginine or an L-arginine derivative is not particularly limited. For example, a carrier (the carrier is a method for purifying the above-mentioned L-arginine binding factor) The carrier-immobilized conjugate was prepared by immobilizing L-arginine or an L-arginine derivative to the immobilized L-arginine or L-arginine derivative and binding the L-arginine binding factor to the immobilized L-arginine derivative. Examples thereof include a method of mixing the carrier-immobilized conjugate with a test substance, a method of filling the column with the carrier-immobilized conjugate and passing a sample through the column, a batch method, and a competitive inhibition method. The carrier-immobilized conjugate can be mixed with the test substance by, for example, mixing the carrier-immobilized conjugate with the test substance in an appropriate buffer and maintaining this mixed state for a certain period of time (this In this case, the mixed state may be maintained while stirring and shaking.) The time for maintaining the mixed state is not particularly limited, but is preferably about 1 minute to 10 hours. Moreover, the temperature at this time is not particularly limited, but is preferably about 4 to 37 ° C.
L-アルギニン又はL-アルギニン誘導体からL-アルギニン結合因子が解離したかどうかは、例えば、L-アルギニン結合因子を標識（放射性標識や蛍光標識など）しておき、その標識の検出によって判定することができる。解離した因子をSDS電気泳動し、その後銀染色する方法や、もし、同因子に対する抗体が存在すれば、SDS電気泳動後、ウエスタンブロット等によりL-アルギニン結合因子の検出を判定することができる。 Whether L-arginine-binding factor is dissociated from L-arginine or L-arginine derivative is determined by, for example, labeling L-arginine-binding factor (such as radioactive label or fluorescent label) and detecting the label. Can do. Detection of L-arginine-binding factor can be determined by SDS electrophoresis of the dissociated factor, followed by silver staining, and if an antibody against the factor is present, after SDS electrophoresis, Western blot or the like.
以下、実施例により本発明を更に詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
〔実験方法〕
材料
L-アルギニン、L-アルギニンメチルエステル、Nω-ニトロ-L-アルギニンメチルエステル、キモトリプシンはSigma-Aldrich(ST Louis, MO, USA)より購入した。2,3-Diaminonaphthalene、succinic anhydride、triethylamine、dithiothreitol、iodoacetamideはナカライより購入した。L-フェニルアラニン、ethyleneglycol diglycidyl ether(EDGE)は和光純薬より購入した。N-Hydroxysuccinimed(NHS)はペプチド研究所より購入した。TrypsinはPromegaより購入した。HeLa細胞及びNIT-1細胞は共にATCCより購入した。293FT細胞はInvitrogenより購入した。
〔experimental method〕
material
L-arginine, L-arginine methyl ester, Nω-nitro-L-arginine methyl ester, chymotrypsin were purchased from Sigma-Aldrich (ST Louis, MO, USA). 2,3-Diaminonaphthalene, succinic anhydride, triethylamine, dithiothreitol and iodoacetamide were purchased from Nacalai. L-phenylalanine and ethyleneglycol diglycidyl ether (EDGE) were purchased from Wako Pure Chemical. N-Hydroxysuccinimed (NHS) was purchased from Peptide Institute. Trypsin was purchased from Promega. Both HeLa cells and NIT-1 cells were purchased from ATCC. 293FT cells were purchased from Invitrogen.
インスリン産生解析
L-アルギニン又はL-アルギニンメチルエステルによるインスリン分泌促進は、Weinhausら1997に従って測定した。簡単には、NIT-1細胞を培養した後、培地をperfusion solution(10mM HEPES-NaOH pH7.4、145mM NaCl、5mM KCl、1mM CaCl2、10mM Glucose)に10分間置き換えた。最終濃度(0, 0.2, 0.6, 2.0, 20.0 mM)になるようにL-アルギニン又はL-アルギニンメチルエステルを加えて１０分間後、perfusion solotionを回収した。インスリン濃度をinsulin ELISA kit（シバヤギ）にて測定した。実験値は、４回の独立した実験の平均値±標準誤差で表示した。
Insulin production analysis
Insulin secretion enhancement by L-arginine or L-arginine methyl ester was measured according to Weinhaus et al. 1997. Briefly, after NIT-1 cells were cultured, the medium was replaced with a perfusion solution (10 mM HEPES-NaOH pH 7.4, 145 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 10 mM Glucose) for 10 minutes. L-arginine or L-arginine methyl ester was added to a final concentration (0, 0.2, 0.6, 2.0, 20.0 mM), and after 10 minutes, perfusion solotion was recovered. Insulin concentration was measured with an insulin ELISA kit (Shibagoat). Experimental values are expressed as the mean value ± standard error of four independent experiments.
NO産生解析
293FT細胞(5.0x105細胞)へ80%以上の効率でiNOS発現ベクターを導入した。24時間後、培養上清を回収し、NOS活性を測定した(Miskoら1993)。簡単には、10μLの用事調整の2,3-Diaminonapphthalene (50μg/mL in 620mM HCl)を100μLの培地に加えよく撹拌し、室温で10分間インキュベートした後、5μLの2.8N NaOHにて反応を止め、産生された2,3-diaminonaphthotriazoleを蛍光プレートリーダー（励起波長：365nm、発光波長：450nm）にて測定した。結果は平均値±標準誤差で表示した。統計学的有意（p<0.005）はt-testにて判定した。
NO production analysis
The iNOS expression vector was introduced into 293FT cells (5.0 × 10 5 cells) with an efficiency of 80% or more. After 24 hours, culture supernatants were collected and NOS activity was measured (Misko et al. 1993). Briefly, add 10 μL of conditioned 2,3-Diaminonapphthalene (50 μg / mL in 620 mM HCl) to 100 μL medium, stir well, incubate for 10 minutes at room temperature, then stop the reaction with 5 μL 2.8N NaOH. The produced 2,3-diaminonaphthotriazole was measured with a fluorescent plate reader (excitation wavelength: 365 nm, emission wavelength: 450 nm). The results are expressed as mean ± standard error. Statistical significance (p <0.005) was determined by t-test.
放射線標識した遺伝子組み換えタンパク質の作製
T7プロモーターをつけたcDNA(iNOS, PPARg, PFK, RBL1, RBL2)断片をPCRにて増幅した。PCRに用いた一方のプライマーは、各cDNAの5’側配列にT7プロモーターを付けたものであり、他方のプライマーは、各cDNAの3’側配列の相補配列にポリTを付けたものである。これらの増幅したDNA断片を用いてPromega社の転写／翻訳キットで35S標識した組換えタンパク質を作製した。
Production of radiolabeled recombinant protein
CDNA fragments with T7 promoter (iNOS, PPARg, PFK, RBL1, RBL2) were amplified by PCR. One primer used for PCR is a 5 'sequence of each cDNA with a T7 promoter, and the other primer is a sequence with a poly T added to the complementary sequence of the 3' sequence of each cDNA. . Using these amplified DNA fragments, a 35 S-labeled recombinant protein was prepared with a transcription / translation kit from Promega.
SDS電気泳動
SDS-PAGEは、Laemmli 1970に従い、キモトリプシン消化解析法、L-アルギニン結合解析法、及びアフィニティ精製したL-アルギニン結合因子の評価用のタンパク質解析法に用いた。サンプルはSDS-PAGE loading buffer (50mM Tris6.8, 2% SDS, 100mM b-ercaptoethanol, 10% glycerol, 0.01% bromophenolblue)で希釈後、98℃、5分間の加熱で変性させた。このサンプルを5-20%勾配ゲル（和光純薬）にアプライし、DPE-1020 Cassette Electrophoresis Unit(第一)を用いて、200V、60分間泳動させた。
SDS electrophoresis
SDS-PAGE was used in chymotrypsin digestion analysis method, L-arginine binding analysis method, and protein analysis method for evaluation of affinity-purified L-arginine binding factor according to Laemmli 1970. The sample was diluted with SDS-PAGE loading buffer (50 mM Tris 6.8, 2% SDS, 100 mM b-ercaptoethanol, 10% glycerol, 0.01% bromophenolblue) and then denatured by heating at 98 ° C. for 5 minutes. This sample was applied to a 5-20% gradient gel (Wako Pure Chemical Industries, Ltd.) and electrophoresed at 200 V for 60 minutes using a DPE-1020 Cassette Electrophoresis Unit (first).
キモトリプシン消化解析法
protease消化アッセイは、Allanら1992の方法を変更して行った。簡単には、放射線標識したタンパク質(iNOS, PFK, RBL1, RBL2)を100μMの化合物(L-アルギニン, L-アルギニンメチルエステル, L-フェニルアラニン)と氷中で10分間混合し、種々の濃度（0, 1, 3, 10 μg/mL）のキモトリプシンで氷中10分間部分消化を行った。タンパク質消化反応は、SDS-PAGE loading bufferの添加と５分間の煮沸で停止させた。消化産物は、SDS-PAGEで分離した。ゲルは乾燥させ、放射線標識した消化産物をオートラジオグラフィーにより解析した。
Chymotrypsin digestion analysis method
The protease digestion assay was performed by changing the method of Allan et al. 1992. Briefly, radiolabeled protein (iNOS, PFK, RBL1, RBL2) was mixed with 100 μM compound (L-arginine, L-arginine methyl ester, L-phenylalanine) in ice for 10 minutes to various concentrations (0 , 1, 3, 10 μg / mL) was partially digested in ice for 10 minutes. The protein digestion reaction was stopped by adding SDS-PAGE loading buffer and boiling for 5 minutes. Digested products were separated by SDS-PAGE. The gel was dried and the radiolabeled digestion product was analyzed by autoradiography.
L-アルギニンメチルエステル固定化ナノビーズ
FGビーズはNishioら2008の論文に従って作製した。FGビーズのエポキシ基はNH4OHでaminolyseし、ethyleneglycol diglycidyl ether (EGDE)と反応させFGNEGDEビーズを作製した(Shimizuら2000)。FGNEGDEビーズのエポキシ基はNH4OHでamiloyseさせ(FGNEGDEN)、succinie anhydrideでcarboxyle化させ(FGNEGDENS)、N-Hydroxysuccinimed (NHS)で活性化させてNHS活性化FGNEGDENSビーズを作製した(Ohtsuら2005)。
L-arginine methyl ester immobilized nanobeads
FG beads were made according to the paper by Nishio et al. 2008. The epoxy group of FG beads was aminolyse with NH 4 OH and reacted with ethyleneglycol diglycidyl ether (EGDE) to produce FGNEGDE beads (Shimizu et al. 2000). Epoxy group FGNEGDE beads were amiloyse with NH 4 OH (FGNEGDEN), was carboxyle of at succinie anhydride (FGNEGDENS), N- Hydroxysuccinimed by (NHS) according to the supplier to produce a NHS activated FGNEGDENS beads (Ohtsu et al. 2005) .
NHS活性化FGNEGDENSビーズ2.5mgを500μLのDMSO（10% triethylamine）中にて1.5mM L-アルギニンメチルエステルと37℃3時間反応させた。結合反応中の遊離したNHSを結合していないL-アルギニンメチルエステルとともに回収した。結合収率は、symmetryC18カートリッジ, 5μm（Waters社）上でビーズより遊離したNHSによって決定した。通常L-アルギニンメチルエステルの40%がビーズに固定化される。ビーズ上に固定化されたL-アルギニンメチルエステルの量は固定反応に加えたL-アルギニンメチルエステルの量で制御することができる。未反応のNHS活性化カルボキシル基は、1Mの2-ethanolamine(pH8.0)による4℃24時間の処理でマスクされる。L-アルギニンメチルエステル固定化ビーズは蒸留水にて懸濁し、4℃で使用まで保存した。 NHS-activated FGNEGDENS beads (2.5 mg) were reacted with 1.5 mM L-arginine methyl ester in 500 μL of DMSO (10% triethylamine) at 37 ° C. for 3 hours. Free NHS during the coupling reaction was recovered along with unbound L-arginine methyl ester. The binding yield was determined by NHS released from the beads on a symmetry C18 cartridge, 5 μm (Waters). Usually 40% of L-arginine methyl ester is immobilized on the beads. The amount of L-arginine methyl ester immobilized on the beads can be controlled by the amount of L-arginine methyl ester added to the immobilization reaction. Unreacted NHS activated carboxyl groups are masked by treatment with 1M 2-ethanolamine (pH 8.0) at 4 ° C. for 24 hours. The L-arginine methyl ester-immobilized beads were suspended in distilled water and stored at 4 ° C. until use.
L-アルギニン結合アッセイ
L-アルギニンメチルエステル固定化ビーズ又はコントロールビーズ(200μg)はbinding buffer(20mM HEPES-NaOH pH7.9, 10% glycerol, 200mM KCl, 1mM MgCl2, 0.2mM CaCl2, 0.2mM EDTA, 1mM DTT, 0.2mM PMSF)で平衡化させ、200μLの放射線標識したタンパク質(iNOS, PPARg, PFK, RBL1, RBL2)と混合後4℃で4時間RT-50 ローテーター(15 rpm, TAITEC)で反応させた。Binding bufferを洗浄後、結合したタンパク質をSDS-PAGEサンプルバッファーにて５分間煮沸することにより溶出させた。溶出分、反応インプットはSDS-PAGEを行った。ゲルは乾燥後オートラジオグラフィーし、放射線標識タンパク質を可視化させた。
L-arginine binding assay
L-arginine methyl ester immobilized beads or control beads (200 μg) are binding buffer (20 mM HEPES-NaOH pH 7.9, 10% glycerol, 200 mM KCl, 1 mM MgCl 2 , 0.2 mM CaCl 2 , 0.2 mM EDTA, 1 mM DTT, 0.2 The mixture was equilibrated with mM PMSF), mixed with 200 μL of radiolabeled protein (iNOS, PPARg, PFK, RBL1, RBL2), and then reacted at 4 ° C. for 4 hours with an RT-50 rotator (15 rpm, TAITEC). After washing the binding buffer, the bound protein was eluted by boiling in SDS-PAGE sample buffer for 5 minutes. SDS-PAGE was used for elution and reaction input. The gel was dried and autoradiographed to visualize the radiolabeled protein.
L-アルギニン結合因子のアフィニティ精製
HeLa細胞の細胞質抽出液は4Lの培養液から作製した（Dignamら1983）。L-アルギニンメチルエステル固定化ナノビーズ又はコントロールビーズ(200μg)をbinding buffer (20mM HEPES-NaOH 7.9, 10% glycerol, 200mM KCl, 1mM MgCl2, 0.2mM CaCl2, 0.2mM EDTA, 1mM DTT, 0.2mM PMSF)にて平衡化し、200μlの細胞質抽出液を4℃で4時間RT-50ローテーター(15rpm, TAITEC)で撹拌した。Binding bufferで洗浄後、結合タンパク質は100mM arginineを含むbinding bufferにて溶出した。
Affinity purification of L-arginine binding factor
The cytoplasmic extract of HeLa cells was prepared from 4 L culture (Dignam et al. 1983). L- arginine methyl ester immobilized nanobeads or control beads (200μg) binding buffer (20mM HEPES -NaOH 7.9, 10% glycerol, 200mM KCl, 1mM MgCl 2, 0.2mM CaCl 2, 0.2mM EDTA, 1mM DTT, 0.2mM PMSF ), And 200 μl of the cytoplasmic extract was stirred with an RT-50 rotator (15 rpm, TAITEC) at 4 ° C. for 4 hours. After washing with the binding buffer, the binding protein was eluted with a binding buffer containing 100 mM arginine.
L-アルギニン結合因子のMass spectrometry解析
アフィニティ精製したL-アルギニン結合因子はSDS-PAGEにより分離し、ゲルを銀染色した(Shevchenkoら1996)。特異的なタンパク質のバンドをゲルから切り出し、55mM iodoacetamideのalkylationののち10mM DTTによる還元反応をした。切り出したバンドはtrypsin(12 ug/mL)で一晩消化し、ZipTip C18 (Millipore)で脱塩した。逆相C18カラム(Magic C18)を用いて、抽出したペプチドはその後nano flow liquid chromatography (LC, Paradigm)で分離した。LC溶出はLCQ Advantage MAXX mass spectrometer (Thermo Electron Corp)付属のmicro-ionsprayに供した。全てのMS/MS spectraはBioWorks 3.2ソフトウエア(Thermo Electron Corp)についているTurboSEQUEST algorithmでサーチした。
Mass spectrometry analysis of L-arginine binding factor Affinity purified L-arginine binding factor was separated by SDS-PAGE and the gel was silver stained (Shevchenko et al 1996). A specific protein band was excised from the gel, and reduced with 10 mM DTT after alkylation of 55 mM iodoacetamide. The excised band was digested overnight with trypsin (12 ug / mL) and desalted with ZipTip C18 (Millipore). The extracted peptides were then separated by nano flow liquid chromatography (LC, Paradigm) using a reverse phase C18 column (Magic C18). LC elution was performed on a micro-ionspray attached to an LCQ Advantage MAXX mass spectrometer (Thermo Electron Corp). All MS / MS spectra were searched with the TurboSEQUEST algorithm supplied with BioWorks 3.2 software (Thermo Electron Corp).
〔結果と考察〕
L-アルギニンメチルエステルの活性評価(図１及び２)
L-アルギニン結合因子を同定するため、複数のL-アルギニン誘導体をスクリーニングしたところ、L-アルギニンメチルエステルが候補化合物として選ばれた（データは示さない）。L-アルギニンは最も強力なインスリン分泌促進活性を持っている（Kahnら2005）。しかしながらその促進メカニズムはわかっていないし、其のL-アルギニン結合因子は同定されていない。実際L-アルギニンは膵臓βNIT-1細胞株のインスリン分泌を促進する（図1Aの○）。（インスリン分泌）促進はL-アルギニン 2mMにて起こる。20mM L-アルギニンは2mM L-アルギニンより低いが有意ではない。L-アルギニンメチルエステルもドーズ依存的にインスリン分泌を促進する[■破線、図1A]。L-アルギニンメチルエステルおよびL-アルギニンはインスリン産生の強力な活性化剤である。
〔Results and discussion〕
Activity evaluation of L-arginine methyl ester (Figures 1 and 2)
When multiple L-arginine derivatives were screened to identify L-arginine binding factors, L-arginine methyl ester was selected as a candidate compound (data not shown). L-arginine has the most potent insulinotropic activity (Kahn et al 2005). However, the promotion mechanism is not known, and the L-arginine binding factor has not been identified. In fact, L-arginine promotes insulin secretion in the pancreatic βNIT-1 cell line (◯ in FIG. 1A). (Insulin secretion) promotion occurs at 2 mM L-arginine. 20 mM L-arginine is lower but not significant than 2 mM L-arginine. L-arginine methyl ester also promotes insulin secretion in a dose-dependent manner (■ broken line, FIG. 1A). L-arginine methyl ester and L-arginine are potent activators of insulin production.
NOおよびシトルリンはNOSによりL-アルギニンから変換される。Dawsonら1991によるとNω-ニトロ-L-アルギニンメチルエステルはNOSに高い親和性で結合し、結合性アンタゴニストである。実際、Nω-ニトロ-L-アルギニンメチルエステルはNO代謝物であるnitrite（亜硝酸塩エステル）およびnitrate（硝酸塩）の産生を抑える（△、図1B）。（NOはすぐに分解されてしまうため、通常はnitrite, nitrateの量を持ってNO産生量とする。）L-アルギニンメチルエステルもNO代謝物の産生を抑える（■破線、図1B）。よってNω-ニトロ-L-アルギニンメチルエステルもL-アルギニンメチルエステルもNOSの結合性アンタゴニストである。 NO and citrulline are converted from L-arginine by NOS. According to Dawson et al. 1991, Nω-nitro-L-arginine methyl ester binds to NOS with high affinity and is a binding antagonist. Indeed, Nω-nitro-L-arginine methyl ester suppresses the production of NO metabolites, nitrite and nitrate (Δ, FIG. 1B). (NO is decomposed immediately, so the amount of nitrite and nitrate is usually used as NO production.) L-arginine methyl ester also suppresses the production of NO metabolites (■ dashed line, Fig. 1B). Thus, Nω-nitro-L-arginine methyl ester and L-arginine methyl ester are both NOS binding antagonists.
リガンド依存的転写因子である核内受容体の場合、リガンドと結合するとその立体構造が変化し、proteaseに対する感受性が変化することが報告されている(Allanら1992、Bergerら1996、Elbrechtら1999)。試験管内にて合成されたiNOSはアミノ酸がない場合、L-アルギニン、L-アルギニンメチルエステル、L-フェニルアラニン存在下でキモトリプシンにより部分消化される(図 2)。L-アルギニンを加えた時、コントロール（非存在下およびL-フェニルアラニン存在下）に比べてiNOSのキモトリプシン感受性が低くなる。L-アルギニンメチルエステルを加えてもL-アルギニンと同様にキモトリプシンへの感受性が低くなる。L-アルギニンおよびL-アルギニンメチルエステル自身がキモトリプシンの活性は変化させない。これはこれらに結合しないと考えられるPPARgに加えてもキモトリプシンの感受性は変化しないからである（データは示さない）。よって、L-アルギニンメチルエステルおよびL-アルギニンは試験管内でiNOSタンパク質に結合し、立体構造を変化させ、キモトリプシンのiNOSへのアクセスを変化させ、iNOSのキモトリプシンの感受性の低下を認めたと推測される。まとめると、L-アルギニンメチルエステルはL-アルギニン結合因子を効果的に精製する際ナノビーズに固定化するのに適した化合物であることが分かった。 In the case of nuclear receptors, which are ligand-dependent transcription factors, it has been reported that binding to a ligand changes its conformation and changes its sensitivity to proteases (Allan et al. 1992, Berger et al. 1996, Elbrecht et al. 1999). . In the absence of amino acids, iNOS synthesized in vitro is partially digested with chymotrypsin in the presence of L-arginine, L-arginine methyl ester, and L-phenylalanine (FIG. 2). When L-arginine is added, iNOS is less sensitive to chymotrypsin compared to controls (in the absence and in the presence of L-phenylalanine). Even when L-arginine methyl ester is added, the sensitivity to chymotrypsin is reduced in the same manner as L-arginine. L-arginine and L-arginine methyl ester itself do not change the activity of chymotrypsin. This is because the sensitivity of chymotrypsin does not change when added to PPARg, which is thought not to bind to them (data not shown). Thus, it is speculated that L-arginine methyl ester and L-arginine bound in vitro to iNOS protein, altered conformation, altered chymotrypsin access to iNOS, and reduced iNOS chymotrypsin sensitivity. . In summary, L-arginine methyl ester was found to be a suitable compound for immobilization on nanobeads when effectively purifying L-arginine binding factors.
L-アルギニンメチルエステル固定化ビーズの作製（図 3）
L-アルギニン結合因子を精製することを目的としてL-アルギニンメチルエステル固定化ビーズを作製した。L-アルギニンではなくL-アルギニンメチルエステルを選択した。なぜならば、ビーズへの固定化反応の時L-アルギニンはそのカルボキシル基とアミノ基で自己縮合がおきることを防ぐ意味がある。
Preparation of L-arginine methyl ester immobilized beads (Fig. 3)
For the purpose of purifying L-arginine binding factor, L-arginine methyl ester immobilized beads were prepared. L-arginine methyl ester was selected instead of L-arginine. This is because L-arginine has a meaning to prevent self-condensation from occurring at its carboxyl group and amino group during the immobilization reaction to beads.
図 3にL-アルギニンメチルエステルとFGビーズの結合のスキームを示した。簡単に書くと、FGビーズのエポキシ基はNH4OHによりaminolyseされ、EGDEと結合し、FGNEGDEビーズを作製した。EDGEはスペーサーでsteric hindranceを抑えるのに有効である。FGNEGDEビーズのエポキシ基はNH4OHによりaminolyseされ、succinic anhydrideによりcarboxylateされ、NHSにより活性化されたNHS-活性化FGNEGDENSビーズを作製した。L-アルギニンメチルエステルはそれからNHS活性化FGNEGDENSビーズへ結合させた。本発明者はNHS活性化FGNEGDENSビーズをキャリアーとして選択した。なぜなら、L-アルギニンメチルエステル固定化ビーズはこの方法で作製すると、非特異的なタンパク質の吸着が少ない（理由はL-アルギニンメチルエステルのアミノ基とNHS活性化FGNEGDENSビーズのCarboxy基によるカップリング反応から得られるアミド基は中性であるかである。(データは示さない)）。加えて、NHS活性化FGNEGDENSビーズとL-アルギニンメチルエステルのカップリング反応のはL-アルギニンメチルエステル固定化量の測定にも使えた。 Fig. 3 shows the scheme for binding L-arginine methyl ester and FG beads. Briefly, the epoxy group of FG beads was aminolyseed with NH 4 OH and combined with EGDE to produce FGNEGDE beads. EDGE is effective in suppressing steric hindrance with a spacer. The epoxy group of FGNEGDE beads was aminolyse with NH 4 OH, carboxylated with succinic anhydride, and NHS-activated FGNEGDENS beads activated with NHS were prepared. L-arginine methyl ester was then bound to NHS activated FGNEGDENS beads. The inventor selected NHS activated FGNEGDENS beads as a carrier. This is because when L-arginine methyl ester-immobilized beads are prepared by this method, non-specific protein adsorption is low (the reason is the coupling reaction between the amino group of L-arginine methyl ester and the Carboxy group of NHS-activated FGNEGDENS beads. The amide group obtained from is neutral (data not shown)). In addition, the coupling reaction between NHS-activated FGNEGDENS beads and L-arginine methyl ester could be used to measure the amount of L-arginine methyl ester immobilized.
L-アルギニンメチルエステル固定化ビーズの評価（図 4）
L-アルギニン結合因子（例えばiNOS）やL-アルギニン結合因子以外のタンパク質(例えば PPARg)に対するL-アルギニンメチルエステル固定化ビーズの結合能を解析した（図 4）。これら放射線標識したタンパク質をL-アルギニンメチルエステル固定化ビーズ（AL）、コントロールビーズ(L)と混合後、ビーズへの結合タンパク質をSDS-PAGEで解析した。iNOSはL-アルギニンメチルエステル固定化ビーズに結合し、コントロールビーズには結合しなかった。L-アルギニンメチルエステル固定化ビーズへのiNOSの結合回収率は9.6%で、L-アルギニンメチルエステルとiNOSの結合は一時的であるため、合理的な値である。PPARgはL-アルギニンメチルエステル固定化ビーズ、コントロールビーズともに結合しなかった。これらの結果は、このシステムではL-アルギニン結合因子は精製されるが、L-アルギニン結合因子以外のタンパク質は精製されないことを示した。
Evaluation of L-arginine methyl ester immobilized beads (Figure 4)
The binding ability of the L-arginine methyl ester-immobilized beads to L-arginine binding factor (eg, iNOS) and proteins other than L-arginine binding factor (eg, PPARg) was analyzed (FIG. 4). These radiolabeled proteins were mixed with L-arginine methyl ester immobilized beads (AL) and control beads (L), and then the proteins bound to the beads were analyzed by SDS-PAGE. iNOS bound to L-arginine methyl ester immobilized beads and not to control beads. The recovery rate of iNOS binding to L-arginine methyl ester-immobilized beads is 9.6%, and the binding between L-arginine methyl ester and iNOS is temporary, which is a reasonable value. PPARg did not bind to either L-arginine methyl ester immobilized beads or control beads. These results indicated that L-arginine binding factor was purified in this system, but proteins other than L-arginine binding factor were not purified.
L-アルギニンメチルエステル固定化ビーズによるL-アルギニン結合因子の精製・同定(図 ５及び６)
遺伝子組み換えタンパク質の代わりにHeLa細胞由来粗細胞質抽出液をL-アルギニンメチルエステル固定化ビーズと混合し、HeLa細胞からの未知L-アルギニン結合因子の精製を試みた。結合タンパク質をSDS-PAGEで分離し、銀染色で可視化した。結合タンパク質のパターンは4回の独立した精製でほぼ同じであった（データは示さない）。４回の精製から得られた結合因子を混ぜてSDS-PAGE/銀染色した（図 5A）。特異的なバンドの中で３つのバンド（80, 51, 50 kDa）からタンパク質の同定に成功した。Mass解析により各々phosphofructokinase (PFK), RuvB-like 2 (RBL2), RuvB-like 1 (RBL1)であった(図 5B)。予想又は仮想タンパク質と同定された他のバンドに関しては解析を行わなかった。
Purification and identification of L-arginine binding factor using L-arginine methyl ester immobilized beads (Figures 5 and 6)
HeLa cell-derived crude cytoplasmic extract was mixed with L-arginine methyl ester-immobilized beads instead of the recombinant protein, and purification of unknown L-arginine binding factor from HeLa cells was attempted. Bound proteins were separated by SDS-PAGE and visualized by silver staining. The pattern of binding protein was almost the same in 4 independent purifications (data not shown). The binding factors obtained from the four purifications were mixed and subjected to SDS-PAGE / silver staining (FIG. 5A). Proteins were successfully identified from three of the specific bands (80, 51, 50 kDa). Mass analysis revealed phosphofructokinase (PFK), RuvB-like 2 (RBL2), and RuvB-like 1 (RBL1) (FIG. 5B). No analysis was performed on other bands identified as expected or hypothetical proteins.
PFK、RBL1、及びRBL2の放射線標識した遺伝子組み換えタンパク質を調製し、L-アルギニン結合活性を確かめた。まず、L-アルギニンメチルエステル固定化ビーズ(AL)又はコントロールビーズ(L)と混合後結合タンパク質をSDS-PAGEにて解析した（図 6A）。これら３タンパク質はL-アルギニンメチルエステル固定化ビーズに結合し、コントロールビーズには結合しなかった。回収率はRBL1, RBL1, PFK固定化ビーズ各々2.2%, 1.4%, 1.2%であった。これらの回収率からは、PFK, RBL1, RBL2はiNOSに比べて回収率が低いこと、及びL-アルギニンメチルエステル固定化ビーズは低いアフィニティのタンパク質も回収できることが示唆される。さらには、キモトリプシン消化アッセイも行った。放射線標識タンパク質をアミノ酸非存在下、L-アルギニン, L-フェニルアラニン存在下でキモトリプシン消化を行った(図 6B)。L-アルギニン存在下ではキモトリプシン感受性が陰性コントロール（非存在下ないしはL-フェニルアラニン存在下）に比べて低下したり、増加したりした。 Radiolabeled recombinant proteins of PFK, RBL1, and RBL2 were prepared and L-arginine binding activity was confirmed. First, after binding with L-arginine methyl ester immobilized beads (AL) or control beads (L), the binding protein was analyzed by SDS-PAGE (FIG. 6A). These three proteins bound to L-arginine methyl ester immobilized beads and did not bind to control beads. The recovery rates were 2.2%, 1.4%, and 1.2% for RBL1, RBL1, and PFK-immobilized beads, respectively. These recovery rates suggest that PFK, RBL1, and RBL2 have lower recovery rates than iNOS, and that L-arginine methyl ester-immobilized beads can recover proteins with low affinity. In addition, a chymotrypsin digestion assay was also performed. The radiolabeled protein was digested with chymotrypsin in the absence of amino acids and in the presence of L-arginine and L-phenylalanine (FIG. 6B). In the presence of L-arginine, chymotrypsin sensitivity decreased or increased compared to the negative control (in the absence or in the presence of L-phenylalanine).
まとめると、L-アルギニンメチルエステル固定化ビーズはHeLa細胞粗細胞質抽出液から直接L-アルギニン結合因子(PFK, RBL1, RBL2)を精製することに成功した。一般的には数種類のカラムクロマトグラフィーが粗細胞抽出液から精製するためには必要である。加えて磁性担体はオートマティックハイスループットスクリーニングシステムに適している。この実験に使用した最近開発した磁性担体は、市販されている磁性体に比べて効率が良かった（Nishioら2008）。それゆえ本実験で開発したL-アルギニンメチルエステル固定化ビーズは、種々の細胞から、極めて広い条件で、いろいろな試薬の条件においても高効率にL-アルギニン結合因子を精製できるものである。 In summary, L-arginine methyl ester-immobilized beads succeeded in purifying L-arginine binding factors (PFK, RBL1, RBL2) directly from HeLa cell crude cytosolic extract. In general, several types of column chromatography are required for purification from crude cell extracts. In addition, the magnetic carrier is suitable for an automatic high throughput screening system. The recently developed magnetic carrier used for this experiment was more efficient than commercially available magnetic materials (Nishio et al. 2008). Therefore, the L-arginine methyl ester-immobilized beads developed in this experiment can purify L-arginine-binding factor from various cells with high efficiency and under various reagent conditions.
〔結論〕
ヒトにおいて準必須アミノ酸であるL-アルギニンは代謝面で多機能である。また、L-アルギニンはUrea, NO, polyamines, Proline, glutamate, creatine, agmatineの前駆体である。L-アルギニンは複合体により代謝され、多くの不明の多数の機能やシグナルにより制御されている。
[Conclusion]
L-arginine, which is a semi-essential amino acid in humans, is multifunctional in terms of metabolism. L-arginine is a precursor of Urea, NO, polyamines, Proline, glutamate, creatine, and agmatine. L-arginine is metabolized by the complex and is controlled by many unknown functions and signals.
L-アルギニン結合因子を精製し、同定し、研究するためにL-アルギニンメチルエステル固定化ビーズを開発した。このシステムは粗細胞抽出液から直接L-アルギニン結合因子を高効率で精製できることを示した。L-アルギニンメチルエステル固定化ビーズはL-アルギニン結合因子精製に強力なツールであると結論した。L-アルギニンメチルエステル固定化ビーズは、例えば膵臓β細胞、下垂体、内皮等の種々の細胞からL-アルギニン結合因子を精製できるであろう。それら因子の同定・解析はL-アルギニンの種々の機能（糖によるインスリン分泌促進、成長ホルモンの分泌促進、内皮細胞の機能）のメカニズムを明らかにするであろう。 L-arginine methyl ester immobilized beads have been developed to purify, identify and study L-arginine binding factors. This system showed that L-arginine binding factor could be purified with high efficiency directly from crude cell extract. We conclude that L-arginine methyl ester immobilized beads are a powerful tool for L-arginine binding factor purification. L-arginine methyl ester immobilized beads could purify L-arginine binding factor from various cells such as pancreatic β cells, pituitary, endothelium. Identification and analysis of these factors will clarify the mechanisms of various functions of L-arginine (stimulation of insulin secretion by sugar, promotion of growth hormone secretion, function of endothelial cells).
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Tong BC and Barbul A. Cellular and physiological effects of arginine.Mini Reviews in Medicinal Chemistry 2004; 4: 823-832.
Wada T, Watanabe H, Kawaguchi H and Handa H. DNA affinity chromatography. Methods in Enzymology 1995; 254: 595-604.
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本発明は、インスリン分泌促進作用を持つ化合物など医薬として利用可能な化合物を取得する手段を提供する。従って、本発明は、製薬などの産業分野において利用可能である。 The present invention provides a means for obtaining a pharmaceutically usable compound such as a compound having an insulin secretion promoting action. Therefore, the present invention can be used in industrial fields such as pharmaceuticals.
Claims (8)
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07316023A (en) * | 1994-05-26 | 1995-12-05 | Shiseido Co Ltd | Hair tonic |
| JPH09255589A (en) * | 1996-03-28 | 1997-09-30 | Shiseido Co Ltd | Stress suppressant |
| JP2001512471A (en) * | 1997-02-21 | 2001-08-21 | バイヤースドルフ・アクチエンゲゼルシヤフト | Preparations for the treatment of rosacea |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH07316023A (en) * | 1994-05-26 | 1995-12-05 | Shiseido Co Ltd | Hair tonic |
| JPH09255589A (en) * | 1996-03-28 | 1997-09-30 | Shiseido Co Ltd | Stress suppressant |
| JP2001512471A (en) * | 1997-02-21 | 2001-08-21 | バイヤースドルフ・アクチエンゲゼルシヤフト | Preparations for the treatment of rosacea |
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