JP3789134B2 - Plant cell growth factor - Google Patents

Description

（式中、Ｒ¹又はＲ²は同一又は異なってSO₃H又はＨを表し、Ｘはα−アミノ酸又は単結合を表し、Ｚ¹およびＺ²は同一又は異なったα−アミノ酸を表し、ＹはOH又はNH₂を表す）で表されるペプチドに関する。
以下、式（Ｉ）で表される化合物を化合物（Ｉ）と称す。
式（Ｉ）の各基の定義において、α−アミノ酸とは、グリシン、アラニン、バリン、ロイシン、イソロイシン等の脂肪族アミノ酸類、セリン、トレオニン等のオキシアミノ酸類、システイン、シスチン、メチオニン等の含イオウアミノ酸類、アスパラギン酸、グルタミン酸等の酸性アミノ酸類、アスパラギン、グルタミン等のアミドアミノ酸類、リジン、アルギニン、オルニチン等の塩基性アミノ酸類、フェニルアラニン、チロシン等の芳香族アミノ酸類、ヒスチジン、トリプトファン、プロリン、オキシプロリン等の複素環アミノ酸類があげられる。Ｘにおいては前記α−アミノ酸の中でもアミドアミノ酸類が好ましく、とりわけグルタミンが好ましい。Ｚ¹においては前記α−アミノ酸の中でも脂肪族アミノ酸が好ましく、なかでもバリン、イソロイシンがとりわけ好ましい。Ｚ²においては前記α−アミノ酸の中でもオキシアミノ酸類が好ましい。
化合物（Ｉ）は、高等植物からの抽出あるいは通常のペプチド合成法で得ることができる植物細胞増殖因子である。
本発明における細胞増殖因子を抽出する植物としては、アスパラガス、イネ、トウモロコシ等の単子葉植物であればどのようなものでもよく、抽出は、植物体からの水性媒体による抽出法（植物細胞組織培養、原田および駒嶺編、１９７９年、理工学社刊、３８２頁）、培養細胞の培養液からの回収法（植物細胞組織培養、原田および駒嶺編、１９７９年、理工学社刊、２７頁）により行なうことができる。
本発明における植物細胞増殖因子は、すべての植物の細胞増殖を促進するが、とくにアスパラガス、イネ、トウモロコシ等の単子葉植物の細胞増殖促進に優れている。
高等植物からの抽出法は、例えば常法により培養する細胞を採取し（植物細胞組織培養、原田および駒嶺編、１９７９年、理工学社刊、２７頁）、採取した細胞を通常の植物細胞培養に用いる培地（プラントサイエンス、６５巻、１１１〜１７１頁、１９８９年）に移植して、２０〜３０℃、好ましくは２４〜２８℃の温度で８〜１６日間、好ましくは９〜１１日間振盪培養する。振盪培養終了後に細胞と培養液を遠心分離等の操作で分離し、調製培養液（Conditioned Medium、以下にＣＭという）を得る。
DEAE Sephadex A-25、DEAEセルロース、DEAE Sepharose等陰イオン交換樹脂を１０〜１００mMのトリス塩酸緩衝液、リン酸緩衝液、炭酸ナトリウム-炭酸緩衝液等の緩衝液（ｐＨ6.5〜8.0）を用いて膨潤させた後、カラム法又はバッチ法によりＣＭ中の植物細胞増殖因子を吸着させる。次に塩化カリウム、炭酸ナトリウム等の塩濃度を順次１０〜２０００mMまで増加させて溶出を行なうことにより植物細胞増殖因子を回収することができる。植物細胞増殖因子は上記の溶出画分のうち８００〜２０００mMの塩濃度好ましくは１０００〜１２５０mMの塩濃度の領域に溶出される。
得られた画分を、更に透析等により脱塩した後、好ましくはBio-Gel P2、Bio-Gel P4（以上、バイオラド社製）、セファデックスG25（以上、ファルマシア社製）等のゲル浸透カラムにかける。
透析、ゲル浸透画分等で脱塩した活性画分をヌクレオシル-100-C18（ナーゲル社製）、ミクロゾルブPR18（メルク社製）等を担体とする逆層高速液体クロマトグラフィー（逆相ＨＰＬＣ）で分離し、トリフルオロ酢酸（ＴＦＡ）を含有した水−アセトニトリル、水−メタノール、水−エタノール等の溶媒を用いて、保持時間８〜１２分の間に溶出した画分から精製した植物細胞増殖因子を得る。
また、化合物（Ｉ）は“ペプチド合成”泉屋信夫ら著、昭和５０年丸善刊等に記載されたペプチド合成法によりペプチド骨格を合成した後、次いでアリルスルフォトランスフェラーゼ等、スルホン酸基をチロシン残基の側鎖に結合させうる酵素によるスルホン酸化を行い所望のペプチドを製造することができる。
化合物（１）は、植物成長剤として以下のような形態で使用できる。
▲１▼液剤
防腐剤、ｐＨ調整剤を含む水溶液に、化合物（Ｉ）を０．０００１〜１％溶解して植物細胞成長促進剤を製造する。防腐剤としては、ホウ酸、さらし粉、安息香酸、サリチル酸、ソルビン酸、デヒドロ酢酸、プロピオン酸、イソシアヌル酸、亜塩素酸、次亜塩素酸、パラオキシ安息香酸およびそのエステル、ラウリルトリメチルアンモニウム−２，４，５−トリクロロカルボニライト、トリブロモサリチルアニライド，３，４，４′−トリクロロカルボニライド、ヘキサクロロフェン、ビチオノール、クロラミン−Ｔ、クロラミンＢハラゾーン等好ましくはソルビン酸があげられる。ｐＨ調整剤としてはクエン酸塩、リン酸塩等一般に用いられているものを、単独あるいは組み合わせて使用できる。
前記のように製造した液剤を水に１００−１００００倍、好ましくは１０００倍に希釈したものに植物の種子あるいは差し穂等の種苗を浸漬するか、または水耕培の培養液に当該ペプタイドの最終濃度が０．００１から１０ＰＰＭになるよう添加することにより、植物成長剤として用いることができる。
▲２▼ペースト製剤
当該ペプチドを０．０１〜１０ＰＰＭペースト材料に練りこみ植物成長促進剤を製造する。ペースト材料としては脂肪、脂肪油、ラノリン、ワセリン、パラフィン、ろう、樹脂、プラスチック、グリコール、高級アルコール類、グリセリン等、好ましくはワセリン、ラノリンがあげられる。
前記のように製造したペースト製剤を接ぎ木された植物の接ぎ木部分、あるいは果実の果柄部分、あるいは切断時の切断面等に塗布することにより植物成長剤として用いることができる。
化合物（１）の具体例として

等が例示される。
【図面の簡単な説明】
図１はアスパラガス由来の培養細胞のコロニー形成率に対するＣＭ濃度の影響を示す。
図中の符号は、--□-- ＣＭ 25.０％、−◇− ＣＭ 12.５％、−○− ＣＭ 6.３％、−△− ＣＭ 3.１％、−■− ＣＭ 1.６％、−◆− コントロールを表す。
図２はアスパラガス由来の培養細胞のコロニー形成率に対する化合物（Ｉ−１）、化合物（Ｉ−２）、化合物（Ｉ−３）、化合物（Ｉ−６）、化合物（Ｉ−７）、化合物（Ｉ−８）の各種濃度の影響を示す。
図中の符号は、−□− 化合物（Ｉ−１）、−◇− 化合物（Ｉ−２）、−○− 化合物（Ｉ−３）、−△− 化合物（Ｉ−６）、−■− 化合物（Ｉ−７）、−◆− 化合物（Ｉ−８）、
図３は（ａ）DEAE Sephadex A-25イオン交換クロマトグラフィー、（ｂ）Bio-Gel P-2 extra fineクロマトグラフィー、（ｃ）Devlosil ODS-HG-5逆層ＨＰＬＣにおける植物細胞増殖因子の溶出挙動を示す。
図中の符号は、□ コロニー形成率、−吸光度（２２０nm）を表す。
発明を実施するための最良の形態
以下に本願発明の植物細胞増殖因子の植物細胞増殖活性を試験例で示す。
試験例1.
実施例（２）の方法により得られたアスパラガスの遊離細胞を実施例（３）の方法により調製した培地に移植し、本発明の植物細胞増殖因子を添加して培養し、培養アスパラガス細胞の増殖に対する植物細胞増殖因子の影響をコロニー形成率の変化を測定することにより調べた。
（ｉ）細胞の培養
細胞培養には24穴のマイクロプレート（IWAKI 3820-024）を用いた。マイクロプレートの１穴あたり、あらかじめ目的とする細胞密度の２倍密度となるように調製したアスパラガスの遊離単離細胞懸濁液250μl、４倍濃度の液体培地125μl、滅菌蒸留水125μl、または濾過滅菌(ADVANTEC DISMIC-13cp 0.20μm)後希釈した実施例（４）で得られたＣＭ 125μlを加えよく攪拌し、蒸散防止のためビニルテープでシールドした後、暗黒下、２８℃、120rpmの条件で振盪培養(TAITEC BR-300L)した。
（ｉｉ）細胞の観察
１穴ごとに１００倍の倒立顕微鏡(OLYMPUS CK2)下で、視野内に存在する生存細胞数（コロニー形成細胞を含む）、死細胞数、およびコロニー形成細胞数をカウントし、３穴以上の観察結果をもとにして、以下の計算式に従いコロニー形成率(colony formation frequency)、および細胞生存率を算出した。
Ｃ（％）＝ａ／ｂＸ１００
Ｃ：コロニー形成率 ａ：コロニー形成細胞数 ｂ：生存細胞数
Ｌ（％）＝〔ｂ／（ｂ＋ｄ）〕×１００
Ｌ：細胞生存率 ｂ：生存細胞数 ｄ：死細胞数
（iii）ＣＭがコロニー形成に及ぼす効果
5.０×１０⁴cells/mlおよび2.5×１０⁴cells/mlのアスパラガスの遊離細胞培養液中に、ＣＭを最終濃度２5.０％、１2.５％、6.３％、3.１％、1.６％となるように添加し培養した。各ＣＭ添加濃度におけるアスパラガス培養細胞のコロニー形成率を測定した。結果を図１に示す。
以下、上記方法を植物細胞増殖因子の生物検定として示す。
その結果図１によれば、ＣＭの濃度依存的に著しいコロニー形成の増加がみられ、ＣＭが植物細胞増殖活性を持つことがわかる。
試験例2.
実施例（７）で得た化合物（Ｉ−１）、化合物（Ｉ−２）、化合物（Ｉ−３）、化合物（Ｉ−６）、化合物（Ｉ−７）または化合物（Ｉ−８）（１０^-5Ｍ〜１０^-9Ｍ）を用いる以外は試験例１と同様の方法により細胞増殖活性を測定した。結果を図２に示した。
実施例
Ａ．〔ＣＭの抽出とその理化学的性質〕
（１）植物材料
アスパラガス（Asparagus officinalis L. cv. Mary Washington 500W、タキイ種苗）の種子を、クレハ培養土（クレハ化学工業）を入れた育苗用ポット（直径９cm）に播種後、人工気象室（小糸工業）内で育成した。人工気象室内の照度は葉面で２０,９９９Lux、日長は１日１６時間明期８時間暗期、温度は昼間２２℃夜間１８℃、湿度は約８０％とした。種子は通常播種後約３週間で発芽し、播種後６週目までに３本程度の芽条を生じるので、随時直径２１cmの植木鉢に移植した。実験には、播種後９週間以降の株から採集した葉状茎を用いた。
（２）葉肉細胞由来細胞の調製
長さ約１０cmのアスパラガス葉状茎を、生物検定には１本、調製培養液（ＣＭ）の調製には２００mlあたり４本使用した。採集した葉状茎は、７０％エタノールに３０秒間浸漬後、１０倍希釈アンチホルミンに１００mlあたり２滴のTween 20を加えた溶液中で１０分間滅菌し、さらに滅菌蒸留水で３回洗浄した。次に、クリーンベンチ内にてガラス製ホモジェナイザー（22×167mm、岩城硝子）を用い、滅菌蒸留水中で葉状茎を破砕後、破砕液を３７μmのステンレス製メッシュ（飯田製作所）で濾過し、濾液を遠心分離（100×g、3min、Kubota KS-5000)することにより遊離単細胞を沈殿させた。沈殿した遊離単細胞は再度滅菌蒸留水に懸濁させ、遠心分離後に上清を除去する操作を３回繰り返し、完全に夾雑物を除いた。
（３）培地の調製
培地組成を第１表に示す。

第１表に示した液体培地を、使用直前に保存液を目的濃度の４倍濃度となるように蒸留水で希釈して調製し、1.０N KOHでｐＨ5.８とした後、滅菌フィルター(ADV ANTEC DISMIC-25cs 0.２０μm）を用いて濾過滅菌した。
（４）ＣＭの採取
遊離単細胞懸濁液を、ビュケルチュルク血球計算盤（日本臨床器械工業）を用いて、約5.０×１０⁵cells/mlとなるように調製し、その５０mlと２倍濃度の液体培地５０mlの合計１００mlを３００mlの三角フラスコに加え、シリコン栓をかぶせて、暗黒下、２８℃、１２０rpmの条件で振盪培養(高崎科学器械 TB-25R)した。細胞増殖が最大となる培養開始１０日目に、吸引濾過(ADVANTEC No.2）によりＣＭを回収し、−３０℃で凍結保存した。
以下に、得られたＣＭの理化学的性質について詳述する。
（ａ）熱安定性
培養１０日目に採取したＣＭ（以下同じＣＭを使用する）1.５mlを蒸留水で2倍希釈し、沸騰水浴中で１０分間加熱後直ちに氷冷した。同様に、ＣＭ 1.５mlを蒸留水で２倍希釈し、１２１℃、２０分間のオートクレーブ処理を行なった。以上の２サンプルについて生物検定を行なった。
ＣＭを沸騰水浴中で１０分間加熱した場合には、細胞増殖活性の７０％は保持されたが、１２１℃、２０分間のオートクレーブ処理では、完全に失活した。
（ｂ）ｐＨ安定性
ＣＭ 1.０mlを蒸留水で4.０mlに希釈後、0.１N HNO₃または0.１N KOHを用いｐＨ3.０、ｐＨ5.０、ｐＨ7.０、ｐＨ9.０、ｐＨ11.０とし、４℃にて２４時間放置した。次に0.１N KOHまたは0.１N HNO₃でｐＨ5.８とした後、2.０mlに濃縮し、それぞれのサンプルについて生物検定を行なった。なお、ＣＭ自体のｐＨは約5.０であった。
本発明の植物細胞増殖因子はｐＨ3.０、ｐＨ5.０、ｐＨ7.０、ｐＨ9.０においては概ね安定であったが、ｐＨ11.０では６０％程度活性が低下した。
（ｃ）ＣＭの溶媒分画
ＣＭ2.０mlを蒸留水で１０mlに希釈し、0.１N HNO₃を用いてｐＨ3.０にした後、5.０mlの酢酸エチルで３回抽出した。得られた水層は、0.１N KOHでｐＨ5.８とした後4.０mlに濃縮した。酢酸エチル層は硫酸ナトリウムで乾燥させた後、蒸留乾固し、4.０mlの蒸留水で溶解した。同様に、ＣＭ2.０mlを蒸留水で１０mlに希釈し、0.１N KOHでｐＨ11.０にした後、5.０mlのジエチルエーテルで３回抽出した。得られた水層は、0.１N HNO₃でｐＨ5.８とした後4.０mlに濃縮した。エーテル層は硫酸ナトリウムで乾燥させた後、蒸発乾固し、4.０mlの蒸留水に溶解した。以上の４サンプルについて生物検定を行なった。
酸性条件、塩基性条件のいずれにおいても、細胞増殖促進活性は水層に残った。
（ｄ）逆相担体に対する吸脱着実験
Cosmosil 75C₁₈-OPN(Nacalai tesque)１０ｇをメタノールに懸濁し、真空下で脱気後カラム（1.７×８cm、１８ml）に充填した。担体が完全に充填された後に溶出液を蒸留水に置換し、１００mlの蒸留水でカラムを洗浄した。次に、ＣＭ5.０mlをカラムに添加し、蒸留水１００ml、３０％ＣＨ₃ＣＮ １００ml、６０％ ＣＨ₃ＣＮ １００mlにて順次溶出（流速６０ml/h）し、各画分をエバポレーターで蒸発乾固後、１０mlの蒸留水に溶解して生物検定を行なった。同様に、Diaion HP-20（三菱化成）１０ｇをメタノールに懸濁し、真空下で脱気後カラムに充填した後に、海砂Ｂを５mm厚い重層し、担体の浮き上がりを防いだ上で、溶出液を蒸留水に置換し、１００mlの蒸留水でカラムを洗浄した。次に、ＣＭ5.０mlをカラムに添加し、蒸留水１００ml、３０％ＣＨ₃ＣＮ １００ml、６０％ＣＨ₃ＣＮ １００mlにて順次溶出（流速６０ml/h）し、各画分をエバポレーターで蒸発乾固後、１０mlの蒸留水に溶解して生物検定を行なった。
Cosmosil 75C₁₈-OPNおよびDiaion HP-20のいずれの担体に対しても細胞増殖因子は保持されず、蒸留水によって溶出された。したがって、本発明の植物細胞増殖因子は比較的極性が高いことがわかる。
（ｅ）活性炭に対する吸脱着実験
活性炭（和光純薬工業）5.０ｇを、１５％酢酸１００ml中で１００℃、３０分間加熱して不純物を除き、さらに蒸留水５００mlで洗浄後、蒸留水に懸濁してカラム（1.７×１１cm、２５ml）に充填した。次にＣＭ5.０mlをカラムに添加し、蒸留水１００ml、１５％エタノール１００ml、３０％エタノール１００ml、アセトン１００mlにて順次溶出（流速６０ml/h）し、各画分をエバポレーターで蒸発乾固後、１０mlの蒸留水に溶解して生物検定を行なった。
本発明の植物細胞増殖因子は活性炭に極めて強く吸着され、１５％エタノール、３０％エタノールおよびアセトンによっても全く溶出されなかった。この条件では、中性のオリゴ糖や一部の酸性糖が溶出されることが知られている。
（ｆ）イオン交換樹脂に対する吸脱着実験
DEAE Sephadex A-25(Pharmacia LKB Biotechnology）0.８ｇを５００mM Tris-HCl緩衝液（ｐＨ7.４）５０ml中、室温で膨潤（２４時間）させた後、２０mMの同緩衝液に懸濁しカラム（1.２×3.５cm、4.０ml）に充填した。ＣＭ １０mlを凍結乾燥し、１０mlの同緩衝液に溶解した後、カラム上に添加し、同緩衝液２０ml、２５０mM ＫＣｌを含む同緩衝液２０ml、５００mM ＫＣｌを含む同緩衝液２０ml、１０００mM ＫＣｌを含む同緩衝液２０mlで順次溶出（流速１５ml/h）した。次に、各溶出画分を１０mlに濃縮し、透析チューブ(Spectra/Por 7 MWCO:1000)に注入し、両端をクローサーで閉じた後、３０００mlの蒸留水中で４℃において２４時間透析することにより脱塩を行なった。透析内液を濃縮して１０mlとし、各画分について生物検定を行なった。
同様に、ＣＭ Sephadex C-25 0.８ｇを５００mM KH₂PO₄-KOH緩衝液ｐＨ6.０ ５０ml中で、室温で膨潤（２４時間）させた後、２０mMの同緩衝液に懸濁しカラム（1.２×3.５cm、4.０ml）に充填した。ＣＭ １０mlを凍結乾燥し、１０mlの同緩衝液に溶解した後、カラム上に添加し、同緩衝液２０ml、２５０mM ＫＣｌを含む同緩衝液２０mlで溶出（流速１５ml/h）した。透析により脱塩後、各画分について生物検定を行なった。
本発明の細胞増殖因子はDEAE Sephadex A-25に極めて強く吸着され、１０００mMのＫＣｌによって溶出された。一方、ＣＭ Sephadex C-25には該細胞増殖因子は全く吸着されず、２０mM KH₂PO₄-KOH緩衝液によって溶出された。この結果から、該細胞増殖因子は酸性物質であることがわかる。
（ｇ）各種加水分解酵素による活性物質の失活試験
非特異的ペプチド分解酵素であるPronase E(Sigma)3.０mgを、２０mM KH₂PO₄-KOH緩衝液(pH7.5)3.０mlに溶解し、セルロースアセテートフィルター(ADVANTEC DISMIC-13cp 0.２０μm)で不溶物を濾過して酵素溶液とした。酵素溶液のうち1.０mlは、沸騰水浴中で１０分間加熱することにより失活酵素液とした。試験管に同緩衝液を0.９ml、ＣＭまたは蒸留水を1.０ml、酵素溶液または失活酵素液または同緩衝液を１００μl、それぞれ加え、恒温振盪器(TAITEC, Personal-11)を用い３７℃、１７０rpmにおいて３時間振盪した。酵素反応後、反応液を0.１N HNO₃でpH5.８とし、沸騰水浴中で１０分間加熱後直ちに氷冷することにより酵素を失活させた後、生物検定を行なった。
同様に、数種類の糖鎖の加水分解酵素混合物であるGlycosidases “Mixed”（生化学工業）3.０mgを、２０mM Glutamic acid-KOH緩衝液（pH4.０）3.０mlに溶解し、セルロースナイトレートフィルターで不溶物を濾過して酵素溶液とした。酵素溶液のうち1.０mlは、沸騰水浴中で１０分間加熱することにより失活させ、失活酵素液とした。試験管に同緩衝液を0.９ml、ＣＭまたは蒸留水を1.０ml、酵素溶液または失活酵素液または同緩衝液を１００μl、それぞれ加え、恒温振盪器を用い３７℃、１７０rpmにおいて３時間振盪した。酵素反応後、反応液を0.１N KOHでpH5.８とし、沸騰水浴中で１０分間加熱後直ちに氷冷することにより酵素を失活させた後、生物検定を行なった。
本発明の植物細胞増殖因子は、ペプチド分解酵素であるPronase Eを作用させることにより、完全に失活した。したがって、該細胞増殖因子は分子内にペプチド構造を持っており、この部分が活性の発現に重要であることがわかる。
一方、糖鎖の加水分解酵素混合物であるGlycosidases “Mixed”を作用させても、細胞増殖因子の活性は保持された。
以上のＣＭの理化学的性質をまとめると以下のようになる。

Ｂ．〔化合物（Ｉ）の抽出、合成、構造決定〕
（１）DEAE Sephadex A-25 3.０ｇを５００mM Tris-HCl緩衝液(pH7.4)５０ml中、室温で膨潤（２４時間）させた後、２０mMの同緩衝液に懸濁しカラム(1.7×8.０cm、１８ml）に充填した。次に、ＣＭ １００mlをエバポレーターにより５０mlに濃縮後、Trisを最終濃度２０mMとなるように加え、6N HClでpH7.４としてカラム上に添加し、同緩衝液３０ml、２５０mM ＫＣｌを含む同緩衝液３０ml、５００mM ＫＣｌを含む同緩衝液３０ml、７５０mM ＫＣｌを含む同緩衝液３０ml、１０００mM ＫＣｌを含む同緩衝液３０ml、１２５０mM ＫＣｌを含む同緩衝液３０ml、１５００mM ＫＣｌを含む同緩衝液３０mlで順次溶出した。この際、ＵＶ２２０nmにおける吸収により活性分画を推定した。
植物細胞増殖活性を示す分画が、１０００mMおよび１２５０mMのＫＣｌによって溶出された（図３−ａ）。１２５０mM ＫＣｌ溶出画分のみを凍結乾燥し秤量したところ、9.０２mgであった。
各溶出フラクションを透析（スペクトラ／Por MWCO:1000)により脱塩化した後、５０mlまで濃縮した。
（２）ゲル浸透カラム
DEAE Sephadexカラムからの脱塩化溶出液(1000mM KCl、1250mM KCl溶出フラクション）を凍結乾燥した後、1.０mlの２０mM、KH₂PO₄-KOH-緩衝液(pH5.8)に溶解した。つづいて得られた溶液を、あらかじめ上記の溶解液に用いた緩衝液で平衡化したBio-Gel P-2 extra fineカラム(1.7×37cm)に注入した。ＵＶ２２０nmにおける吸収を測定しながら該緩衝液を流速１５mh^-1で溶出させた（図3-b）。
５mlずつにフラクションを採取し、各々のフラクションの活性をバイオアッセイで測定した。
（３）逆相ＨＰＬＣカラム
Bio-Gelカラムからの溶出活性画分を凍結乾燥させた後、１０μｌの１０％アセトニトリル含有0.１％ＴＦＡに溶解した。得られた溶液をDevelosil ODS-HG-5カラムに注入した（4.６×２５０nm、野村化学社製）た後、0.１％ＴＦＡを含んだ１０％アセトニトリルのイソクラチィック溶出（isocratic elution）により分離した（流速1.０ml/m、モニターＵＶ２２０nm）。フラクション（各２ml）を採取し、各フラクションの活性をバイオアッセイで検討した（図３−ｃ）。活性を持つ２本のフラクションを採取した。６００mlのＣＭからの収量は化合物（Ｉ−１）で２μg、化合物（Ｉ−２）で１０μｇであった。ＣＭから１０⁷倍の精製が行われ、活性の回収％は１０％であった。
以下に上記の溶出フラクションのアミノ酸組成を検討した。
（４）アミノ酸組成
ガス層アミノ酸シークエンサーによるアミノ酸の解析は化合物（Ｉ−１）の場合はTyr-Ile-Tyr-Thr-Glnであった。しかしＦＡＢ−質量分析法による分子量は８４６でありTyr-Ile-Tyr-Thr-Glnの分子量よりも分子量が160大きかった。
更に、〔Ｍ−２Ｈ＋Ｋ〕^-に相当する疑似分子イオンm/z883で観測され、〔Ｍ−Ｈ＋８０〕^-に相当するフラグメントイオンがm/z765で観測された。
上記の結果から化合物（Ｉ−１）のアミノ酸は化学的に修飾されており、その修飾は、アミノ酸構造分析等の条件下では容易に離脱することが示された。
ＦＡＢ−質量分析実験における〔Ｍ−Ｈ＋８０〕^-フラグメントイオンから化合物（Ｉ−１）は硫酸化合物であり〔ロジカースら、カルボハイドロ リサーチ、179巻、７−19頁（1988）〕、アミノ酸構造を元に算出した推定分子量より多い化合物（Ｉ−１）の１６０マスユニットは、２つのチロシン残基のＯＨ基にスルフォン酸が置換されているためであると推定された。スルフォン酸の置換したチロシンはDEAE Sephadexに吸着されること、アミノ酸構造決定条件下ではスルフォン酸が離脱すること等により化合物（Ｉ）の構造が決定された。
上記構造を確認するため、上記化合物（Ｉ−１）の合成を行った。スルフォン酸を持たないペプチドをペプチド合成機により合成し、得られたペプチドにアリルスルフォトランスペプチダーゼによりスルフォン酸基を置換した〔村松ら、ヨーロピアン ジャーナル オブ バイオケミストリー、223巻、243-248頁（1994）〕。得られたペプチドは、逆相ＨＰＬＣにおいて化合物（Ｉ−１）と同じ保持時間で溶出され、化合物（Ｉ−１）に特有のマススペクトルをFAB-MSによる分析で示した。更に生物活性を示す濃度も同一であった。
よって化合物（Ｉ−１）の構造がH-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-Gln-OHであることが合成法によっても確認された。さらに、化合物（Ｉ−１）のＣ末端カルボン酸をアミド化したアミド化体も合成した〔化合物（Ｉ−３）〕。
上記と同様の試験により化合物（Ｉ−２）の構造もH-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-Gln-OHであると決定された。
さらに、上記のペプチド合成法に従い、化合物（Ｉ−１）のイソロイシンの代わりにバリンを置換した構造〔H-Tyr(SO₃H)-Val-Tyr(SO₃H)-Thr-Gln-OH〕を持つ化合物（Ｉ−４）〔配列番号：４〕(FAB-MS m/z 831(M-H)^-）、化合物（Ｉ−１）のスレオニンの代わりにセリンを置換した構造〔H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Ser-Gln-OH〕を持つ化合物（Ｉ−５）〔配列番号：５〕(FAB-MS m/z 831(M-H)^-)、また、化合物（Ｉ−１）のスルフォチロシンのひとつをチロシンに置換した構造〔H-Tyr(SO₃H)-Ile-Try-Thr-Gln-OH〕を持つ化合物（Ｉ−６）〔配列番号：６〕(FAB-MS 765(M-H)^-）および〔H-Tyr-Ile-Tyr(SO₃H)-Thr-Gln-OH〕を持つ化合物（Ｉ−７）〔配列番号：７〕(FAB-MS 765(M-H)^-）も合成した。また、化合物（Ｉ−１）の二つのスルフォチロシンを共にチロシンに置換した〔H-Tyr-Ile-Tyr-Thr-Gln-OH〕化合物（Ｉ−８）〔配列番号：８〕も合成した。
産業上の利用分野
本発明は、植物細胞成長促進剤として用いることができる植物細胞増殖因子に関する。
配列表
配列番号：１
配列の長さ：５
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
起源：
生物名：アスパラガス
細胞の種類：葉肉細胞
配列の特徴
特徴を表す記号：Modified-site
存在位置：１
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
特徴を表す記号：Modified-site
存在位置：３
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
配 列

配列番号：２
配列の長さ：４
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
起源：
生物名：アスパラガス
細胞の種類：葉肉細胞
配列の特徴
特徴を表す記号：Modified-site
存在位置：１
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
特徴を表す記号：Modified-site
存在位置：３
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
配 列

配列番号：３
配列の長さ：５
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
配列の特徴
特徴を表す記号：Modified-site
存在位置：１
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
特徴を表す記号：Modified-site
存在位置：３
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
特徴を表す記号：Modified-site
存在位置：５
特徴を決定した方法：Ｅ
他の情報：Xaaはグルタミンアミドを表す。
配 列

配列番号：４
配列の長さ：５
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
配列の特徴
特徴を表す記号：Modified-site
存在位置：１
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
特徴を表す記号：Modified-site
存在位置：３
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
配 列

配列番号：５
配列の長さ：５
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
配列の特徴
特徴を表す記号：Modified-site
存在位置：１
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
特徴を表す記号：Modified-site
存在位置：３
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
配 列

配列番号：６
配列の長さ：５
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
配列の特徴
特徴を表す記号：Modified-site
存在位置：１
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
配 列

配列番号：７
配列の長さ：５
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
配列の特徴
特徴を表す記号：Modified-site
存在位置：３
特徴を決定した方法：Ｅ
他の情報：XaaはＯ−スルフォチロシンを表す。
特徴を表す記号：Modified-site
配 列

配列番号：８
配列の長さ：５
配列の型：アミノ酸(amino acid)
鎖の数：一本鎖(single)
配列の種類：ペプチド(peptide)
配列の特徴
配 列

Technical field
The present invention relates to peptides having properties as plant cell growth factors.
Background art
Plant-derived cell growth factors include barley-derived fat-soluble fatty acids with a molecular weight of 600 or less (Journal of Plant Physiology, 121, 181-191, 1985), pine-derived oligosaccharides with a molecular weight of 1000 or less. Factor (Plant Cell, Tissue and Organ Culture, 26, 53-59, 1991), a heat-resistant growth factor of about 700 molecular weight derived from carrot (Plant Science, 51, 83-91, 1987), Growth factor derived from black Mexican corn, having a molecular weight of 1350 or less, oligosaccharide-like characteristics, and non-adsorbing to anion exchange resin and cation exchange resin in pH 5 buffer [Journal of Plant Physiology, Vol. 132, 316-321, 1988].
Known plant-derived cell growth factors are difficult to isolate and purify, and techniques for producing such factors in large quantities are not known. In order to use plant cell growth factors as plant cell growth agents, it is necessary to search for plant cell growth factors that are easily mass-produced.
Disclosure of the invention
The present invention relates to a compound of formula (I)

(Wherein R¹Or R²Are the same or different SO_ThreeH or H, X represents an α-amino acid or a single bond, Z¹And Z²Represents the same or different α-amino acids, Y represents OH or NH₂Represents a peptide represented by:
Hereinafter, the compound represented by formula (I) is referred to as compound (I).
In the definition of each group of formula (I), α-amino acid includes aliphatic amino acids such as glycine, alanine, valine, leucine and isoleucine, oxyamino acids such as serine and threonine, cysteine, cystine, methionine and the like. Sulfur amino acids, acidic amino acids such as aspartic acid and glutamic acid, amide amino acids such as asparagine and glutamine, basic amino acids such as lysine, arginine and ornithine, aromatic amino acids such as phenylalanine and tyrosine, histidine, tryptophan and proline And heterocyclic amino acids such as oxyproline. In X, among the α-amino acids, amide amino acids are preferable, and glutamine is particularly preferable. Z¹Among them, aliphatic amino acids are preferable among the α-amino acids, and valine and isoleucine are particularly preferable. Z²Among them, oxyamino acids are preferable among the α-amino acids.
Compound (I) is a plant cell growth factor that can be obtained by extraction from higher plants or by ordinary peptide synthesis methods.
The plant for extracting the cell growth factor in the present invention may be any monocotyledonous plant such as asparagus, rice, corn, etc., and the extraction may be carried out by an extraction method using an aqueous medium from plant bodies (plant cell tissue). Culture, edited by Harada and Komazaki, 1979, published by Science and Engineering, page 382), recovery method of cultured cells from the culture medium (plant cell tissue culture, edited by Harada and Komazaki, 1979, published by science and engineering, page 27) Can be performed.
The plant cell growth factor in the present invention promotes cell growth of all plants, and is particularly excellent in promoting cell growth of monocotyledonous plants such as asparagus, rice and corn.
For extraction from higher plants, for example, cells to be cultured are collected by a conventional method (plant cell tissue culture, edited by Harada and Komazaki, 1979, published by Rigaku Corporation, page 27), and the collected cells are subjected to normal plant cell culture. Transplanted to the medium used in the above (Plant Science, 65, 111-171, 1989) and shake culture at a temperature of 20-30 ° C., preferably 24-28 ° C., for 8-16 days, preferably 9-11 days To do. After completion of the shaking culture, the cells and the culture solution are separated by an operation such as centrifugation to obtain a prepared culture solution (Conditioned Medium, hereinafter referred to as CM).
Anion exchange resin such as DEAE Sephadex A-25, DEAE cellulose, DEAE Sepharose, etc., using a buffer solution (pH 6.5-8.0) such as 10-100 mM Tris-HCl buffer, phosphate buffer, sodium carbonate-carbonate buffer Then, the plant cell growth factor in the CM is adsorbed by a column method or a batch method. Next, the plant cell growth factor can be recovered by elution by increasing the salt concentration of potassium chloride, sodium carbonate or the like successively to 10 to 2000 mM. The plant cell growth factor is eluted in a region having a salt concentration of 800 to 2000 mM, preferably 1000 to 1250 mM in the above elution fraction.
The obtained fraction is further desalted by dialysis or the like, and is preferably a gel permeation column such as Bio-Gel P2, Bio-Gel P4 (manufactured by Biorad), Sephadex G25 (manufactured by Pharmacia) or the like. Call it.
The active fraction desalted by dialysis, gel permeation fraction, etc. is subjected to reverse phase high performance liquid chromatography (reverse phase HPLC) using nucleosyl-100-C18 (Nagel), microsolbu PR18 (Merck) etc. as a carrier. Using a solvent such as water-acetonitrile, water-methanol, water-ethanol, etc. containing trifluoroacetic acid (TFA), the plant cell growth factor purified from the fraction eluted during a retention time of 8-12 minutes obtain.
Compound (I) was synthesized from peptide skeleton by the peptide synthesis method described in “Peptide Synthesis” by Nobuo Izumiya et al., 1975 Maruzen, etc., and then allylic sulfotransferase etc. A desired peptide can be produced by sulfonation with an enzyme capable of binding to the side chain of the group.
Compound (1) can be used as a plant growth agent in the following forms.
(1) Liquid agent
A plant cell growth promoter is produced by dissolving 0.0001 to 1% of compound (I) in an aqueous solution containing a preservative and a pH adjuster. Examples of preservatives include boric acid, bleached powder, benzoic acid, salicylic acid, sorbic acid, dehydroacetic acid, propionic acid, isocyanuric acid, chlorous acid, hypochlorous acid, p-hydroxybenzoic acid and esters thereof, lauryltrimethylammonium-2,4. , 5-trichlorocarbonitrite, tribromosalicylanilide, 3,4,4'-trichlorocarbonide, hexachlorophene, bithionol, chloramine-T, chloramine B halazone and the like, preferably sorbic acid. As the pH adjuster, commonly used ones such as citrate and phosphate can be used alone or in combination.
Soaking seedlings such as plant seeds or ears in a solution prepared as described above in water diluted 100-10000 times, preferably 1000 times, or the final of the peptide in the culture medium of hydroponic culture By adding so that the concentration is 0.001 to 10 PPM, it can be used as a plant growth agent.
(2) Paste formulation
The said peptide is kneaded in 0.01-10PPM paste material, and a plant growth promoter is manufactured. Examples of the paste material include fat, fatty oil, lanolin, petrolatum, paraffin, wax, resin, plastic, glycol, higher alcohols, glycerin and the like, preferably petrolatum and lanolin.
The paste preparation produced as described above can be used as a plant growth agent by applying to a grafted plant part of a grafted plant, a fruit handle, or a cut surface at the time of cutting.
As a specific example of compound (1)

Etc. are exemplified.
[Brief description of the drawings]
FIG. 1 shows the effect of CM concentration on the colony formation rate of cultured cells derived from asparagus.
The symbols in the figure are:-□-CM 25.0%,-◇-CM 12.5%, -O- CM 6.3%, -Δ- CM 3.1%,-■-CM 1. 6%,-◆-represents control.
FIG. 2 shows compound (I-1), compound (I-2), compound (I-3), compound (I-6), compound (I-7) and compound with respect to the colony formation rate of cultured cells derived from asparagus. The influence of various concentrations of (I-8) is shown.
The symbols in the figure are-□-Compound (I-1),-◇-Compound (I-2),-○-Compound (I-3), -Δ- Compound (I-6),-■-Compound (I-7),-◆-Compound (I-8),
Fig. 3 shows (a) DEAE Sephadex A-25 ion exchange chromatography, (b) Bio-Gel P-2 extra fine chromatography, (c) Elution behavior of plant cell growth factor in Devlosil ODS-HG-5 reverse layer HPLC. Indicates.
The symbols in the figure represent □ colony formation rate and -absorbance (220 nm).
BEST MODE FOR CARRYING OUT THE INVENTION
The plant cell growth activity of the plant cell growth factor of the present invention is shown below as test examples.
Test example 1.
Asparagus free cells obtained by the method of Example (2) were transplanted to the medium prepared by the method of Example (3), cultured with the addition of the plant cell growth factor of the present invention, and cultured asparagus cells. The effect of plant cell growth factor on the growth of the plant was examined by measuring the change in colony formation rate.
(I) Cell culture
A 24-well microplate (IWAKI 3820-024) was used for cell culture. 250 μl of asparagus free isolated cell suspension prepared in advance to be twice the target cell density per well of microplate 250 μl of 4 times concentration liquid medium, 125 μl of sterile distilled water, or filtration After sterilization (ADVANTEC DISMIC-13cp 0.20μm), diluted with 125μl of CM obtained in Example (4), stirred well, shielded with vinyl tape to prevent transpiration, then in the dark at 28 ° C and 120rpm. Shake culture (TAITEC BR-300L) was performed.
(Ii) Observation of cells
Count the number of viable cells (including colony-forming cells), dead cells, and colony-forming cells in the field of view under an inverted microscope (OLYMPUS CK2) at a magnification of 100 times for each well. Based on the results, the colony formation frequency and the cell viability were calculated according to the following formula.
C (%) = a / bX100
C: Colony formation rate a: Number of colony forming cells b: Number of viable cells
L (%) = [b / (b + d)] × 100
L: Cell viability b: Number of living cells d: Number of dead cells
(Iii) Effect of CM on colony formation
5.0 × 10^Fourcells / ml and 2.5 × 10^FourIn a cell / ml asparagus free cell culture, CM was added to a final concentration of 25.0%, 12.5%, 6.3%, 3.1%, 1.6% and cultured. . The colony formation rate of cultured asparagus cells at each CM addition concentration was measured. The results are shown in FIG.
Hereinafter, the above method is shown as a bioassay for plant cell growth factor.
As a result, according to FIG. 1, a significant increase in colony formation was observed depending on the concentration of CM, indicating that CM has plant cell proliferation activity.
Test example 2.
Compound (I-1), Compound (I-2), Compound (I-3), Compound (I-6), Compound (I-7) or Compound (I-8) obtained in Example (7) ( 10^-FiveM-10^-9Cell proliferation activity was measured by the same method as in Test Example 1 except that M) was used. The results are shown in FIG.
Example
A. [CM extraction and its physicochemical properties]
(1) Plant material
Seeds of asparagus (Asparagus officinalis L. cv. Mary Washington 500W, Takii seedlings) are seeded in a seedling pot (9 cm in diameter) containing Kureha culture soil (Kureha Chemical Industry), then in an artificial weather room (Koito Industry) I grew up with. The illuminance in the artificial weather room was 20,999 Lux on the foliage, the day length was 16 hours a day, the light period was 8 hours dark, the temperature was 22 ° C during the day, 18 ° C at night, and the humidity was about 80%. The seeds normally germinate about 3 weeks after sowing, and about 3 shoots are formed by 6 weeks after sowing, so that the seeds were transplanted to a flower pot having a diameter of 21 cm as needed. In the experiment, foliate stems collected from strains after 9 weeks after sowing were used.
(2) Preparation of mesophyll cell-derived cells
About 10 cm long asparagus foliate stems were used for bioassay and 4 per 200 ml for the preparation of the prepared culture solution (CM). The collected leaf stalks were immersed in 70% ethanol for 30 seconds, sterilized in a solution obtained by adding 2 drops of Tween 20 per 100 ml to 10-fold diluted antiformin, and further washed three times with sterile distilled water. Next, using a glass homogenizer (22 x 167 mm, Iwaki Glass) in a clean bench, crush the foliate stalks in sterile distilled water, and then filter the crushed liquid through a 37 μm stainless steel mesh (Iida Seisakusho) Free single cells were precipitated by centrifuging the filtrate (100 × g, 3 min, Kubota KS-5000). The precipitated free single cells were suspended again in sterilized distilled water, and the operation of removing the supernatant after centrifugation was repeated three times to completely remove impurities.
(3) Preparation of medium
The medium composition is shown in Table 1.

The liquid medium shown in Table 1 was prepared by diluting the stock solution with distilled water to a concentration 4 times the target concentration immediately before use, adjusting the pH to 5.8 with 1.0 N KOH, and then sterilizing filter ( The solution was sterilized by filtration using ADV ANTEC DISMIC-25cs (0.20 μm).
(4) Collecting CM
The free single-cell suspension was about 5.0 × 10 × using a Bukerturk hemocytometer (Nippon Clinical Instrument Co., Ltd.).^FivePrepare a total of 100 ml of 50 ml and 50 ml of double concentration liquid medium in a 300 ml Erlenmeyer flask, cover with a silicon stopper, and shake culture under dark conditions at 28 ° C. and 120 rpm. Takasaki Scientific Instruments TB-25R). On the 10th day from the start of the culture when cell growth was maximized, CM was collected by suction filtration (ADVANTEC No. 2) and stored frozen at −30 ° C.
The physicochemical properties of the obtained CM are described in detail below.
(A) Thermal stability
1.5 ml of the CM collected on the 10th day of culture (hereinafter the same CM is used) was diluted 2-fold with distilled water, heated in a boiling water bath for 10 minutes, and then immediately cooled on ice. Similarly, 1.5 ml of CM was diluted 2-fold with distilled water and autoclaved at 121 ° C. for 20 minutes. Bioassay was performed on the above two samples.
When CM was heated in a boiling water bath for 10 minutes, 70% of the cell growth activity was retained, but was completely inactivated by autoclaving at 121 ° C. for 20 minutes.
(B) pH stability
After diluting 1.0 ml of CM to 4.0 ml with distilled water, 0.1N HNO_ThreeAlternatively, 0.1N KOH was used to adjust the pH to 3.0, pH 5.0, pH 7.0, pH 9.0, pH 11.0, and left at 4 ° C. for 24 hours. Then 0.1N KOH or 0.1N HNO_ThreeAfter adjusting the pH to 5.8, the solution was concentrated to 2.0 ml and subjected to bioassay for each sample. The pH of CM itself was about 5.0.
The plant cell growth factor of the present invention was generally stable at pH 3.0, pH 5.0, pH 7.0, and pH 9.0, but its activity decreased by about 60% at pH 11.0.
(C) Solvent fraction of CM
Dilute 2.0 ml of CM to 10 ml with distilled water, 0.1 N HNO_ThreeThe pH was adjusted to 3.0 using and then extracted three times with 5.0 ml of ethyl acetate. The resulting aqueous layer was adjusted to pH 5.8 with 0.1 N KOH and concentrated to 4.0 ml. The ethyl acetate layer was dried over sodium sulfate, distilled to dryness, and dissolved with 4.0 ml of distilled water. Similarly, 2.0 ml of CM was diluted to 10 ml with distilled water, adjusted to pH 11.0 with 0.1 N KOH, and extracted three times with 5.0 ml of diethyl ether. The obtained water layer is 0.1N HNO._ThreeTo pH 5.8 and concentrated to 4.0 ml. The ether layer was dried over sodium sulfate, evaporated to dryness and dissolved in 4.0 ml of distilled water. Bioassay was performed on the above four samples.
The cell growth promoting activity remained in the aqueous layer under both acidic and basic conditions.
(D) Adsorption / desorption experiment on reversed-phase carrier
Cosmosil 75C₁₈-10 g of OPN (Nacalai tesque) was suspended in methanol, degassed under vacuum, and then packed in a column (1.7 × 8 cm, 18 ml). After the carrier was completely packed, the eluate was replaced with distilled water, and the column was washed with 100 ml of distilled water. Next, 5.0 ml of CM was added to the column, 100 ml of distilled water, 30% CH_ThreeCN 100ml, 60% CH_ThreeElution was successively carried out with 100 ml of CN (flow rate 60 ml / h), and each fraction was evaporated to dryness with an evaporator and dissolved in 10 ml of distilled water for bioassay. Similarly, Diagion HP-20 (Mitsubishi Kasei) 10g is suspended in methanol, degassed under vacuum and packed into a column, and then 5mm thick sea sand B is overlaid to prevent the carrier from floating, and the eluate Was replaced with distilled water, and the column was washed with 100 ml of distilled water. Next, 5.0 ml of CM was added to the column, 100 ml of distilled water, 30% CH_ThreeCN 100ml, 60% CH_ThreeElution was successively carried out with 100 ml of CN (flow rate 60 ml / h), and each fraction was evaporated to dryness with an evaporator and dissolved in 10 ml of distilled water for bioassay.
Cosmosil 75C₁₈-Cell growth factor was not retained on either carrier of OPN or Diaion HP-20 and was eluted with distilled water. Therefore, it can be seen that the plant cell growth factor of the present invention has a relatively high polarity.
(E) Adsorption / desorption experiment on activated carbon
Activated charcoal (Wako Pure Chemical Industries) 5.0 g was heated in 100 ml of 15% acetic acid at 100 ° C. for 30 minutes to remove impurities, washed with 500 ml of distilled water, suspended in distilled water and suspended in a column (1.7 × 11 cm, 25 ml). Next, 5.0 ml of CM was added to the column, and eluted successively with 100 ml of distilled water, 100 ml of 15% ethanol, 100 ml of 30% ethanol, and 100 ml of acetone (flow rate 60 ml / h), and each fraction was evaporated to dryness with an evaporator. Bioassay was performed by dissolving in 10 ml of distilled water.
The plant cell growth factor of the present invention was very strongly adsorbed on activated carbon and was not eluted at all by 15% ethanol, 30% ethanol and acetone. Under these conditions, it is known that neutral oligosaccharides and some acidic sugars are eluted.
(F) Adsorption / desorption experiment for ion exchange resin
After 0.8 g of DEAE Sephadex A-25 (Pharmacia LKB Biotechnology) was swollen in 50 ml of 500 mM Tris-HCl buffer (pH 7.4) at room temperature (24 hours), suspended in 20 mM of the same buffer (1 2 × 3.5 cm, 4.0 ml). 10 ml of CM was lyophilized, dissolved in 10 ml of the same buffer, added onto the column, and 20 ml of the same buffer, 20 ml of the same buffer containing 250 mM KCl, 20 ml of the same buffer containing 500 mM KCl, and 1000 mM KCl. Elution was successively carried out with 20 ml of the same buffer (flow rate 15 ml / h). Next, each elution fraction was concentrated to 10 ml, poured into a dialysis tube (Spectra / Por 7 MWCO: 1000), closed at both ends with a closer, and then dialyzed in 3000 ml of distilled water at 4 ° C. for 24 hours. Desalting was performed. The dialyzed solution was concentrated to 10 ml, and bioassay was performed on each fraction.
Similarly, 0.8 g of CM Sephadex C-25 was added to 500 mM KH.₂PO_Four-Swelled at room temperature (24 hours) in 50 ml of KOH buffer pH 6.0, suspended in 20 mM of the same buffer, and packed in a column (1.2 x 3.5 cm, 4.0 ml). 10 ml of CM was lyophilized, dissolved in 10 ml of the same buffer, added onto the column, and eluted with 20 ml of the same buffer and 20 ml of the same buffer containing 250 mM KCl (flow rate 15 ml / h). After desalting by dialysis, bioassay was performed on each fraction.
The cell growth factor of the present invention was very strongly adsorbed on DEAE Sephadex A-25 and eluted with 1000 mM KCl. On the other hand, CM Sephadex C-25 does not adsorb the cell growth factor at all, and 20 mM KH.₂PO_Four-Eluted with KOH buffer. From this result, it can be seen that the cell growth factor is an acidic substance.
(G) Deactivation test of active substances by various hydrolases
Pronase E (Sigma) 3.0 mg, a non-specific peptide degrading enzyme, was added to 20 mM KH.₂PO_Four-Dissolved in 3.0 ml of KOH buffer (pH 7.5) and filtered the insoluble matter with a cellulose acetate filter (ADVANTEC DISMIC-13cp 0.20 μm) to obtain an enzyme solution. 1.0 ml of the enzyme solution was made into an inactivated enzyme solution by heating in a boiling water bath for 10 minutes. Add 0.9 ml of the same buffer, 1.0 ml of CM or distilled water, and 100 μl of the enzyme solution or inactivated enzyme solution or the same buffer to the test tube, and use a constant temperature shaker (TAITEC, Personal-11) 37 The mixture was shaken at 170 ° C. for 3 hours. After the enzyme reaction, the reaction solution is 0.1N HNO._ThreeThe pH was adjusted to 5.8, and the enzyme was inactivated by heating in a boiling water bath for 10 minutes and immediately cooling with ice, and then bioassay was performed.
Similarly, 3.0 mg of Glycosidases “Mixed” (Seikagaku Corporation), which is a hydrolase mixture of several kinds of sugar chains, is dissolved in 3.0 ml of 20 mM Glutamic acid-KOH buffer (pH 4.0) to obtain cellulose nitrate. Insoluble matter was filtered with a filter to obtain an enzyme solution. 1.0 ml of the enzyme solution was inactivated by heating in a boiling water bath for 10 minutes to obtain an inactivated enzyme solution. Add 0.9 ml of the same buffer, 1.0 ml of CM or distilled water, and 100 μl of the enzyme solution or inactivated enzyme solution or the same buffer to the test tube, and shake at 37 ° C. and 170 rpm for 3 hours using a constant temperature shaker. did. After the enzyme reaction, the reaction solution was adjusted to pH 5.8 with 0.1 N KOH, heated for 10 minutes in a boiling water bath, and immediately cooled with ice to inactivate the enzyme, followed by bioassay.
The plant cell growth factor of the present invention was completely inactivated by the action of Pronase E, which is a peptide degrading enzyme. Therefore, it can be seen that the cell growth factor has a peptide structure in the molecule, and this part is important for expression of the activity.
On the other hand, even when Glycosidases “Mixed”, which is a mixture of sugar chain hydrolases, was allowed to act, the activity of cell growth factor was retained.
The physicochemical properties of the above CM are summarized as follows.

B. [Extraction, synthesis and structure determination of compound (I)]
(1) After 3.0 g of DEAE Sephadex A-25 was swollen in 50 ml of 500 mM Tris-HCl buffer (pH 7.4) at room temperature (24 hours), it was suspended in 20 mM of the same buffer and the column (1.7 × 8 0.0 cm, 18 ml). Next, after concentrating 100 ml of CM to 50 ml with an evaporator, Tris is added to a final concentration of 20 mM, and added to the column with 6N HCl to pH 7.4, and 30 ml of the same buffer and 30 ml of the same buffer containing 250 mM KCl. Then, 30 ml of the same buffer containing 500 mM KCl, 30 ml of the same buffer containing 750 mM KCl, 30 ml of the same buffer containing 1000 mM KCl, 30 ml of the same buffer containing 1250 mM KCl, and 30 ml of the same buffer containing 1500 mM KCl were sequentially eluted. At this time, the active fraction was estimated by absorption at UV 220 nm.
Fractions showing plant cell proliferation activity were eluted with 1000 mM and 1250 mM KCl (FIG. 3-a). Only the 1250 mM KCl elution fraction was lyophilized and weighed to be 9.02 mg.
Each elution fraction was dechlorinated by dialysis (Spectra / Por MWCO: 1000) and then concentrated to 50 ml.
(2) Gel permeation column
After lyophilizing the desalted eluate (1000 mM KCl, 1250 mM KCl elution fraction) from the DEAE Sephadex column, 1.0 ml of 20 mM KH₂PO_FourDissolved in -KOH-buffer (pH 5.8). Subsequently, the obtained solution was injected into a Bio-Gel P-2 extra fine column (1.7 × 37 cm) that had been equilibrated in advance with the buffer solution used for the lysis solution. While measuring the absorption at UV 220 nm, the buffer solution was flowed at 15 mh.^-1(Fig. 3-b).
Fractions were collected in 5 ml portions, and the activity of each fraction was measured by a bioassay.
(3) Reversed phase HPLC column
The eluted active fraction from the Bio-Gel column was lyophilized and then dissolved in 10 μl of 0.1% TFA containing 10% acetonitrile. The obtained solution was injected into a Develosil ODS-HG-5 column (4.6 × 250 nm, manufactured by Nomura Chemical Co., Ltd.) and then separated by isocratic elution of 10% acetonitrile containing 0.1% TFA. (Flow rate 1.0 ml / m, monitor UV 220 nm). Fractions (2 ml each) were collected and the activity of each fraction was examined by bioassay (FIG. 3-c). Two fractions with activity were collected. The yield from 600 ml of CM was 2 μg for compound (I-1) and 10 μg for compound (I-2). 10 from CM⁷Double purification was performed and the% recovery of activity was 10%.
The amino acid composition of the above elution fraction was examined below.
(4) Amino acid composition
Analysis of the amino acid by the gas layer amino acid sequencer was Tyr-Ile-Tyr-Thr-Gln in the case of compound (I-1). However, the molecular weight by FAB-mass spectrometry was 846, and the molecular weight was 160 larger than the molecular weight of Tyr-Ile-Tyr-Thr-Gln.
Furthermore, [M-2H + K]^-Observed at the pseudo-molecular ion m / z883 equivalent to [M−H + 80]^-A fragment ion corresponding to was observed at m / z 765.
From the above results, it was shown that the amino acid of the compound (I-1) was chemically modified, and the modification was easily released under conditions such as amino acid structure analysis.
[M−H + 80] in FAB-mass spectrometry experiment^-From the fragment ion, compound (I-1) is a sulfate compound [Rojkers et al., Carbohydro Research, Vol. 179, pp. 7-19 (1988)], which is a compound (I-1 ) 160 mass units were presumed to be due to substitution of sulfonic acid for the OH groups of the two tyrosine residues. The structure of compound (I) was determined by adsorbing tyrosine substituted with sulfonic acid to DEAE Sephadex and releasing sulfonic acid under conditions for determining amino acid structure.
In order to confirm the structure, the compound (I-1) was synthesized. A peptide having no sulfonic acid was synthesized by a peptide synthesizer, and the resulting peptide was substituted with sulfonic acid group by allylsulfotranspeptidase [Muramatsu et al., European Journal of Biochemistry, 223, 243-248 (1994) )]]. The obtained peptide was eluted with the same retention time as that of Compound (I-1) in reverse phase HPLC, and a mass spectrum peculiar to Compound (I-1) was shown by analysis by FAB-MS. Furthermore, the concentration showing biological activity was also the same.
Therefore, the structure of compound (I-1) is H-Tyr (SO_ThreeH) -Ile-Tyr (SO_ThreeH) -Thr-Gln-OH was also confirmed by the synthesis method. Furthermore, an amidated product obtained by amidating the C-terminal carboxylic acid of compound (I-1) was also synthesized [compound (I-3)].
By the same test as above, the structure of the compound (I-2) was also H-Tyr (SO_ThreeH) -Ile-Tyr (SO_ThreeH) -Thr-Gln-OH.
Further, according to the above peptide synthesis method, a structure in which valine is substituted for isoleucine in compound (I-1) [H-Tyr (SO_ThreeH) -Val-Tyr (SO_Three(H) -Thr-Gln-OH] compound (I-4) [SEQ ID NO: 4] (FAB-MS m / z 831 (M-H)^-), A structure in which serine is substituted for threonine in compound (I-1) [H-Tyr (SO_ThreeH) -Ile-Tyr (SO_ThreeH) -Ser-Gln-OH] (SEQ ID NO: 5) (FAB-MS m / z 831 (M-H))^-), And a structure in which one of the sulfotyrosine of compound (I-1) is substituted with tyrosine [H-Tyr (SO_ThreeH) -Ile-Try-Thr-Gln-OH] (SEQ ID NO: 6) (FAB-MS 765 (M-H))^-) And [H-Tyr-Ile-Tyr (SO_Three(H) -Thr-Gln-OH] compound (I-7) [SEQ ID NO: 7] (FAB-MS 765 (M-H)^-) Was also synthesized. In addition, [H-Tyr-Ile-Tyr-Thr-Gln-OH] compound (I-8) [SEQ ID NO: 8] in which two sulfotyrosines of compound (I-1) are both substituted with tyrosine was also synthesized. .
Industrial application fields
The present invention relates to a plant cell growth factor that can be used as a plant cell growth promoter.
Sequence listing
SEQ ID NO: 1
Sequence length: 5
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
origin:
Name of organism: Asparagus
Cell type: mesophyll cells
Sequence features
Symbols representing features: Modified-site
Location: 1
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Symbols representing features: Modified-site
Location: 3
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Arrangement

SEQ ID NO: 2
Sequence length: 4
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
origin:
Name of organism: Asparagus
Cell type: mesophyll cells
Sequence features
Symbols representing features: Modified-site
Location: 1
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Symbols representing features: Modified-site
Location: 3
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Arrangement

SEQ ID NO: 3
Sequence length: 5
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
Sequence features
Symbols representing features: Modified-site
Location: 1
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Symbols representing features: Modified-site
Location: 3
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Symbols representing features: Modified-site
Location: 5
Method for determining characteristics: E
Other information: Xaa represents glutamine amide.
Arrangement

SEQ ID NO: 4
Sequence length: 5
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
Sequence features
Symbols representing features: Modified-site
Location: 1
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Symbols representing features: Modified-site
Location: 3
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Arrangement

SEQ ID NO: 5
Sequence length: 5
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
Sequence features
Symbols representing features: Modified-site
Location: 1
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Symbols representing features: Modified-site
Location: 3
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Arrangement

SEQ ID NO: 6
Sequence length: 5
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
Sequence features
Symbols representing features: Modified-site
Location: 1
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Arrangement

SEQ ID NO: 7
Sequence length: 5
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
Sequence features
Symbols representing features: Modified-site
Location: 3
Method for determining characteristics: E
Other information: Xaa represents O-sulfotyrosine.
Symbols representing features: Modified-site
Arrangement

SEQ ID NO: 8
Sequence length: 5
Sequence type: amino acid
Number of chains: single
Sequence type: Peptide
Sequence features
Arrangement

Claims (9)

formula

Wherein R ¹ and R ² are the same or different and represent SO ₃ H or H, X represents glutamine or a single bond, Z ¹ represents valine or isoleucine, Z ² represents serine or threonine, Y Represents OH or NH ₂ ). The following (a) peptide of claim 1, wherein selected from the group consisting of ~ (h).
(A) a peptide in which R ¹ and R ² are SO ₃ H, X is glutamine, Z ¹ is isoleucine, Z ² is threonine, and Y is OH (b) R ¹ and R ² are Peptide where c is SO ₃ H, X is a single bond, Z ¹ is isoleucine, Z ² is threonine, Y is OH (c) R ¹ and R ² are SO ₃ H, X is Peptide where glutamine, Z ¹ is isoleucine, Z ² is threonine, Y is NH ₂ (d) R ¹ and R ² are SO ₃ H, X is glutamine, Z ¹ is valine A peptide in which Z ² is threonine, Y is OH (e) R ¹ and R ² are SO ₃ H, X is glutamine, Z ¹ is isoleucine, and Z ² is serine Peptide where Y is OH (f) R ¹ is SO ₃ H, R ² is H, X is glutamine, Z ¹ is Peptide (g) R ² is H, R ² is SO ₃ H, X is glutamine, Z ¹ is isoleucine, Z ² is threonine, Y is OH Peptide where ² is threonine and Y is OH (h) R ¹ and R ² are H, X is glutamine, Z ¹ is isoleucine, Z ² is threonine and Y is OH peptide A plant growth agent comprising the peptide according to claim 1 or 2 . formula
H-Tyr-Z ¹ -Tyr-Z ² -XY
( Wherein X represents glutamine or a single bond, Z ¹ represents valine or isoleucine, Z ² represents serine or threonine , and Y represents OH or NH ₂ ) The peptide according to claim 1, wherein sulfonation is performed by an enzyme capable of binding a sulfonic acid group to a side chain of a tyrosine residue (except for a peptide in which R ¹ and R ² are represented by H). ) Manufacturing method. Cells were harvested from plants and transplanted harvested cells into the medium and cultured, and wherein the obtaining a prepared culture broth by separating the cells and culture liquid after completion of the culture, wherein

(In the formula, X represents glutamine or a single bond) . The production method according to claim 5 , wherein the plant is a monocotyledonous plant. The production method according to claim 6 , wherein the monocotyledonous plant is asparagus, rice or corn. Formula obtained by the method according to any one of claims 5 to 7.

Wherein X represents glutamine or a single bond) . The peptides in the preparation culture according to claim 8, wherein adsorbed on the anion exchange resin, by carrying out the dissolution of the peptide by increasing the salt concentration from the resin, and recovering the peptide of formula

(Wherein X represents glutamine or a single bond) .

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