JPH0332748B2 - - Google Patents
Info
- Publication number
- JPH0332748B2 JPH0332748B2 JP56113317A JP11331781A JPH0332748B2 JP H0332748 B2 JPH0332748 B2 JP H0332748B2 JP 56113317 A JP56113317 A JP 56113317A JP 11331781 A JP11331781 A JP 11331781A JP H0332748 B2 JPH0332748 B2 JP H0332748B2
- Authority
- JP
- Japan
- Prior art keywords
- endotoxin
- coagulation system
- fraction
- lysate
- agarose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000002158 endotoxin Substances 0.000 claims description 34
- 238000005345 coagulation Methods 0.000 claims description 24
- 230000015271 coagulation Effects 0.000 claims description 24
- 239000006166 lysate Substances 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 230000002401 inhibitory effect Effects 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 229920000936 Agarose Polymers 0.000 claims description 8
- 239000003112 inhibitor Substances 0.000 claims description 8
- 241001529572 Chaceon affinis Species 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- 239000002808 molecular sieve Substances 0.000 claims description 4
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 4
- 229920000858 Cyclodextrin Polymers 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 2
- 108010059712 Pronase Proteins 0.000 claims description 2
- 108090000787 Subtilisin Proteins 0.000 claims description 2
- 238000005377 adsorption chromatography Methods 0.000 claims description 2
- 229960002897 heparin Drugs 0.000 claims description 2
- 229920000669 heparin Polymers 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 description 15
- 238000000034 method Methods 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 229920006008 lipopolysaccharide Polymers 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 108010048121 pro-clotting enzyme Proteins 0.000 description 5
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 108700023418 Amidases Proteins 0.000 description 3
- 241000239224 Tachypleus tridentatus Species 0.000 description 3
- 102000005922 amidase Human genes 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 210000003918 fraction a Anatomy 0.000 description 3
- 238000001879 gelation Methods 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000239218 Limulus Species 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 210000002196 fr. b Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 101710099705 Anti-lipopolysaccharide factor Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 241000239220 Limulus polyphemus Species 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 241000239221 Tachypleus gigas Species 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 108010071063 butyloxycarbonyl-leucyl-glycyl-arginine-4-nitroanilide Proteins 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 108010072542 endotoxin binding proteins Proteins 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 210000000087 hemolymph Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011046 pyrogen test Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
（産業上の利用分野）
本発明は、カブトガニのアメボサイト・ライゼ
ート中に存在する、エンドトキシンによる凝固系
阻害因子に関するものである。
（従来の技術）
カブトガニ（Limulus polyphemus，
Tachypleus tridentatus，T.gigas等）の血リン
パ液に存在するアメボサイト（血球細胞）より取
得されるライゼートかグラム陰性細菌の表層物質
のエンドトキシン（リポポリサツカライド）と作
用して起る凝固現象を応用して、検体中の微量エ
ンドトキシンを検出する「リムルステスト」は、
動物を用いるパイロジエンテストの代用として医
学・薬学の領域に広く利用されている。しかしア
メボサイト・ライゼートのエンドトキシンによる
ゲル化現象を応用した「リムルステスト」は、そ
れが商業的規模で実用化されるためには多くの問
題点を含んでいる。その問題点の中でも特に重要
なものは、エンドトキシンによる凝固系を阻害す
る因子を除去することである。ライゼート中の凝
固系阻害因子の量は、カブトガニの生息環境や季
節的因子により変動するものであり、これをコン
トロールすることは、アメボサイト・ライゼート
をエンドトキシンの定量に用いるのに信頼性を高
めることになる。
本発明者らは、先にこれらライゼートの凝固機
構の解明を行ない、合成基質を用いるエンドトキ
シンの高感度定量法を開示し（特開昭54−15797
号）、さらには、凝固機作に関与する因子につい
て報告して来た（FEBS.Letters、120、217
（1980）、S.Iwanaga et al）。
これらの検討からゲル化現象によらず、ライゼ
ート中に存在するエンドトキシンに依存する酵素
活性を、その特異的合成基質を用いて測定するこ
とにより高感度且つ定量性の高いエンドトキシン
の検出測定法を提供して来た。しかしながら、こ
の方法は、通常のエンドトキシン量の測定域では
阻害因子の存在による影響はそれほど受けないも
のの、極微量のエンドトキシン量の測定時に、阻
害因子がエンドトキシンの作用を陰覆することか
ら、基本的にライゼート中に阻害因子が存在しな
い方が好ましいことは明白である。阻害因子の概
念については、その存在が支持されており、その
本態及びその作用機作について、酵素説
［Nachum，et al，J.Inv.Patho.、32、51（1978）］
や、エンドトキシン結合性リポタンパク説
［Sullivan et al.，Appl.Microbiol.、28、1023
（1974）］等が提出されてきたが、ゲル化現象に対
する説明のみで充分明確にされているとは考えら
れない。
一方、アメボサイト・ライゼート中から凝固系
阻害因子を除去する方法が検討されてきた（米国
特許第4107077号、Sullivan，et al及び特開昭56
−42590号、アール・クラーク・ブラウンら）。前
者はライゼートを有機溶媒を用いて振盪し、蛋白
変性せしめることにより凝固系阻害因子を除去す
る方法であり、後者はライゼート中の凝固酵素に
影響を与えない程度のアルカリ領域のPHにて凝固
系阻害因子を失活せしめた後に、凝固酵素の作用
域のPHに戻す方法である。これらの方法は、いず
れもライゼート中のエンドトキシン感受性因子等
に影響を与えるという欠点を有する。
（発明の構成）
本発明者らは、ライゼート中の凝固系因子の作
用機作の研究の過程で、エンドトキシンによる凝
固系阻害因子の存在並びにその阻害因子を初めて
採取し、本発明に到達した。
すなわち、ヘパリン・アガロースを用いる特異
的吸着担体を用いて、吸着クロマトグラフイーに
付し、ライゼート中のエンドトキシン感受性因子
と凝固系阻害因子を吸着させ、0.15MNaCl溶液
を用いて、プロクロツテイングエンザイムを脱着
させ、次いで1.0MNaCl溶液を用いて溶出し、エ
ンドトキシン感受性因子と凝固系阻害因子を主成
分とする画分を得る。
この画分をセフアロース
、架橋セフアロース
、セフアデツクス
、セフアクリル
（以上フ
アルマシア社製）等の商標名で知られるアガロー
ス、架橋アガロース、架橋デキストランゲル、架
橋アクリルアミドゲルのような分子篩剤を用いて
分子篩クロマトグラフイーに付し、シクロデキス
トリン（分子量：1100〜900）及びNaCl（分子
量：58）の溶出位置と同じ位置に溶出される画分
を採取することにより、アメボサイト・ライゼー
ト並びに該ライゼート中に存在するエンドトキシ
ン感受性因子のエンドトキシンによる活性化の段
階を強力に阻害するペプチドを得た。このものは
強塩基性を示し、アルカリ処理、プロナーゼ、ス
ブチリシン等の酵素処理で完全に失活する性質を
有する。
以下、本発明を、調製例及び実施例によつてさ
らに詳しく説明する。
調製例
(1) 基質と担体 Boc−Leu−Gly−Arg−
pNA・HClは、生化学工業(株)より購入した。
ヘパリン−アガロースはセフアロース・CL−
6B（フアルマシア社）を用い、マルク
（Marck）らの方法で調製し、20分間オートク
レーブして用いた。
(2) 血球抽出液の調製 日本産カブトガニ
（Tachypleus tridentatus）より公知の方法に
従い採血し、得られた血球を0.05Mトリスー塩
酸緩衝液（PH7.2）を用いて、ヒスコトロン
でホモゲナイスし、800rpm、30分間遠心後、
その上清を用いた。
(3) 活性測定法 クロマトグラフイー後の各画分
の酵素活性および各因子の活性は、37℃の条件
下で下記の方法に従つて測定した。なお、酵素
活性はすべて１分間に1μmolのパラニトロアニ
リンを遊離する酵素量を１単位とした。
() エンドトキシン感受性因子：各画分50μ
とエンドトキシン（LPS）（600nｇ／ml）
30μ並びに0.2Mトリス−塩酸−13ｍＭ
MgCl2緩衝液（PH8.0）100μを混合し、15
分放置後、５ｍＭ Boc−Leu−Gly−Arg
−pNA20μとプロクロツテイングエンザイ
ムを含む画分Ａ（図１）50μの混液を加え、
一定時間後0.6M酢酸0.8mlを加えて反応を停
止し、遊離したパラニトロアニリン量（ε＝
10500）を405nｍで測定した。
() プロクロツテイングエンザイムの活性：
各画分50μと活性型エンドトキシン感受性
因子50μ、トリス−塩酸緩衝液（（ｉ）で
使用したもの）100μを混合し、30分放置
後、２ｍＭ合成基質（（ｉ）で使用したもの）
50μを加え、出現したアミダーゼ活性を測
定した。なお、活性型エンドトキシン感受性
因子は画分B2（図１）400μと0.4Mトリス−
塩酸−26ｍＭ MgCl2緩衝液（PH8.0）200μ
、LPS（400nｇ／ml）200μの混液を15分
放置して調製したものを用いた。
() クロツテイングエンザイムの活性：各画
分50μとトリス−塩酸緩衝液（（ｉ）で使
用したもの）100μ、２ｍＭ 合成基質
（（ｉ）で使用したもの）50μ、生理食塩水
50μを混合し、一定時間後、0.6M酢酸0.8
mlを加えて反応を停止し、パラニトロアニリ
ンの遊離量を測定した。
() 凝固系阻害因子（Anti−LPS factor）
の活性：試料、プロクロツテイングエンザイ
ムを含む画分Ａ、エンドトキシン感受性因子
を含む画分B2、0.4Mトリス−塩酸−26ｍＭ
MgCl2緩衝液（PH8.0）を各50μ、さらに
５ｍＭ合成基質（（ｉ）で使用したもの）
20μとLPS（400nｇ／ml）30μを混合し、
25分放置後、0.6M酢酸0.8mlを加えて反応を
停止し、パラニトロアニリンの遊離量を測定
した。この条件下、エンドトキシン感受性因
子の活性化をLPS2nｇを使用して測定し、
50％阻害する活性を１単位とした。
実施例 １
凝固系阻害因子画分の採取法
無菌的に調製したヘパリン−アガロース・CL
−6Bカラム（５×17cm）に、カブトガニ・アメ
ボサイト・ライゼート340mlを添加し、0.15M
NaClで溶出し、次いで1M NaCl溶液を用いて溶
出を行つた（図１）。ついで、1M NaCl溶出画分
（180〜260管）をあつめ、セフアロース・CL−
6Bカラム（3.5×122.5cm）を用いて、無菌的にゲ
ルろ過を行い、プロクロツテイングエンザイム画
分、エンドトキシン感受性因子画分および凝固系
阻害因子画分をそれぞれの検出法（前述）に従つ
て検出したところ、シクロデキストリン及び食塩
が溶出される195〜204管にエンドトキシン感受性
酵素系を含まない凝固系阻害因子が溶出され、こ
れとは別に125〜150管にプロクロツテイングエン
ザイムとエンドトキシン感受性因子が溶出された
（図２）。
試験例 凝固系阻害因子を含まないライゼートの
効果
実勢例１において得られた各画分を混合して、
アミダーゼ活性を測定したとき、表に示すよう
に、凝固系阻害因子を含んだ混合物ではいずれも
著しい活性阻害がみられたが、凝固系阻害因子を
含まない混合物ではいずれもきわめて高いアミダ
ーゼ活性が保持された。
(Industrial Application Field) The present invention relates to a factor that inhibits the coagulation system caused by endotoxin, which is present in horseshoe crab amebocyte lysate. (Prior art) Horseshoe crab (Limulus polyphemus,
We apply the coagulation phenomenon that occurs when the lysate obtained from amebocytes (blood cells) present in the hemolymph of Tachypleus tridentatus, T. gigas, etc. interacts with endotoxin (lipopolysaccharide), a surface substance of Gram-negative bacteria. The Limulus test, which detects trace amounts of endotoxin in samples, is
It is widely used in the medical and pharmaceutical fields as a substitute for pyrogen tests using animals. However, the Limulus test, which applies the endotoxin-induced gelation phenomenon of amebocyte lysate, has many problems before it can be put to practical use on a commercial scale. One of the most important issues is the removal of factors that inhibit the endotoxin-induced coagulation system. The amount of coagulation system inhibitors in the lysate varies depending on the horseshoe crab's habitat and seasonal factors, and controlling this will improve the reliability of using amebocyte lysate for endotoxin quantification. Become. The present inventors first elucidated the coagulation mechanism of these lysates and disclosed a highly sensitive method for quantifying endotoxin using a synthetic substrate (Japanese Patent Application Laid-open No. 15797-1987).
Furthermore, we have reported on factors involved in the coagulation mechanism (FEBS.Letters, 120 , 217).
(1980), S. Iwanaga et al). Based on these studies, we have provided a highly sensitive and highly quantitative method for detecting endotoxin by measuring enzyme activity that is dependent on endotoxin present in lysate using a specific synthetic substrate, regardless of the gelation phenomenon. I came. However, although this method is not significantly affected by the presence of inhibitors in the normal endotoxin measurement range, the inhibitors override the effects of endotoxin when measuring extremely small amounts of endotoxin, so the basic problem is that Obviously, the absence of inhibitors in the lysate is preferred. The existence of the concept of inhibitory factors is supported, and the enzyme theory [Nachum, et al, J.Inv. Patho., 32 , 51 (1978)]
and the endotoxin-binding lipoprotein theory [Sullivan et al., Appl. Microbiol., 28 , 1023
(1974)], etc., but it is not considered to be sufficiently clear just by explaining the gelation phenomenon. On the other hand, methods for removing coagulation system inhibitory factors from amebocyte lysate have been investigated (US Pat. No. 4,107,077, Sullivan, et al.
−42590, Earl Clark Brown et al.). The former is a method in which coagulation system inhibitory factors are removed by shaking the lysate using an organic solvent to denature proteins, while the latter is a method in which coagulation system inhibitors are removed at a pH in the alkaline range that does not affect the coagulation enzymes in the lysate. This method returns the pH to the action range of coagulation enzymes after deactivating the inhibitory factors. All of these methods have the disadvantage of affecting endotoxin-sensitive factors in the lysate. (Structure of the Invention) In the process of researching the mechanism of action of coagulation system factors in lysate, the present inventors first detected the existence of coagulation system inhibitory factors due to endotoxin and collected the inhibitory factors, thereby arriving at the present invention. That is, using a specific adsorption carrier using heparin/agarose, it was subjected to adsorption chromatography to adsorb endotoxin-sensitive factors and coagulation system inhibitory factors in the lysate, and pro-clotting enzyme was adsorbed using a 0.15M NaCl solution. It is desorbed and then eluted using a 1.0M NaCl solution to obtain a fraction whose main components are endotoxin-sensitive factors and coagulation system inhibitory factors. This fraction was subjected to molecular sieve chromatography using molecular sieve agents such as agarose, cross-linked agarose, cross-linked dextran gel, and cross-linked acrylamide gel, which are known by trade names such as Cepharose, cross-linked Cepharose, Cephadex, and Cephacryl (manufactured by Pharmacia). By collecting the fraction eluted at the same position as that of cyclodextrin (molecular weight: 1100-900) and NaCl (molecular weight: 58), we analyzed the amebocyte lysate and the endotoxin-sensitive factors present in the lysate. We obtained a peptide that strongly inhibits the activation step by endotoxin. This substance exhibits strong basicity and has the property of being completely inactivated by alkaline treatment and enzyme treatment such as pronase and subtilisin. Hereinafter, the present invention will be explained in more detail with reference to Preparation Examples and Examples. Preparation example (1) Substrate and carrier Boc−Leu−Gly−Arg−
pNA/HCl was purchased from Seikagaku Corporation.
Heparin-Agarose is Sepharose/CL-
6B (Pharmacia) according to the method of Marck et al. and autoclaved for 20 minutes before use. (2) Preparation of blood cell extract Blood was collected from Japanese horseshoe crabs (Tachypleus tridentatus) according to a known method, and the obtained blood cells were incubated with a hiscotron using 0.05M Tris-HCl buffer (PH7.2).
After homogenizing and centrifuging at 800 rpm for 30 minutes,
The supernatant was used. (3) Activity measurement method The enzyme activity and activity of each factor in each fraction after chromatography were measured at 37°C according to the following method. In all enzyme activities, 1 unit was defined as the amount of enzyme that released 1 μmol of paranitroaniline per minute. () Endotoxin sensitivity factor: 50μ for each fraction
and endotoxin (LPS) (600ng/ml)
30μ and 0.2M Tris-HCl-13mM
Mix 100μ of MgCl2 buffer (PH8.0), 15
After leaving for a minute, 5mM Boc-Leu-Gly-Arg
- Add a mixture of 20μ of pNA and 50μ of fraction A (Figure 1) containing pro-clotting enzyme,
After a certain period of time, 0.8 ml of 0.6 M acetic acid was added to stop the reaction, and the amount of paranitroaniline released (ε =
10500) was measured at 405 nm. () Pro-clotting enzyme activity:
Mix 50μ of each fraction, 50μ of activated endotoxin-susceptible factor, and 100μ of Tris-HCl buffer (used in (i)), leave for 30 minutes, and mix with 2mM synthetic substrate (used in (i)).
50μ was added, and the appearing amidase activity was measured. The activated endotoxin-sensitive factor is fraction B2 (Fig. 1) 400μ and 0.4M Tris-
Hydrochloric acid-26mM MgCl2 buffer (PH8.0) 200μ
A mixture of 200μ of LPS (400ng/ml) was prepared by allowing it to stand for 15 minutes. () Clot protein enzyme activity: 50μ of each fraction, 100μ of Tris-HCl buffer (used in (i)), 2mM synthetic substrate (used in (i)), 50μ of physiological saline
After a certain period of time, mix 50μ 0.6M acetic acid 0.8
ml was added to stop the reaction, and the amount of free paranitroaniline was measured. () Anti-LPS factor
Activity: sample, fraction A containing pro-clotting enzyme, fraction B 2 containing endotoxin-sensitive factors, 0.4M Tris-HCl-26mM
50μ each of MgCl2 buffer (PH8.0) and 5mM synthetic substrate (used in (i))
Mix 20μ and 30μ of LPS (400ng/ml),
After standing for 25 minutes, 0.8 ml of 0.6M acetic acid was added to stop the reaction, and the amount of paranitroaniline released was measured. Under these conditions, the activation of endotoxin-sensitive factors was measured using 2ng LPS,
The activity inhibiting 50% was defined as 1 unit. Example 1 Collection method of coagulation system inhibitory factor fraction Aseptically prepared heparin-agarose/CL
Add 340 ml of horseshoe crab amebosite lysate to a -6B column (5 x 17 cm) and add 0.15M
Elution was performed with NaCl and then with 1M NaCl solution (Figure 1). Next, the 1M NaCl elution fractions (180 to 260 tubes) were collected and separated into Sepharose/CL-
Gel filtration was performed aseptically using a 6B column (3.5 x 122.5 cm), and the pro-clotting enzyme fraction, endotoxin-susceptible factor fraction, and coagulation system inhibitory factor fraction were detected according to the respective detection methods (described above). Upon detection, coagulation system inhibitory factors that do not contain endotoxin-sensitive enzyme systems were eluted in tubes 195-204, where cyclodextrin and salt are eluted, and pro-clotting enzymes and endotoxin-sensitive factors were separately detected in tubes 125-150. eluted (Figure 2). Test Example: Effect of lysate that does not contain coagulation system inhibitory factors. Each fraction obtained in Practical Example 1 was mixed,
When amidase activity was measured, as shown in the table, a significant inhibition of activity was observed in all mixtures containing coagulation system inhibitors, but extremely high amidase activity was maintained in all mixtures that did not contain coagulation system inhibitors. It was done.
【表】
イト・ラ
イゼート
画分A＋B LPS 335 30 91.0
画分B LPS 120 10 91.7
[Table] Ito Ra
Iseto
Fraction A+B LPS 335 30 91.0
Fraction B LPS 120 10 91.7
Claims (1)
ヘパリン・アガロースを吸着担体とする吸着クロ
マトグラフイーに付し、0.15MNaCl溶液で溶出
した画分を除き、次いで1.0MNaCl溶液で溶出
し、この溶出画分を、さらに分子篩クロマトグラ
フイーに付し、シクロデキストリン乃至塩化ナト
リウムが溶出する位置と同じ位置に溶出する画分
を採取することにより得ることができるペプチド
であつて、次の性質を有するエンドトキシンによ
る凝固系阻害因子。 (a) 強塩基性を示す。 (b) カブトガニ・アメボサイト・ライゼートのエ
ンドトキシンによる凝固系の活性化を阻害す
る。 (c) アルカリ処理、プロナーゼ処理及びスブチリ
シン処理で失活する。 ２ 分子篩クロマトグラフイー用担体として、ア
ガロース、架橋アガロース、架橋デストランゲル
及び架橋アクリルアミドゲルから選ばれた担体を
用いて採取される特許請求の範囲第１項記載の凝
固系阻害因子。[Claims] 1. Horseshoe crab amebosite lysate,
Subjected to adsorption chromatography using heparin/agarose as an adsorption carrier, removed the fraction eluted with a 0.15M NaCl solution, then eluted with a 1.0M NaCl solution, and further subjected to molecular sieve chromatography, A peptide that can be obtained by collecting a fraction that elutes at the same position as that of cyclodextrin or sodium chloride, and has the following properties and is an endotoxin-induced coagulation system inhibitor. (a) Shows strong basicity. (b) Inhibits the activation of the coagulation system by endotoxin in horseshoe crab amebocyte lysate. (c) Inactivated by alkali treatment, pronase treatment, and subtilisin treatment. 2. The coagulation system inhibitory factor according to claim 1, which is collected using a carrier selected from agarose, cross-linked agarose, cross-linked destrange gel, and cross-linked acrylamide gel as a carrier for molecular sieve chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56113317A JPS5813517A (en) | 1981-07-20 | 1981-07-20 | Inhibitory factor on coagulation present in amebocyte lysate of horse crab |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56113317A JPS5813517A (en) | 1981-07-20 | 1981-07-20 | Inhibitory factor on coagulation present in amebocyte lysate of horse crab |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5813517A JPS5813517A (en) | 1983-01-26 |
| JPH0332748B2 true JPH0332748B2 (en) | 1991-05-14 |
Family
ID=14609159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56113317A Granted JPS5813517A (en) | 1981-07-20 | 1981-07-20 | Inhibitory factor on coagulation present in amebocyte lysate of horse crab |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5813517A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1328074C (en) * | 1988-09-01 | 1994-03-29 | Shigenori Tanaka | Horseshoe crab amebocyte lysate factor g inhibitor |
| KR0141685B1 (en) * | 1988-09-01 | 1998-07-01 | 야마따니 와따루 | Crab Amebosite Lysate G Factor Activation Inhibitor |
-
1981
- 1981-07-20 JP JP56113317A patent/JPS5813517A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5813517A (en) | 1983-01-26 |
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