JPH02119775A - Cytotoxic kallikrein, production thereof and use as cytotoxic agent - Google Patents
Cytotoxic kallikrein, production thereof and use as cytotoxic agentInfo
- Publication number
- JPH02119775A JPH02119775A JP63275116A JP27511688A JPH02119775A JP H02119775 A JPH02119775 A JP H02119775A JP 63275116 A JP63275116 A JP 63275116A JP 27511688 A JP27511688 A JP 27511688A JP H02119775 A JPH02119775 A JP H02119775A
- Authority
- JP
- Japan
- Prior art keywords
- cytotoxic
- kallikrein
- mouse
- fraction
- submandibular gland
- 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.)
- Pending
Links
- 102000001399 Kallikrein Human genes 0.000 title claims abstract description 20
- 108060005987 Kallikrein Proteins 0.000 title claims abstract description 20
- 230000001472 cytotoxic effect Effects 0.000 title claims abstract description 20
- 231100000433 cytotoxic Toxicity 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 229940127089 cytotoxic agent Drugs 0.000 title claims description 3
- 239000002254 cytotoxic agent Substances 0.000 title claims description 3
- 231100000599 cytotoxic agent Toxicity 0.000 title claims description 3
- 210000001913 submandibular gland Anatomy 0.000 claims abstract description 5
- 238000005227 gel permeation chromatography Methods 0.000 claims abstract description 4
- 238000004007 reversed phase HPLC Methods 0.000 claims abstract description 3
- 238000000746 purification Methods 0.000 claims description 4
- 238000005194 fractionation Methods 0.000 claims 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract description 6
- 206010020772 Hypertension Diseases 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 2
- 201000011510 cancer Diseases 0.000 abstract description 2
- 230000003449 preventive effect Effects 0.000 abstract description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 abstract 4
- 210000002196 fr. b Anatomy 0.000 abstract 2
- 238000011725 BALB/c mouse Methods 0.000 abstract 1
- 206010059150 Cerebrosclerosis Diseases 0.000 abstract 1
- 208000035473 Communicable disease Diseases 0.000 abstract 1
- 230000005526 G1 to G0 transition Effects 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 238000011067 equilibration Methods 0.000 abstract 1
- 208000015181 infectious disease Diseases 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000006228 supernatant Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 13
- 230000000694 effects Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 210000004907 gland Anatomy 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000012264 purified product Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 206010065559 Cerebral arteriosclerosis Diseases 0.000 description 3
- 102000012479 Serine Proteases Human genes 0.000 description 3
- 108010022999 Serine Proteases Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000009931 harmful effect Effects 0.000 description 3
- 201000005851 intracranial arteriosclerosis Diseases 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- DUUCJSJYISFGCI-WBPXWQEISA-N (2r,3r)-2,3-dihydroxybutanedioic acid;2-(dimethylamino)ethanol Chemical compound CN(C)CCO.OC(=O)[C@H](O)[C@@H](O)C(O)=O.OC(=O)[C@H](O)[C@@H](O)C(O)=O DUUCJSJYISFGCI-WBPXWQEISA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- QCHPKSFMDHPSNR-UHFFFAOYSA-N 3-aminoisobutyric acid Chemical compound NCC(C)C(O)=O QCHPKSFMDHPSNR-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- 240000003473 Grevillea banksii Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000975003 Homo sapiens Kallistatin Proteins 0.000 description 1
- 101001077723 Homo sapiens Serine protease inhibitor Kazal-type 6 Proteins 0.000 description 1
- 229940122920 Kallikrein inhibitor Drugs 0.000 description 1
- 101000878457 Macrocallista nimbosa FMRFamide Proteins 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 101150078410 Odf1 gene Proteins 0.000 description 1
- 102100025286 Outer dense fiber protein 1 Human genes 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100025421 Serine protease inhibitor Kazal-type 6 Human genes 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 210000003323 beak Anatomy 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000003350 kerosene Substances 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000003396 thiol group Chemical class [H]S* 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【発明の詳細な説明】
〈産業１−の利用分野〉
本発明は、医学、生理学および免疫学的研究もしく （
を病気の予防および冶ｆり（こ使用な細胞傷害性のカリ
９しイン（ｋａｌｌ＋ｌ〜ｒｅｉｎ）、その製造法およ
びキ■胞傷害剤とシ／゛この使用（こ関する。[Detailed Description of the Invention] <Field of Application in Industry 1-> The present invention is applicable to medical, physiological and immunological research or (
Related to the prevention and treatment of diseases, including the use of cytotoxic potassium rein, its production process, and its use as a cytotoxic agent.
〈従来の技術〉
カリ９しインは、活性部位ζこ反応性ヒスチジンＪ５よ
ひセ１ｊ：、−残Ｊ、（をイ１する蛋白質分解酵素（セ
リンフ）−ｒ ５２アーセ）であって ヒトのキＩＩ
Ｎ、お、Ｌひ面Ｗ４’４中にり、出され、特にキｆ１織
カリクレイシは脳動脈硬化ｆｊ１＼゛）高血圧Ｌ１’、
ＯＪ Ｊ′−防、））よひ／４１゛た（、１治療ζご
広く臨床利用さ）１．でいる１、一方、免投学的ζ７二
は、セノンフ１コテアー七はキ■胞傷害性′Ｆ袖胞やＮ
Ｋ細胞等の細胞用害作用の重要か因子であることか示
唆されでおり、牛体のキ■胞性免疫に：Ｆ−； Ｇ：ｌ
る季■胞傷害の機構を解明するためにもその分離が求め
られてきた。１ノかし、Ｊ：たその確認もされていなか
った。<Prior art> Cali-9 is a proteolytic enzyme (serinph)-r52ase that has an active site ζ-reactive histidine J5:, - residue J, and a human. Ki II
N, O, L face W4'4, especially Ki f1 Ori Kalikreishi is cerebral arteriosclerosis fj1\゛) Hypertension L1',
1. On the other hand, immunological ζ72, Senonph 1 Coteor 7 is cytotoxic 'F cuff and N.
It has been suggested that it may be an important factor in the harmful effect on cells such as K cells, and in the cystic immunity of cattle: F-; G: l
Its isolation has been sought to elucidate the mechanism of seasonal cell injury. 1 Nokashi, J: It was not even confirmed.
〈発明か解決り、ようとする課題〉
このよう（ごカリクレインは、脳動脈硬化症や高血圧症
用の医療薬剤として才３よひ伺究用試薬として広＜Ｔ；
ザかある。従来の製造方法によると、医療用のものはフ
タの膵臓やヒトの尿からの分離枯製物が利用されている
。しかし、この方法では相科に制限があり、精製工程が
複雑であることから製品の不足と高価格が問題となって
いる。他の動物からの分離精製も試みられており、いく
つかの酵素が単離精製されてヒトのものとの異同も明ら
かになってきた。しかし、い１゛れも収率か低くに程か
複雑であるという難点がある。<Problem to be solved by invention> As described above, kallikrein has been widely used as a research reagent for children aged 3 and 3 as a medical drug for cerebral arteriosclerosis and hypertension.
There is. According to the conventional manufacturing method, for medical use, the dried products separated from pancreas and human urine are used. However, this method has a limited amount of phase, and the purification process is complicated, resulting in product shortages and high prices. Attempts have also been made to isolate and purify enzymes from other animals, and several enzymes have been isolated and purified, and it has become clear that they are similar to humans. However, all of them have the drawbacks of low yield and complexity.
一方、細胞傷害性のセリンブ［ｌテアーセについては、
■−記のようにその存在が確認されるに至っていなかっ
た。On the other hand, regarding the cytotoxic serinbu[ltease],
-As mentioned above, its existence had not yet been confirmed.
本発明は、以りのような１＋’ｆｆ来技術の間頴点を解
決し、精製工程が簡単てあ・ってかつ細胞傷害性を有す
るカリクレインを動物組織からｔ）ることを可能にする
、製造法を提供するものである。The present invention solves the drawbacks of the previous technology as described above, and makes it possible to purify kallikrein, which has cytotoxic properties, from animal tissue with a simple purification process. , provides a manufacturing method.
〈課題を解決するための手段〉
本発明者は、細胞傷害性カリクしインをマウス顎Ｆ腺か
ら分離することに成功Ｌノた。その分離精製法は従来法
に較へて極めて容易であり、分離精製された物質がカリ
クレインであることを同定し・、これか細胞傷害性を有
することを確認した。<Means for Solving the Problems> The present inventors have succeeded in isolating cytotoxic calcinin from the mouse jaw F gland. The separation and purification method is extremely easy compared to conventional methods, and the separated and purified substance was identified as kallikrein, and it was confirmed that it has cytotoxicity.
マウスは、例えばＢＡＩＢ／ｃマウスを用いろ。マウス
の顎下腺を摘出Ｌ／て計量し・、これに１０〜２０羞（
Ｗ／Ｖ）のイークル・−ミニマムエセンシＡ・ルメシウ
ム（卜ＩＥＭ）を加えてホモシフ−卜した後、・１°Ｃ
で１００．０ＯＯＸ ｇて１時間遠心分離し、その−１
１清液の願下線抽出ン１ンを得る。この抽出液をセファ
デックス（Ｊ−５０ゲルクロマトクラフイーで分画して
第１図に示ずようなビークＢの画分を得る。このＢ画分
をざらに逆相高速液体り［１マドクラフイー（１円−Ｃ
）で分画して第２図に示ずようなビークＣの画分を得る
。For example, use a BAIB/c mouse. The submandibular gland of the mouse was removed and weighed, and 10 to 20 g
After homosizing by adding A. lumesium (W/V), the mixture was heated to 1°C.
Centrifuge at 100.0OOX g for 1 hour, then -1
1. Obtain 1 liter of liquid. This extract was fractionated using Sephadex (J-50 gel chromatography) to obtain a peak B fraction as shown in Figure 1. This B fraction was roughly separated by reverse phase high-performance liquid chromatography [1 MAD chromatography]. (1 yen-C
) to obtain a beak C fraction as shown in FIG.
このビークＣの両分は、セリンプロテアーゼであること
、その中のカリクレインであること、さらに細胞傷害性
を有ずろこと、を次のようにして確認した。It was confirmed in the following manner that both components of Beak C were serine proteases, kallikrein among them, and had cytotoxicity.
（１）セリンブｌ＝１テアーセの基質であるｌ・シルア
ルキニンメチルエステル（ＴＡＭＥ）ここよってセリン
プロテアーゼの活性が認められた。さらに、セリンプロ
テアーセ阻害剤であるシイソプロビルフルオルホスファ
ターセ（ＤＦＰ）およびファニルメチルスルフォニルフ
ルオライド（ＰＭＳＦ）を用いてセリンプロテアーセ作
用が隋書されることを確認した。(1) l.sylalkinine methyl ester (TAME), which is a substrate for selimb l=1-tease.The activity of serine protease was therefore observed. Furthermore, it was confirmed that the serine protease action was inhibited using the serine protease inhibitors diisoprobyl fluorophosphatase (DFP) and phanylmethylsulfonyl fluoride (PMSF).
（２） ＳＯＳポリアクリルアミドゲル電気泳動を行
い、分子ｉ ２７ 、０００の牟−ハントを得た。ざら
にメルカプト」−タノールｌ呑ノ用ゲルを用いて、１７
．００（ｌおよび１０，０００の二つのバンドを得た。(2) SOS polyacrylamide gel electrophoresis was performed to obtain a molecule of i 27 ,000. 17 using Zarani Mercapto-tanol drinking gel.
．． Two bands were obtained: 00(l and 10,000).
（３） （２）の単一・ハンドのタンパク質のアミノ
酸配列を調へてこれがカリクレインと同一・であること
を確認した。(3) The amino acid sequence of the single hand protein in (2) was examined and it was confirmed that it was the same as kallikrein.
（１１）胸腺細胞を標的細胞として用いるトリバンブル
ー色素排除試験によって細胞傷害性を確認した。この他
に、標的細胞としては、牌細胞、Ｔリンパ球、］３リン
パ球、腫瘍化マウス線維等細胞（１９２９株）等を用い
て細胞傷害性が認められた。(11) Cytotoxicity was confirmed by a trivan blue dye exclusion test using thymocytes as target cells. In addition, cytotoxicity was observed using target cells such as tile cells, T lymphocytes, ]3 lymphocytes, and tumorigenic mouse fibrous cells (1929 strain).
以下に実施例を示す。特に記載がない限りパーセント表
示は重量パーセントを表す。Examples are shown below. Unless otherwise specified, percentages represent weight percentages.
実施例１；
Ｆｉ週令の雄のＢＡＬＤ／ Ｃマウスの顎丁腺を摘出し
て（４，０ｇ）、これニ２０％（ｗ／ｖ） （７）ＭＥ
Ｍを加えテン０〜１３０分間ホモシネ−・１・した。得
られた均ｆｆｌ？１ｔ２（１，５ｇタノバク質ンを／１
’Ｃ，：で１００．０００Ｘ ｇて１時間遠心分離し
て、肖られた上清を、ｌｌＥＰＥｓｉ５０ｍＭ ＮａＣ
ＩｔｌＮ衝液（ｐＨ７，２’）で平衡にしておいたセフ
ァデックスＣニー５０ゲルクロマトグラフィー （ファ
ルマシア社製、φ４．Ｏｃｎ＋Ｘ ］０００ｃｍζこて
分画した。溶出は同緩衝液で行った。第１図ピーク１３
に相当する両分を集め、次いでこれを直接に、０．１％
トリフルオロ酢酸（ＴＦＡ）で平衡にしておいたカラノ
、を用いた逆相１１ＰＬｃ （ＴＯ８（１＋１製、＋’
１ＤＳ−１２０Ｔカラム、 φ２．］５ｃｍＸ３０ｃｍ
）に供して、分画した。溶出は０暑％’ｒト”Ａを含む
７０％（ｖ／ｖ、’）アセトニトリル配にて流速１ｍ１
／分て行った。第２図ピークＣに相当する両分を回収し
て、８４３０ノＪ．ｇタンパク質の精製分離物を得た。Example 1; The maxillary gland of a Fi-week-old male BALD/C mouse was removed (4.0 g), and 20% (w/v) (7) ME
M was added and the mixture was homogenized for 0 to 130 minutes. The average ffl obtained? 1t2 (1.5g tanobacterium/1
Centrifuged at 100.000Xg for 1 hour at
Sephadex C Knee 50 gel chromatography (manufactured by Pharmacia, φ4.Ocn + peak 13
Collect the amount equivalent to 0.1% and then directly add this to 0.1%
Reversed phase 11PLc (TO8 (manufactured by 1+1, +'
1DS-120T column, φ2. ]5cmX30cm
) and fractionated. Elution was carried out using a 70% (v/v,') acetonitrile solution containing 0% 'r'A at a flow rate of 1 ml.
I went / minutes. Both portions corresponding to peak C in FIG. 2 were collected and 8430 J. A purified isolate of g protein was obtained.
得られた精製物の性質を次のような確認試験等を行って
確認した。The properties of the obtained purified product were confirmed by conducting the following confirmation tests.
々 ｌ ゝ
ＳｒｌＳポリケルアクリルアミドゲル濃度は、、ａｅｍ
ｍｌｉらの方法ｒ：Ｕ． Ｋ． Ｌａｅｍｍｌｉ、Ｎａ
ｔｕｒｅ， ’２１：１（ｉ８０−６８５．（１９７０
）］ （ご′Ｃ行−った（アクリル−／ミド淵）朗２０
％、Ｓｌ）！ＪＪｊ度２％、ケルＰ５２１１１ｍ）。ざ
１．りこメルカフトエタノー・ル）不順試料の場合には
メルカーノト］−タノールＦ）％を加えた。試料な２％
５ｔ）Ｓを含む試料用緩衝液（５％メルカフトエタノ・
−ルｌ奈加ンＩＳたは無添加緩衝液）！こ各々溶解−５
分間煮沸処哩して電気泳動ζこ供した。泳動は５０〜（
ｉｏｍ八て１１−）人・。l ゝSrlS polykelacrylamide gel concentration is, aem
mli et al.'s method: U. K. Laemmli, Na.
ture, '21:1 (i80-685. (1970
)] (Go'C line (acrylic/Midobuchi) Akira 20
%, Sl)! JJj degree 2%, Kel P52111m). Za1. In the case of nonconforming samples, % Mercanot]-tanol F) was added. 2% sample
5t) Sample buffer containing S (5% mercaft ethanol,
-Lnakan IS or non-additive buffer)! Dissolve each of these-5
The mixture was boiled for a minute and subjected to electrophoresis. The electrophoresis is from 50 to (
iom eight 11-) people.
分子積標準マーカー（こは、５Ｉ）Ｓ−６［シクマ比製
、ウシ面詰アルフミン（分子・ψ６Ｇ、０００）、卵白
アルフミン（同４５，０００）、パパイン（同３Ｌ７０
０） ＰＭＳＩ・処理トリアジノ・−りン（＋１ｊｌ
２４ 、０００ ）、β ラクトり１７ブリン（同１８
９．り００）、リソチーム（同１４，３００）および炭
酸脱水素酵素（同２眠０００）を含む］、−Ｔ゛トクロ
ーｉＣ（同１２，４００）およびアブ［−１チニン〈同
（ｉ、５００）を用いた。・その結果、メルカプ゛ｌ・
エタノール無添加ケルここて分−（−ｉ−２７，０００
の牟−ハントか得られた。メルカプトＪ−夕７ノールを
奈加り゛ルＣ゛は分１’量２７．０００． Ｖ７，００
０および１０ 、０００の三つの単副ハントか１１＋ら
れた。これは、ＳＳ結合したサフユニツトからなるカリ
クレインの分子−構成と同一である。Molecular volume standard marker (Koha, 5I) S-6 [Shikumahi, bovine stuffed alhumin (molecular ψ6G, 000), egg white alhumin (45,000), papain (3L70)
0) PMSI-treated triazino-phosphorus (+1jl
24,000), β-lacti 17 brine (18
9. 00), lysozyme (14,300) and carbonic anhydrase (2000); was used.・As a result, Mercapil・
Ethanol-free kerosene - (-i-27,000
I got the hunt. Mercapto J-Y7 Nord is 1 minute amount of 27.000. V7,00
Three single minor hunts of 0 and 10,000 or 11+ were made. This is identical to the molecular structure of kallikrein, which consists of SS-bonded suffunits.
アーミ＝Ｊ鍍配列−θし一定
？ｉに法（ごて還元してカルオ、キシメチル化した試本
・Ｉの一／Ｓ〕酸配列を１ｌｃｔｓ＋ｃｌ、の方ｉ、！
′、ｃ、Ｊ、 Ｂｉｏｌ、 ＣＩ＋ｅ＋ｎ。Army = J plate arrangement - θ constant? i method (sample reduced with a trowel and converted to caro-oxymethyl, 1/S of I) acid sequence to 1 lcts + cl, the one i,!
', c, J, Biol, CI+e+n.
２別７！Ｊ９０−７！１９７ （＋り８１）］ ！こて
液相アミノ酸シクエンザー（Ａ 円Ｊｌ ｉ ｃ〔ｌ
Ｒｉ ｏｓｙｓ Ｌｅｍｓ？−１′製、４７０Ａ！ｔ２
）で調へた。2 different 7! J90-7!197 (+ri81)]! Trowel liquid phase amino acid sequencer (A circle Jl ic [l
Ri osys Lems? -1' made, 470A! t2
).
以トの分子量およびアミノ酸配列分析の結果から、精製
分離タンパ７質ａｔ力リクレイノ（ご属するものである
ことが６１１１月された。Based on the results of molecular weight and amino acid sequence analysis, it was determined that the purified isolated protein 7 protein belongs to Lykleino (6111).
組胞−イ■〜割性會舌−性謙−１ｕ’ｌ一定試トｌをＲ
１）ト１ｆ−１６４０−１Ｂ地（キップ！社製）て−噌
透析する。種）２の潤度に調製した透１１ｉ試料の各１
０７Ｊ、ｌを、胸Ｉ！細１抱の１０％加熱処理胎児ウシ
血清（ハイク１フーン社製）含有量培地？″′；＝遊液
仁こ最終容１１００７ｚ（細胞数約ｌＸｌ０）になる様
に加える。浮遊ｊ音は９（１−ウｊ：ルンイグロプレ−
１−（：２＝二シンク製）にｒめ分？４’、 ＬｙでＪ
′５＜。これを「逼％炭酸カスを含む加湿雰囲気中で１
３７°Ｃて３（４間培養ずろ。各培養の生細胞をトリパ
ンフルー色素排除試験で計数する。Histocytosis - I■ ~ Climax - Seiken - 1 u'l constant test l R
1) Dialyze using 1F-1640-1B (manufactured by Kip!). Species) 1 of each of the transparent 11i samples prepared to a moisture content of 2.
07J, l, chest I! A medium containing a small amount of 10% heat-treated fetal bovine serum (manufactured by Haiku Hoon Co., Ltd.)? ″';=Final volume of liquid liquid is 11007z (number of cells approximately 1X10).
1-(:2=Made by Nisink) rth minute? 4', Ly in J
'5<. This is carried out in a humidified atmosphere containing 1% carbon dioxide scum.
Incubate for 3 (4) days at 37°C. Live cells from each culture are counted by trypan flu dye exclusion test.
細胞傷害活性は下記の式で示すＡ１１ｌ胞傷害活性（％
）で表した。The cytotoxic activity is expressed by the following formula: A11 cytotoxic activity (%
).
細胞傷害活性（％）＝
（１−試験培養の細胞数／対照培養の細胞数）Ｘ１００
その結果、本精製物（５０）１８／ ｍ ｌ ）の活性
５０．２％が認められた。Cytotoxic activity (%) = (1 - number of cells in test culture/number of cells in control culture) x 100
As a result, the activity of this purified product (50) (18/ml) was found to be 50.2%.
セ１゛ ロー” −゛Ｉ！１リ−しｔ
セリンブＤテアーセ活性の測定は、Ｍｏｒｉｗａｋｉ（
＋）方法［薬学雑誌、９１４１３−４１６ （１９７１
）］に準しで′ｒ Ａ ｐ＋ １：、を基質に用いで行
った。試；ｊａｉｌ Ｏ、１１ｎ＋ｎ ｌを０．１ｍｌ
の０、団Ｔ Ａ旨Ｅおよ０Ｏ６１ト１燐酸緩南液（１＋
ｌｌ ８．０）と混合し、；３０°（゛て３０分間イン
ギ：Ｌヘートする。反応を０．２ｍｌの１５％（ｗ／ｖ
） Ｉ・リクＤル酢酸および帆１ｍｌの２％（ｗ／ν）
マンガンカリウノ、を加えて反応を停止［−させる、１
分後、０．１ｍｌの１０％（ＩＪ／ｖ）重炭酸ナトリウ
１、を加え、次いて４ｍｌの０．／１％クロモトローフ
酸の６７％硫酸溶液とよく混合する。混合液の吸光度を
５８Ｏｎｍて測定する。その結果、本精製物は特異的な
カリクレイン活性（８（ｉ、７７Ｊ、ｍｏｌｅｓ／ｍｉ
ｎ／ｍｇ）を有することか証明された。Serimbu D teaase activity was measured using Moriwaki (
+) Method [Pharmaceutical Journal, 91413-416 (1971
)] using 'r A p+ 1: as the substrate. Test; jail O, 11n+n l 0.1ml
0, group T A to E and 0O61 to 1 phosphoric acid softening solution (1+
8.0) and heat at 30° for 30 minutes.
) 2% (w/ν) of I.LikuDruacetic acid and 1 ml of
Stop the reaction by adding manganese kaluno [-, 1
After 0.1 ml of 10% (IJ/v) sodium bicarbonate was added, followed by 4 ml of 0.1 ml of 10% (IJ/v) sodium bicarbonate. /1% chromotrophic acid in 67% sulfuric acid and mix well. The absorbance of the mixture is measured at 58 Onm. As a result, this purified product showed specific kallikrein activity (8(i, 77J, moles/mi)
n/mg).
宝 −イーンー活オＬの一阻
７七−イ１ユｊ１］
試料（３，′７ｘｌＯ−’計１）をＤ Ｆｌ）またはＰ
Ｍ Ｓ Ｆ’の３．７×１０−２ｕｎｉ、３．７Ｘ
ＩＱ−’ｍＭまたは３．７ｍＭｃ７）溶液（コ各々加え
て、３７℃で６０分間インキュベートシた。基質ＴＡ阻
：を用いて−１−記の方法でカリクレイン活性を測定し
た。ドロ害剤を加えかい対！！何と比較したカリクレー
イン活性をト１］害率（％）で表した。その結果、本精
製物のニス：Ｊ−ル分解作用はノｌリクレイン阻害剤（
こよって確実むこｒ口害されることか証明された（表１
）。Treasure - Een - Active O L 77 - I 1 Yuj1] D Fl) or P the sample (3,'7xlO-'total 1)
M S F' 3.7 x 10-2 uni, 3.7X
IQ-'mM or 3.7mM Mc7) solution (each) was added and incubated at 37°C for 60 minutes. Kallikrein activity was measured using the substrate TA inhibitor by the method described in -1. The activity of kallikrein was expressed as a harm rate (%) compared to that of the kallikrein inhibitor.
This proves that people will definitely be harassed (Table 1)
).
一−−−／
／、−、、−、−、−−−−
表−１−
セリンフロテアーセ阻害剤ζごよるセリンプ１−１テア
ーセ活性の阻害作用
団害剤 試料／ド■１害剤比
阻害率
無しく月明）Ｏ
ＤＦＰ １０−”
：３．１１０ ’ ４
２．３１０−’ ９５．４）’Ｍ
ＳＦ １０−／
Ｌ５１０−’ ３８．’Ｊ１
０’＝ ８４．７実施例２：
ＢＡＬＢ／ ｃ７１ブスの雌の５週令を用いて実施例と
同様に精製物を調製した。督られた調製物ｃｉ、セリン
ブロテアーセ活性が６．７μ…ｏｌｅｓ／ｍ団／ｎ１８
、細胞傷害性活性が３２．３％（２３７１ｇ／ｍｌ）で
あって、アミノ酸配列はカリクレインのｍ Ｇ Ｋ −
（ｉと同・てあ・旬こ。----/ /, -,, -, -, ---- Table 1 - Inhibitory effect of serine phlotease inhibitor ζ on selymp 1-1 tease activity Group harmful agent Sample/d ■ 1 harmful agent ratio Moonlight without inhibition rate) ODFP 10-”
:3.110' 4
2.310-'95.4)'M
SF 10-/
L510-' 38. 'J1
0'=84.7 Example 2: A purified product was prepared in the same manner as in Example using a 5-week-old female BALB/c71 female. The prepared preparation ci had a serine brotease activity of 6.7 μoles/m group/n18
, the cytotoxic activity was 32.3% (2371 g/ml), and the amino acid sequence was m G K - of kallikrein.
(Same as i, Thea, Shunko.
本発明の好ましい実施態様をここに詳細ζこ記述しＩた
が、腫／、７の変更が本発明の精神または添付した特許
請求の範囲に反するごとなく１テわれろことは当業者に
理解されよう。Although preferred embodiments of the invention have been described in detail herein, those skilled in the art will appreciate that modifications may be made without departing from the spirit of the invention or the scope of the appended claims. It will be.
く本発明の効果〉
以北のようζこ、本発明ζこよれは、細胞傷害性カック
レイ〉・をマウス顎Ｆ腺から容易に高収率で製造するこ
とかｉＪｉ能にん゛す、臨床用もしくは実験研究用の細
胞傷害性カリクレインを大量に安価に提供することかで
きる。Effects of the Present Invention The present invention has the ability to easily produce cytotoxic pharyngeal glands from mouse jaw F glands in high yields, and has been shown to be clinically viable. It is possible to provide cytotoxic kallikrein in large quantities at low cost for use in commercial or experimental research.
また、本発明もこまって製造されるカリクレインは高い
細胞傷害性活性をイ１することから、カリクレインを１
．Ｙ来の脳動脈硬化症や高血圧症の予防治療のみならず
、鞘Ｉ胞、例えば、自ｒｆｎ球、癌細胞、ウイルズ感染
斤■胞等の破壊にこれを使用することかでき、諸疾唐、
のｊ−防治療にきわめて有用である。In addition, since kallikrein produced according to the present invention has high cytotoxic activity, kallikrein is
．． It can be used not only for the preventive treatment of cerebral arteriosclerosis and hypertension, but also for the destruction of I-sheath cells, such as autologous RFN bulbs, cancer cells, virus-infected cells, etc., and is effective against various diseases. ,
It is extremely useful for J-preventive treatment.
第１図は、本発明の細胞傷害性カリクレインの調製工程
ζこおけるケルクロマトクラ゛フィーの溶出パターンを
示す、説明図である。
第２図は、本発明の細胞傷害性）１リクレインの調製工
程における逆相高速？（１体り１コマトクラーノ７−の
溶出パターンを示す、説明図である。FIG. 1 is an explanatory diagram showing the elution pattern of Kel chromatography in the preparation step ζ of the cytotoxic kallikrein of the present invention. Figure 2 shows the high-speed reversed phase preparation process of cytotoxic) 1 recrein of the present invention. (This is an explanatory diagram showing the elution pattern of 1 column of 1 column of matoclano 7.
Claims (1)
。 ２、マウス顎下腺をホモジネートして、遠心分離し、ゲ
ルクロマトグラフィー分画の後、さらに逆相高速液体ク
ロマトグラフィーで分画して精製することからなる、細
胞傷害性カリクレインの製造法。 ３、請求項１に記載の細胞傷害性カリクレインの、細胞
傷害剤としての使用。[Claims] 1. Cytotoxic kallikrein isolated from mouse submandibular gland. 2. A method for producing cytotoxic kallikrein, which comprises homogenizing mouse submandibular gland, centrifuging, gel chromatography fractionation, and further fractionation and purification by reverse phase high performance liquid chromatography. 3. Use of the cytotoxic kallikrein according to claim 1 as a cytotoxic agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63275116A JPH02119775A (en) | 1988-10-31 | 1988-10-31 | Cytotoxic kallikrein, production thereof and use as cytotoxic agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63275116A JPH02119775A (en) | 1988-10-31 | 1988-10-31 | Cytotoxic kallikrein, production thereof and use as cytotoxic agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02119775A true JPH02119775A (en) | 1990-05-07 |
Family
ID=17550945
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63275116A Pending JPH02119775A (en) | 1988-10-31 | 1988-10-31 | Cytotoxic kallikrein, production thereof and use as cytotoxic agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02119775A (en) |
-
1988
- 1988-10-31 JP JP63275116A patent/JPH02119775A/en active Pending
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