JPH08781B2 - Glucagon-like bioactive agent - Google Patents
Glucagon-like bioactive agentInfo
- Publication number
- JPH08781B2 JPH08781B2 JP61307504A JP30750486A JPH08781B2 JP H08781 B2 JPH08781 B2 JP H08781B2 JP 61307504 A JP61307504 A JP 61307504A JP 30750486 A JP30750486 A JP 30750486A JP H08781 B2 JPH08781 B2 JP H08781B2
- Authority
- JP
- Japan
- Prior art keywords
- glucagon
- gsp
- bioactive agent
- synthesis
- glycogen
- 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
- 239000012867 bioactive agent Substances 0.000 title claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 17
- 238000003786 synthesis reaction Methods 0.000 claims description 17
- 229920002527 Glycogen Polymers 0.000 claims description 11
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 11
- 239000004202 carbamide Substances 0.000 claims description 11
- 229940096919 glycogen Drugs 0.000 claims description 11
- 230000001737 promoting effect Effects 0.000 claims description 6
- GIQZFLZPSASIEJ-UHFFFAOYSA-N Ala-Val-Pro-Tyr-Pro-Gln-Arg Natural products CC(N)C(=O)NC(C(C)C)C(=O)N1CCCC1C(=O)NC(C(=O)N1C(CCC1)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(O)=O)CC1=CC=C(O)C=C1 GIQZFLZPSASIEJ-UHFFFAOYSA-N 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 102000051325 Glucagon Human genes 0.000 description 16
- 108060003199 Glucagon Proteins 0.000 description 16
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 16
- 229960004666 glucagon Drugs 0.000 description 16
- 238000000034 method Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 206010020575 Hyperammonaemia Diseases 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 208000013016 Hypoglycemia Diseases 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 230000002218 hypoglycaemic effect Effects 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- -1 t-butoxycarbonyl group Chemical group 0.000 description 2
- 230000004143 urea cycle Effects 0.000 description 2
- 239000005541 ACE inhibitor Substances 0.000 description 1
- 101710129690 Angiotensin-converting enzyme inhibitor Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101710086378 Bradykinin-potentiating and C-type natriuretic peptides Proteins 0.000 description 1
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 1
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 1
- 102000007390 Glycogen Phosphorylase Human genes 0.000 description 1
- 108010046163 Glycogen Phosphorylase Proteins 0.000 description 1
- 108010001483 Glycogen Synthase Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 108060000200 adenylate cyclase Proteins 0.000 description 1
- 102000030621 adenylate cyclase Human genes 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- FSEUPUDHEBLWJY-HWKANZROSA-N diacetylmonoxime Chemical compound CC(=O)C(\C)=N\O FSEUPUDHEBLWJY-HWKANZROSA-N 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、高アンモニア血症，低血糖症等に有効な、
下記の構造からなるグルカゴン様生理活性剤（以下、GS
Pと記す）に関するものである。Ala−Val−Pro−Tyr−P
ro−Gln−Arg（Alaはアラニン、Valはバリン、Proはプ
ロリン、Tyrはチロシン、Glnはグルタミン、Argはアル
ギニンを示す。） （従来技術） 生体内では、アミノ酸，蛋白質の代謝の一環として、
生成されるアンモニアを尿素に転換し、体外へ排泄する
尿素サイクルという機構が作働しているが、この尿素サ
イクルの酵素系の欠陥あるいは促進ホルモンの不足など
により高アンモニア血症が生じる場合がある。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is effective for hyperammonemia, hypoglycemia, etc.
Glucagon-like bioactive agent consisting of the following structure (hereinafter GS
P)). Ala-Val-Pro-Tyr-P
ro-Gln-Arg (Ala is alanine, Val is valine, Pro is proline, Tyr is tyrosine, Gln is glutamine, and Arg is arginine.) (Prior art) In vivo, as part of metabolism of amino acids and proteins,
The mechanism of the urea cycle, which converts the produced ammonia into urea and excretes it to the outside of the body, operates, but hyperammonemia may occur due to defects in the enzyme system of this urea cycle or lack of stimulating hormones. .
従来、この高アンモニア血症に有効なペプチドホルモ
ンとしてグルカゴンが知られている。正常肝臓の培養細
胞には尿素合成の機能があるが、この培養細胞にグルカ
ゴンを加えることにより、尿素合成は促進される。Heretofore, glucagon has been known as a peptide hormone effective for this hyperammonemia. Although cultured cells of normal liver have a function of urea synthesis, addition of glucagon to the cultured cells promotes urea synthesis.
また、グルカゴンは、アデニルサイクラーゼを活性化
し、サイクリックAMP依存性プロテインキナーゼ，グリ
コーゲンホスホリラーゼを介してグリコーゲン分解を促
進し、一方で、グリコーゲン合成酵素の活性抑制により
グリコーゲン合成を抑制する。このことから、グルカゴ
ンは、経口糖尿病薬やインシュリンによる低血糖症の治
療などにも用いられている。In addition, glucagon activates adenyl cyclase and promotes glycogen degradation via cyclic AMP-dependent protein kinase and glycogen phosphorylase, while suppressing glycogen synthesis by suppressing the activity of glycogen synthase. For this reason, glucagon is also used for oral diabetes drugs and treatment of hypoglycemia with insulin.
（発明が解決しようとする問題点） しかしながら、グルカゴンを膵臓からの抽出によって
得る場合、得られるグルカゴンは微量であり、かつ操作
は多工程を要し繁雑である。合成によって得る場合は、
グルカゴンはアミノ酸29個からなる長いペプチドである
ため、合成する時間、費用等がかかり経済的に有利でな
い。(Problems to be solved by the invention) However, when glucagon is obtained by extraction from the pancreas, the amount of glucagon obtained is very small, and the operation requires many steps and is complicated. If you get it by synthesis,
Since glucagon is a long peptide consisting of 29 amino acids, it takes time and cost to synthesize it, which is not economically advantageous.
（問題を解決するための手段） 本発明者らは、グルカゴンと同様の効果を有する物質
を経済的に有利にかつ容易な方法で得ることを目的に鋭
意研究を重ねた結果、牛由来カゼインをトリプシンなど
で分解することにより得られたペプチドがグルカゴン様
生理活性を有することを見い出し、この知見に基づいて
本発明に至った。すなわち本発明は、下記の構造からな
るグルカゴン様生理活性剤である。(Means for Solving the Problem) The present inventors have conducted extensive studies to obtain a substance having the same effect as glucagon by an economically advantageous and easy method, and as a result, obtain bovine casein. It was found that the peptide obtained by decomposing with trypsin etc. has glucagon-like physiological activity, and the present invention was completed based on this finding. That is, the present invention is a glucagon-like bioactive agent having the following structure.
Ala−Val−Pro−Tyr−Pro−Gln−Arg本発明のGSPと同
一のペプチドは、アンジオテンシン転換酵素阻害剤とし
て知られているものであるが（特公昭61−51562）、こ
のペプチドがグルカゴン様生理活性を有することは、本
発明者らが始めて見い出した知見である。Ala-Val-Pro-Tyr-Pro-Gln-Arg The same peptide as GSP of the present invention is known as an angiotensin converting enzyme inhibitor (Japanese Patent Publication No. 61-51562), but this peptide is glucagon-like. Having physiological activity is a finding that the present inventors first discovered.
本発明によるGSPを得るには、特公昭61−51562に記載
されているように、牛由来カゼインをpH5.5〜9.0の条件
下、トリプシンにより分解し、分解物を100℃程度の加
熱処理又は酸を加えて処理することによりトリプシン及
び未分解のカゼインを沈澱させ、この沈澱物を遠心分離
などにより除去する。このようにして得た母液を水酸化
ナトリウムなどのアルカリで中和したのち、減圧下で２
〜３倍に濃縮する。このようにして得た濃縮液を精製し
て製品を得る。To obtain GSP according to the present invention, as described in JP-B-61-51562, cattle-derived casein is decomposed with trypsin under conditions of pH 5.5 to 9.0, and the decomposed product is subjected to heat treatment at about 100 ° C. or Trypsin and undecomposed casein are precipitated by adding an acid to the precipitate, and the precipitate is removed by centrifugation or the like. The mother liquor thus obtained is neutralized with an alkali such as sodium hydroxide, and then it is concentrated under reduced pressure.
Concentrate ~ 3 times. The concentrate thus obtained is purified to obtain a product.
また、公知のペプチド合成手段を利用して得ることも
可能である。It can also be obtained by utilizing a known peptide synthesis means.
すなわち、一方のアミノ酸のアミノ基をベンジルオキ
シカルボニル基又は、ｔ−ブトキシカルボニル基等で保
護、他方のアミノ酸又はペプチドのカルボキシル基をベ
ンジルエステル等で保護し、DCC（N,N′−ジシクロヘキ
シルカルボジイミド）等でカップリングさせる。この操
作を繰り返し、保護基を脱離させ、精製して製品を得る
ことができる。That is, the amino group of one amino acid is protected with a benzyloxycarbonyl group or a t-butoxycarbonyl group, the carboxyl group of the other amino acid or peptide is protected with a benzyl ester, and DCC (N, N'-dicyclohexylcarbodiimide) Etc. This operation can be repeated to remove the protecting group, and the product can be obtained by purification.
（効果） 本発明のGSPは、経済的に有利に、かつ容易な方法で
得ることができ、尿素合成促進作用及びグリコーゲン合
成抑制作用を有し、高アンモニア血症および低血糖症に
対して有効である。(Effect) The GSP of the present invention is economically advantageous and can be obtained by an easy method, has a urea synthesis promoting action and a glycogen synthesis inhibiting action, and is effective against hyperammonemia and hypoglycemia. Is.
またGSPは、単独でも尿素合成促進作用及びグリコー
ゲン分解促進作用を表わすが、グルカゴンと併用するこ
とにより、GSPはグルカゴンの該二作用を増強する作用
もある。GSP alone exhibits urea synthesis promoting action and glycogen decomposition promoting action, but when used in combination with glucagon, GSP also has the action of enhancing the dual action of glucagon.
（実施例） 次に実施例により本発明のGSPの生理作用について説
明する。(Example) Next, the physiological action of GSP of the present invention will be described with reference to Examples.
実施例１ GSPの尿素合成に及ぼす影響 ウィスター（Wistar）系雄ラット（体重約250g）よ
り、セグレンらの方法（P.O.Seglen,Methods in Cell B
iology 13,29（1976））を改良した中村らの方法（蛋
白質核酸酵素 別冊No.24動物実験の手技手法,P.55）に
より、肝臓細胞を単離し、10％仔牛血清を含むウィリア
ムズＥ培地（Flow Laboratories社製）で、１×105細胞
／培養皿底面1cm2（培養液125μを含む）の濃度で24
時間培養した後、血清およびアルギニンを含まないウィ
リアムズＥ培地でさらに24時間培養した。Example 1 Effect of GSP on urea synthesis From a Wistar male rat (body weight: about 250 g), the method of Seglen et al. (POSeglen, Methods in Cell B) was used.
( 13 ), 29 (1976)), a modified method of Nakamura et al. (Protein / Nucleic Acid and Enzyme, Separate Volume No. 24, Techniques for Animal Experiments, P.55), to isolate liver cells and Williams E medium containing 10% fetal calf serum. (Flow Laboratories) at a concentration of 1 × 10 5 cells / bottom of culture dish 1 cm 2 (including 125 μl of culture solution).
After culturing for an hour, the cells were further cultivated for 24 hours in Williams E medium containing no serum and arginine.
培養３日目の細胞（２×105細胞／培養皿底面2cm
2（培養液250μを含む））に、図１−1,1−２に示し
たようなGSPの各濃度を加え、６時間後に培養上清を取
り、ジェームズらによるジアセチルモノオキシム法（W.
James and D.Danica,Anal.Biochem.,37,412（1971））
による540nmの吸光度を測定し、尿素の相対合成量とし
た。Cells on day 3 of culture (2 x 10 5 cells / bottom 2 cm of culture dish)
2 (including 250 μl of culture broth), each concentration of GSP as shown in FIGS. 1-1 and 1-2 was added, and after 6 hours, the culture supernatant was taken and the diacetyl monooxime method (W.
James and D. Danica, Anal. Biochem., 37 , 412 (1971))
The absorbance at 540 nm was measured and used as the relative amount of urea synthesized.
また、グルカゴンの一定量（10-7M）をGSPの各濃度と
併用したものについても同様の方法で実施した。In addition, the same method was performed using a certain amount of glucagon (10 −7 M) in combination with each concentration of GSP.
結果を図１−１に示す。 The results are shown in Figure 1-1.
次に、同様の方法でグルカゴンの各濃度に一定量のGS
Pを加えたものおよびグルカゴン単独のものについても
実施した。Next, in the same way, a certain amount of GS was added to each concentration of glucagon.
It was also carried out for those with P added and for glucagon alone.
結果を図１−２に示す。 The results are shown in Figure 1-2.
図１−１および図１−２から、GSPが尿素合成を促進
していること、およびグルカゴンの尿素合成促進作用を
増強していることがわかる。特に、グルカゴンの濃度が
10-7M付近のときにGSPはグルカゴンを最も強く増強して
いる。It can be seen from FIGS. 1-1 and 1-2 that GSP promotes urea synthesis and that glucagon enhances the urea synthesis promoting action. Especially when the concentration of glucagon is
At around 10 -7 M, GSP strengthens glucagon most strongly.
実施例２ GSPのグリコーゲン合成に及ぼす影響 実施例１と同様の方法で培養した培養３日目のラット
の肝臓細胞（１×106細胞／培養皿底面10cm2（培養液1m
lを含む））に、GSPの各濃度および14C−Ｄ−グルコー
ス（0.5μCi）を加え、３時間培養後、上清を捨て、30
％水酸化カリウムで細胞を可溶化後（100℃,30分）、冷
エタノールでグリコーゲンを沈澱させた。次に、冷エタ
ノールで沈澱を２回洗浄した後、液体シンチレーション
カウンターにより、14C取り込み量（DPM）を測定し、グ
リコーゲンの相対合成量とした。Example 2 Effect of GSP on glycogen synthesis Rat liver cells cultured on day 3 in the same manner as in Example 1 (1 × 10 6 cells / bottom 10 cm 2 of culture dish (culture medium 1 m
l))), each concentration of GSP and 14 C-D-glucose (0.5 μCi) were added, the mixture was incubated for 3 hours, and the supernatant was discarded.
After solubilizing the cells with 100% potassium hydroxide (100 ° C., 30 minutes), glycogen was precipitated with cold ethanol. Next, after washing the precipitate twice with cold ethanol, the amount of 14 C uptake (DPM) was measured by a liquid scintillation counter and used as the relative amount of glycogen synthesized.
また、GSPにグリコーゲン合成促進作用を有するイン
シュリン（2nM）を加えたものについても同様に実施し
た。The same procedure was performed for GSP to which insulin (2 nM) having a glycogen synthesis promoting action was added.
結果を図２に示す。 The results are shown in Figure 2.
図２より、GSPは、インシュリンの存在にかかわら
ず、グリコーゲンの合成を抑制することがわかる。From FIG. 2, it can be seen that GSP suppresses glycogen synthesis regardless of the presence of insulin.
図１−１および図１−２は、本発明のGSPの、尿素合成
に及ぼす作用を540nmにおける吸光度によって示した図
である。 図２は、本発明のGSPの、グリコーゲン合成に及ぼす作
用を、液体シンチレーションカウンターにより測定した
14C取り込み量（DPM）によって示した図である。FIG. 1-1 and FIG. 1-2 are diagrams showing the action of GSP of the present invention on urea synthesis by the absorbance at 540 nm. FIG. 2 shows the effect of GSP of the present invention on glycogen synthesis, which was measured by a liquid scintillation counter.
It is the figure shown by the amount of 14 C uptake (DPM).
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ａ６１Ｋ 37/02 ＡＥＥ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI technical display area A61K 37/02 AEE
Claims (3)
剤Ala−Val−Pro−Tyr−Pro−Gln−Arg1. A glucagon-like bioactive agent having the following structure: Ala-Val-Pro-Tyr-Pro-Gln-Arg
用である特許請求の範囲第１項記載の生理活性剤2. The bioactive agent according to claim 1, wherein the glucagon-like bioactivity is an action of promoting the synthesis of urea.
抑制作用である特許請求の範囲第１項記載の生理活性剤3. The bioactive agent according to claim 1, wherein the glucagon-like bioactivity is an action of inhibiting glycogen synthesis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61307504A JPH08781B2 (en) | 1986-12-23 | 1986-12-23 | Glucagon-like bioactive agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61307504A JPH08781B2 (en) | 1986-12-23 | 1986-12-23 | Glucagon-like bioactive agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63159323A JPS63159323A (en) | 1988-07-02 |
| JPH08781B2 true JPH08781B2 (en) | 1996-01-10 |
Family
ID=17969878
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61307504A Expired - Lifetime JPH08781B2 (en) | 1986-12-23 | 1986-12-23 | Glucagon-like bioactive agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08781B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2197471A2 (en) * | 2007-09-11 | 2010-06-23 | Mondobiotech Laboratories AG | Use of the peptides maippkknqdk (cow kappa casein 106-116) and/or ygfqna (serorphin) as therapeutic agents |
-
1986
- 1986-12-23 JP JP61307504A patent/JPH08781B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63159323A (en) | 1988-07-02 |
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