JPH0236229B2 - KOSOKATSUSEIZODAIHOHO - Google Patents
KOSOKATSUSEIZODAIHOHOInfo
- Publication number
- JPH0236229B2 JPH0236229B2 JP2533386A JP2533386A JPH0236229B2 JP H0236229 B2 JPH0236229 B2 JP H0236229B2 JP 2533386 A JP2533386 A JP 2533386A JP 2533386 A JP2533386 A JP 2533386A JP H0236229 B2 JPH0236229 B2 JP H0236229B2
- Authority
- JP
- Japan
- Prior art keywords
- pyruvate kinase
- reaction
- dimethyl sulfoxide
- activity
- present
- 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
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 17
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 17
- 230000000694 effects Effects 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 238000006911 enzymatic reaction Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000002777 nucleoside Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 3
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- -1 nucleoside diphosphate Chemical class 0.000 description 3
- 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 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- 239000001177 diphosphate Substances 0.000 description 2
- 235000011180 diphosphates Nutrition 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 2
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000001226 triphosphate Substances 0.000 description 2
- 235000011178 triphosphate Nutrition 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- 108010092060 Acetate kinase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
Description
【発明の詳細な説明】
産業上の利用分野
本発明はピルビン酸キナーゼを用いる酵素反応
において、酵素活性を増大させる方法に関するも
のである。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for increasing enzyme activity in enzymatic reactions using pyruvate kinase.
ピルビン酸キナーゼは、生体内における物質代
謝に係る重要な酵素であつて、近年、アデノシン
二リン酸の定量、アデノシン三リン酸加水分解酵
素の定量、酢酸及び酢酸キナーゼ酵素の定量な
ど、医療や発酵工学などの分野において広く用い
られている。本発明は、このピルビン酸キナーゼ
の酵素活性の増大を図り、前記分野において、効
果的にピルビン酸キナーゼを利用することを目的
とするものである。 Pyruvate kinase is an important enzyme involved in the metabolism of substances in living organisms, and in recent years, it has been used in medical and fermentation applications, such as the determination of adenosine diphosphate, the determination of adenosine triphosphate hydrolase, and the determination of acetic acid and acetate kinase enzymes. It is widely used in fields such as engineering. The present invention aims to increase the enzymatic activity of pyruvate kinase and to effectively utilize pyruvate kinase in the above field.
従来の技術
従来、ピルビン酸キナーゼは主にウサギなどの
動物の筋肉組織から抽出精製され、そのまま用い
られている。このようにピルビン酸キナーゼは生
体から抽出される酵素であるため、特にその精製
標品は高価であり、したがつて、できる限り有効
に用いることが望ましい。しかしながら、ピルビ
ン酸キナーゼの有効利用のために、その酵素活性
を人為的に増大させることは、これまで行われて
いなかつた。PRIOR ART Conventionally, pyruvate kinase has been mainly extracted and purified from the muscle tissues of animals such as rabbits, and used as is. Since pyruvate kinase is an enzyme extracted from living organisms, its purified preparation is particularly expensive, and therefore it is desirable to use it as effectively as possible. However, for the effective use of pyruvate kinase, artificially increasing its enzymatic activity has not been attempted so far.
発明が解決しようとする問題点
本発明の目的は、このような事情のもとに、ピ
ルビン酸キナーゼを用いる酵素反応において、従
来使用されている量より少ない量のピルビン酸キ
ナーゼを用いて、該反応を効果的に行わせるため
に、その酵素活性を人為的に増大させる方法を提
供することにある。Problems to be Solved by the Invention Under these circumstances, an object of the present invention is to solve the problem by using a smaller amount of pyruvate kinase than the amount conventionally used in an enzymatic reaction using pyruvate kinase. The object of the present invention is to provide a method for artificially increasing the enzyme activity in order to carry out the reaction effectively.
問題点を解決するための手段
本発明者らは鋭意研究を重ねた結果、反応溶液
中に所定量のジメチルスルホキシドを存在させる
ことにより前記目的を達成しうることを見出し、
この知見に基づいて本発明をなすに至つた。Means for Solving the Problems As a result of extensive research, the present inventors discovered that the above object could be achieved by making a predetermined amount of dimethyl sulfoxide exist in the reaction solution.
Based on this knowledge, the present invention was accomplished.
すなわち、本発明は、ピルビン酸キナーゼを用
いる酵素反応において、反応溶液中にジメチルス
ルホキシド５〜20容量％を存在させることを特徴
とする酵素活性増大方法を提供するものである。 That is, the present invention provides a method for increasing enzyme activity, which is characterized in that in an enzyme reaction using pyruvate kinase, 5 to 20% by volume of dimethyl sulfoxide is present in the reaction solution.
本発明方法におけるピルビン酸キナーゼとして
は、通常哺乳動物の骨格筋由来のもの、例えば市
販のウサギ筋肉由来のものが用いられる。 The pyruvate kinase used in the method of the present invention is usually derived from mammalian skeletal muscle, such as commercially available rabbit muscle.
また、ピルビン酸キナーゼを用いる酵素反応と
しては、例えば次の反応
ヌクレオシド二リン酸＋ホスホエノールピルビン酸→
ヌクレオシド三リン酸＋ピルビン酸
を基盤としたヌクレオシド二リン酸の定量、ヌク
レオシド三リン酸加水分解酵素の定量、次の反応
アデノシン三リン酸＋酢酸→アデノシン二リン酸＋ア
セチルリン酸
を触媒する酢酸キナーゼ活性の定量、及び酢酸の
定量などが挙げられる。 In addition, as an enzymatic reaction using pyruvate kinase, for example, the following reaction nucleoside diphosphate + phosphoenolpyruvate →
Quantification of nucleoside diphosphate based on nucleoside triphosphate + pyruvate, quantification of nucleoside triphosphate hydrolase, following reaction Adenosine triphosphate + acetic acid → acetic acid catalyzing adenosine diphosphate + acetyl phosphate Examples include quantification of kinase activity and quantification of acetic acid.
このような酵素反応においては、ピルビン酸キ
ナーゼは通常0.05〜100μｇ／mlの範囲の濃度にな
るように加えられる。本発明においては、この
際、ジメチルスルホキシドを反応溶液に対して５
〜20容量％の範囲の濃度になるように加えること
が必要である。この濃度が５容量％未満では酵素
活性の向上が不十分であり、また20容量％を超え
ると、むしろ酵素活性が低下する。 In such enzymatic reactions, pyruvate kinase is usually added at a concentration ranging from 0.05 to 100 μg/ml. In the present invention, at this time, dimethyl sulfoxide is added to the reaction solution at 5%
It is necessary to add it to a concentration in the range of ~20% by volume. When this concentration is less than 5% by volume, the enzyme activity is insufficiently improved, and when it exceeds 20% by volume, the enzyme activity is rather decreased.
また、この酵素反応は、通常PH６〜９、温度20
〜37℃の範囲で行われ、さらに添加剤として塩化
カリウム５〜200mmol／、塩化マグネシウム
１〜10mmol／が用いられる。 In addition, this enzymatic reaction is usually performed at a pH of 6 to 9 and a temperature of 20
The reaction is carried out at a temperature of -37° C., and potassium chloride (5 to 200 mmol) and magnesium chloride (1 to 10 mmol) are used as additives.
発明の効果
本発明方法によると、ピルビン酸キナーゼの酵
素活性を約50％上昇させることができるので、ピ
ルビン酸キナーゼを用いる前記の各種酵素反応に
おいて、その使用量は従来より大幅に減少するこ
とができて、経済的であり、今後さらに該酵素反
応の幅広い応用が期待できる。Effects of the Invention According to the method of the present invention, the enzymatic activity of pyruvate kinase can be increased by about 50%, so the amount used in the various enzyme reactions mentioned above using pyruvate kinase can be significantly reduced compared to conventional methods. It is easy to carry out and is economical, and it is expected that the enzymatic reaction will be widely applied in the future.
実施例
次に実施例により、本発明をさらに詳細に説明
する。Examples Next, the present invention will be explained in more detail with reference to Examples.
実施例 １
トリス塩酸20mmol／（PH8.2）、塩化カリウ
ム30mmol／、塩化マグネシウム3mmol／、
ホスホエノールピルビン酸（シグマ社製）
0.5mmol／、アデノシン二リン酸（ベーリンガ
ー社製）0.5mmol／の溶液に、ピルビン酸キナ
ーゼ（ウサギ筋肉由来、ベーリンガー社製）を
0.05mg／、及びジメチルスルホキシド（試薬特
級を蒸留したもの）を種々の濃度になるように加
え、25℃にて10分間保温した。反応は最終
50mmol／の塩酸を加えて停止し、酵素反応に
よつて生じたピルビン酸をレイナードらの方法
〔A.M.Reynard、L.F.Hass、D.D.Jacobsen＆P.
D.Boyer；Journal of Biological Chemistry、
236巻、2277〜2283ページ（1961年）〕によつて定
量し、酵素活性を算出した。その結果を添付図面
に実線グラフとして示す。Example 1 Tris hydrochloric acid 20 mmol/(PH8.2), potassium chloride 30 mmol/, magnesium chloride 3 mmol/,
Phosphoenolpyruvate (manufactured by Sigma)
Pyruvate kinase (derived from rabbit muscle, manufactured by Boehringer) was added to a solution of 0.5 mmol/adenosine diphosphate (Boehringer).
0.05 mg/dimethyl sulfoxide (distilled from special reagent grade) were added at various concentrations, and the mixture was kept at 25° C. for 10 minutes. reaction is final
50 mmol of hydrochloric acid was added to stop the reaction, and the pyruvate produced by the enzymatic reaction was removed using the method of Reynard et al. [AMReynard, LFHass, DD Jacobsen & P.
D.Boyer；Journal of Biological Chemistry,
236, pp. 2277-2283 (1961)], and the enzyme activity was calculated. The results are shown in the accompanying drawings as a solid line graph.
このグラフから明らかなように、ジメチルスル
ホキシド濃度の広い範囲で活性の上昇がみられ、
ジメチルスルホキシドの濃度が15容量％のとき最
大（46％の活性増大）であつた。 As is clear from this graph, an increase in activity was observed over a wide range of dimethyl sulfoxide concentrations;
The maximum (46% increase in activity) was found when the concentration of dimethyl sulfoxide was 15% by volume.
実施例 ２
実施例１においてトリス塩酸20mmol／（PH
8.2）の代りに、リン酸10mmol／（PH6.8）を
用いる以外は実施例１と同様にして酵素反応を行
わせた。その結果を添付図面に破線グラフとして
示す。Example 2 In Example 1, Tris-HCl 20mmol/(PH
An enzyme reaction was carried out in the same manner as in Example 1 except that 10 mmol/(PH6.8) of phosphoric acid was used instead of 8.2). The results are shown in the attached drawing as a dashed line graph.
このグラフから明らかなように、実施例１と同
様に、ジメチルスルホキシド濃度の広い範囲で活
性の上昇がみられ、ジメチルスルホキシドの濃度
が15容量％のとき最大の活性を示した。 As is clear from this graph, similarly to Example 1, an increase in activity was observed over a wide range of dimethyl sulfoxide concentrations, and the maximum activity was exhibited when the dimethyl sulfoxide concentration was 15% by volume.
図はジメチルスルホキシドの濃度と酵素活性と
の関係を示すグラフであり、実線及び破線はそれ
ぞれ実施例１及び２におけるデータである。図に
おいて横軸はジメチルスルホキシド濃度を表わ
し、縦軸は、ジメチルスルホキシド無添加の場合
の酵素活性を100％とした酵素活性を表わす。
The figure is a graph showing the relationship between the concentration of dimethyl sulfoxide and enzyme activity, and the solid line and broken line are the data in Examples 1 and 2, respectively. In the figure, the horizontal axis represents the dimethyl sulfoxide concentration, and the vertical axis represents the enzyme activity with the enzyme activity in the case of no dimethyl sulfoxide being added as 100%.
Claims (1)
て、反応溶液中にジメチルスルホキシド５〜20容
量％を存在させることを特徴とする酵素活性増大
方法。1. A method for increasing enzyme activity, which comprises adding 5 to 20% by volume of dimethyl sulfoxide to the reaction solution in an enzyme reaction using pyruvate kinase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2533386A JPH0236229B2 (en) | 1986-02-06 | 1986-02-06 | KOSOKATSUSEIZODAIHOHO |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2533386A JPH0236229B2 (en) | 1986-02-06 | 1986-02-06 | KOSOKATSUSEIZODAIHOHO |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62181782A JPS62181782A (en) | 1987-08-10 |
| JPH0236229B2 true JPH0236229B2 (en) | 1990-08-16 |
Family
ID=12163000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2533386A Expired - Lifetime JPH0236229B2 (en) | 1986-02-06 | 1986-02-06 | KOSOKATSUSEIZODAIHOHO |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0236229B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4963021B2 (en) | 2005-09-06 | 2012-06-27 | 独立行政法人産業技術総合研究所 | Semiconductor structure |
-
1986
- 1986-02-06 JP JP2533386A patent/JPH0236229B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62181782A (en) | 1987-08-10 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |