JPS623800A - Determination of ammonia or atp - Google Patents
Determination of ammonia or atpInfo
- Publication number
- JPS623800A JPS623800A JP14124185A JP14124185A JPS623800A JP S623800 A JPS623800 A JP S623800A JP 14124185 A JP14124185 A JP 14124185A JP 14124185 A JP14124185 A JP 14124185A JP S623800 A JPS623800 A JP S623800A
- Authority
- JP
- Japan
- Prior art keywords
- atp
- ammonia
- kinase
- reaction
- glutamine synthetase
- 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
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- Life Sciences & Earth Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は酵素法によるアンモニアまたはＡＴＰの定量法
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 quantifying ammonia or ATP using an enzymatic method.
従来・アンモニアの定量法としては・インドフェノール
法という化学的測定法が主に用いられている。また、酵
素的測定法としては１グルタミン酸脱水素酵素を用いる
方法（例えば特公昭５７−２１９９５号参照）が知られ
ている０この方法では、用いられるグルタミン酸脱水素
酵素のアンモニアに対するＫｍ値が大きいため１測定に
多量のグルタミン酸脱水素酵素を必要とする問題を有し
ている。さらに他の酵素的測定法としてはカルバメート
キナーゼを用いる方法（特開昭５９−２１３３９９号参
照）、またはカルバモイルリン酸合成酵素を用いる方法
（特開昭６０−４７６９８号参照）が知られている。Conventionally, a chemical measurement method called the indophenol method has been mainly used to quantify ammonia. Furthermore, as an enzymatic measurement method, a method using 1-glutamic acid dehydrogenase (for example, see Japanese Patent Publication No. 57-21995) is known. The problem is that a large amount of glutamic acid dehydrogenase is required for one measurement. Furthermore, as other enzymatic measurement methods, a method using carbamate kinase (see JP-A-59-213399) or a method using carbamoyl phosphate synthase (see JP-A-60-47698) is known.
前者の方法では用いられるカルバメートキナーゼが一般
的に不安定で、透析などの処理により失活すると報告さ
れている。それ故、還元型グルタチオン、２−メルカプ
トエタノールなどのＳＨ基保護剤の存在下で酵素を精製
あるいは保存する必要がある。ところが、これらのＳＨ
基保護剤を含むカルバメートキナーゼ標品を用いて・被
検液中のアンモニアを酸化酵素−ヘルオキシダーゼー水
素供与体色源体反応系を含む系で定量する場合、ＳＨ基
保護剤の存在はペルオキシダーゼ水素供与体の発色系を
阻害するので、正確な測定値が得られず負の誤差を与え
ることになる。後者の方法では用いられるカルバモイル
リン酸合成酵素が動物臓器由来のものしか入手できない
ため、酵素の大量かつ安価な供給という点で問題を有し
ている。It has been reported that the carbamate kinase used in the former method is generally unstable and is inactivated by treatments such as dialysis. Therefore, it is necessary to purify or preserve the enzyme in the presence of an SH group protecting agent such as reduced glutathione or 2-mercaptoethanol. However, these SH
When quantifying ammonia in a test solution using a carbamate kinase preparation containing a group-protecting agent and using a system that includes an oxidase-heroxidase-hydrogen donor chromogen reaction system, the presence of an SH group-protecting agent is due to peroxidase. Since the color development system of the hydrogen donor is inhibited, accurate measurement values cannot be obtained and a negative error is given. Since the carbamoyl phosphate synthase used in the latter method can only be obtained from animal organs, there is a problem in supplying the enzyme in large quantities and at low cost.
また、ＡＴＰの定量法としては、従来ダルコー素を作用
させ、生成したＮＡＤＰＨを３４０ｎｍで測定する方法
〔メソッヅ・オプ・エンザイマティツク・アナリシス、
第７巻、筒３４６頁（１９８５））、またはルシフェリ
ンおよびＭｇ２＋の存在下でＡＴＥにルシフェラーゼを
作用させ、発生する５６２ｎｍの光強度よりＡＴＰを定
量する方法が知られている〔同第７巻、第３５７頁（１
９８５））。In addition, as a method for quantifying ATP, there is a conventional method in which darcochloride is applied and the generated NADPH is measured at 340 nm [Methods Op Enzymatic Analysis,
Vol. 7, p. 346 (1985)), or a method is known in which luciferase is allowed to act on ATE in the presence of luciferin and Mg2+, and ATP is quantified from the generated light intensity at 562 nm [Vol. 7, p. 346 (1985)]. Page 357 (1
985)).
しかし前者の方法では、ＡＴＥ １分子よりＮＡＤＰＨ
１分子しか生成しないため感度が低く１また・後者の方
法ではルシフェリンおよヒルシフェラーゼが高価である
という問題を有している。However, in the former method, more NADPH than one ATE molecule
Since only one molecule is produced, the sensitivity is low, and the latter method has problems in that luciferin and luciferase are expensive.
本発明の目的は１上記現状に鑑み１アンモニアまたはＡ
ＴＰの高感度で簡便かつ安価な定量法を提供することに
ある。The purpose of the present invention is to: 1. In view of the above-mentioned current situation, 1. ammonia or A.
The object of the present invention is to provide a highly sensitive, simple, and inexpensive method for quantifying TP.
本発明を概説すれば、本発明はアンモニアまたはＡＴＰ
の定量法に関するものであり、被検液中のアンモニアま
たはＡＴＰを定量するに当り、被定量成分ではないもう
一方の物質およびＬ−グルタミン酸の存在下で被検液に
グルタミン合成酵素を作用させ、生成したＡＤＰとキナ
ーゼ基質用リン化合物にキナーゼを作用させて、生成し
たキナーゼ反応生成物を定量することを特徴とする。上
記被定量成分ではないもう一方の物質とは、アンモニア
定量の場合ＡＴＰを、またＡＴＰ定量の場合アンモニア
を表わすものとする。To summarize the present invention, the present invention provides ammonia or ATP.
In quantifying ammonia or ATP in a test solution, glutamine synthetase is allowed to act on the test solution in the presence of L-glutamic acid and another substance that is not the component to be quantified, The method is characterized in that a kinase is made to act on the generated ADP and a phosphorus compound for a kinase substrate, and the generated kinase reaction product is quantified. The other substance that is not the component to be quantified is ATP in the case of ammonia quantification, and ammonia in the case of ATP quantification.
本発明はサイクリング反応を利用した定量法であり高感
度であること１比色定量が可能であう。と、グルタミン
合成酵素が微生物より安価かつ大量に調整可能であるこ
と、とくにマイクロコツカス属およびブレビバクテリウ
ニ属のグルタミン合成酵素は菌体内タンパク質の２〜３
％にまで達し、また非常に安定である、などの特徴を有
しており、臨床検査分野に新規なアンモニアまたはＡＴ
Ｐの定量法を提供するものである。The present invention is a quantitative method that utilizes a cycling reaction, and is highly sensitive.1 Colorimetric determination is possible. In particular, the glutamine synthetase of Micrococcus spp. and Brevibacterium spp.
% and is extremely stable, making it a new ammonia or AT product in the field of clinical testing.
This provides a method for quantifying P.
本発明に供される被検液としては、アンモニアまたはＡ
Ｔ］’のいずれか一方を含むものであればよく、アンモ
ニアまたはＡＴＰを予め含む被検液や、酵素反応により
生成されたアンモニアまたはＡＴＰを含む被検液がある
。酵素反応によりアンモニアを生成する反応には、以下
に例示するものがあるが、それらの酵素活性測定、基質
または生成物の定ｍを行うことが可能である。The test liquid used in the present invention includes ammonia or A
It is sufficient that the test liquid contains either one of T]', and examples thereof include a test liquid that contains ammonia or ATP in advance, and a test liquid that contains ammonia or ATP produced by an enzyme reaction. Examples of reactions that produce ammonia by enzymatic reactions include those listed below, and it is possible to measure their enzyme activity and determine the amount of substrates or products.
１．７スパラギナーゼ（ＥＣ！３．５．１．１）Ｌ−ア
スパラギン＋ＨＩＯ−＋ Ｊ、−アスパラギン僧＋Ｎ
Ｈ３２、アスパルターゼ（ＩＣＯ４，３，１，１）Ｌ−
アスパラギン酸→ フマール酸十ＮＨ。1.7 Sparaginase (EC! 3.5.1.1) L-asparagine + HIO-+ J, -asparagine + N
H32, aspartase (ICO4,3,1,1)L-
Aspartic acid → fumaric acid 1NH.
３、アデニンデアミナーゼ（Ｍｇ３．５．４．２）アデ
ニン十ＨｚＯ→ ヒボキサンチン十ＮＨ３４、アデノシ
ンデアミナーゼ（ｔｏ３．５．４．４）アデノシン十Ｈ
ｘＯ→イノシン十ＮＨｓ５、アデノシンモノリン酸デア
ミナーゼ（ＥＣ３・５・４・６）ＡＭＰ ＋ Ｈｔｏ
→工ＭＰ ＋ ＭＥ ｓ６、アデノシンＩＪ ン酸テ
アミナーゼ（ｔｏ３．５．４．１７）アデノシンリン酸
＋Ｈ，Ｏ→ イノシンリン酸＋ｍＨｓ７、−ｙミンデヒ
ドａゲナーゼ（ＫＯｌ、４．９９．３）８、アルギニ＞
デアミナーゼ（ｔｏ３．５．３．６）Ｌ−アルギニン→
Ｌ−シトルリン十ＮＨｓ９、アミダーゼ（ＫＯ３，５，
１，４）モノカルボン酸アミド＋Ｊ（，０→モノカルボ
ンｍ＋ｙｎｓ１０、ω−アミダーゼ（ＥＣ３，５，１，
３）２−ケトグルタラミン酸＋Ｈ，Ｏ→ ２−ケトグル
タル酸十ＮＨ３１１、ウレアーゼ（ＫＯ３、５、１、５
）尿素十Ｈ！Ｏ→２ ＮＨｚ ＋ ＯＯ！１２、ウレイ
ドスクシナーゼ（Ｂ：０３．５．１．７）１３、グアニ
ンデアミナーゼ（ＫＯ３，５，４，３）グアニン＋Ｈ，
Ｏ→キサンチン＋ＮＨ。3. Adenine deaminase (Mg3.5.4.2) Adenine 10HzO → Hyboxanthin 10NH34, Adenosine deaminase (to3.5.4.4) Adenosine 10H
xO→Inosine 1NHs5, adenosine monophosphate deaminase (EC3/5/4/6) AMP + Hto
→ Engineering MP + ME s6, adenosine IJ acid theaminase (to3.5.4.17) adenosine phosphate + H, O → inosine phosphate + mHs7, -yminedehyde a genease (KOl, 4.99.3) 8, arginine>
Deaminase (to3.5.3.6) L-arginine →
L-citrulline NHs9, amidase (KO3,5,
1,4) Monocarboxylic acid amide + J (,0 → monocarboxylic m + yns10, ω-amidase (EC3,5,1,
3) 2-ketoglutamic acid + H, O → 2-ketoglutaric acid + NH311, urease (KO3, 5, 1, 5
) Urea 10H! O → 2 NHz + OO! 12, ureidosuccinase (B:03.5.1.7) 13, guanine deaminase (KO3,5,4,3) guanine + H,
O → xanthine + NH.
１４、グアノシンデアミナーゼ（ＩＯ３，５，４，１５
）グアノシン＋Ｈ，Ｏ→キサントシン＋ＮＨ３ニコチン
アミド十Ｈ，Ｏ→ニコチンｍ＋ＮＨｓＬ−ヒスチジン→
ウロカニン酸十ＮＨ。14, Guanosine deaminase (IO3,5,4,15
) Guanosine + H, O → Xanthosine + NH3 Nicotinamide 1 H, O → Nicotine m + NHsL-Histidine →
Urocanic acid 1NH.
また、酵素反応によりＡＴＰを生成する反応としては以
下に例示するものがあるが、それらの。In addition, there are the following examples of reactions that generate ATP by enzymatic reactions.
酵素活性測定・基質または生成物の定量を行うことが可
能である。It is possible to measure enzyme activity and quantify substrates or products.
Ｌ−チロシン＋ＡＴＰ −）−ｔＲＮＡ２．７セチルー
ＯｏＡシンセターゼ（ＥＯ６，２，１，１）ＡＭＰ−１
−ＰＰｉ＋アセチル−０ｏＡコ ＡＴＰ＋酢酸＋ＯｏＡ
ＳＨＡＭＰ＋ビロリン酸＋ＡＭＰ ２ ＡＴＰ＋デアミ
ド−ＮＡＤ＋ＮＨ。L-Tyrosine+ATP-)-tRNA2.7 Cetyl-OoA Synthetase (EO6,2,1,1)AMP-1
-PPi+acetyl-0oA ATP+acetic acid+OoA
SHAMP + birophosphoric acid + AMP 2 ATP + deamide-NAD + NH.
以上これらは例示であり、何ら本発明の対象を限定する
ものではない。These are merely examples and do not limit the scope of the present invention in any way.
次に、本発明に用いられるグルタミン合成酵素は各種高
等動物の脳や肝臓、マメの種子、大腸菌その他の微生物
に存在するが、大社かつ安価に供給できるという点でマ
イクロコツカス属およびブレビバクテリウム属より選ば
れたグルタミン合成酵素生産菌より取得される酵素（こ
のグルタミン合成酵素については特開昭５７−３３５９
４号参照）を使用するのが有利である０まず・本発明に
使用されるグルタミン合成酵素の各性質を示す。Next, although the glutamine synthetase used in the present invention is present in the brains and livers of various higher animals, bean seeds, Escherichia coli, and other microorganisms, Micrococcus spp. and Brevibacterium spp. Enzymes obtained from glutamine synthetase-producing bacteria selected from the genus
First, the properties of the glutamine synthetase used in the present invention will be shown.
グルタミン合成酵素の酵素化学的および理化学的性質
（１）作 用：
本酵素は下式のようにＬ−グルタミン酸とアンモニアよ
り、ＡＴＰの化学エネルギーを利用してグルタミンを合
成する反応を触媒する。Enzyme-chemical and physicochemical properties of glutamine synthetase (1) Action: This enzyme catalyzes the reaction of synthesizing glutamine from L-glutamic acid and ammonia using the chemical energy of ATP, as shown in the following formula.
Ｌ−グルタミン酸＋アンモニア＋ＡＴＰ →Ｌ−グル
タミン＋ＡＤＰ＋無機リン酸
（２）基質特異性ニ
アミノ基受容体としては、本酵素はＬ−グルタミン酸に
極めて高い特異性を示す。塩化アンモニウムの代りにヒ
ドロキシルアミンを用いた場合にも、約３０％の活性が
認められた。L-glutamic acid + ammonia + ATP → L-glutamine + ADP + inorganic phosphate (2) Substrate specificity As a niamin group receptor, this enzyme shows extremely high specificity for L-glutamic acid. Approximately 30% activity was also observed when hydroxylamine was used instead of ammonium chloride.
（Ｊ至適ｐＨおよびｐＨ安定性：
本酵素の至適ｐＨは７．０〜８．０である。また・本酵
素を５０’Ｃにおいて、それぞれのｐｋｌで１０分間処
理したとき、ｐＨ６，０〜９．０の範囲で安定であるＣ
［４）至適温度および熱安定性：
本酵素の至適温度は５０°Ｃ付近にあり、ｐａ７．０に
おいて１それぞれの温度で１０分間処理したとき５０°
Ｃまで安定である。(J optimal pH and pH stability: The optimal pH of this enzyme is 7.0 to 8.0. Also, when this enzyme was treated with each pkl at 50'C for 10 minutes, the pH was 6.0 to 8.0. C that is stable in the range of ~9.0
[4) Optimal temperature and thermostability: The optimal temperature of this enzyme is around 50°C, and when treated at each temperature for 10 minutes at pa 7.0, the temperature of 50°C
It is stable up to C.
（ω分子社：
本酵素の分子量は沈降平衡法により、偏比容を０４７５
と仮定したときに約５０万である。(ω Molecule Company: The molecular weight of this enzyme was determined by the sedimentation equilibrium method, and the partial specific volume was determined to be 0475.
Assuming that, it is approximately 500,000.
また、５ＤＳ−ポリアクリルアミドゲル電気泳動法では
約６万〜６，５万であることから、本酵素は同一のサブ
ユニット８個からなる８量体である。Moreover, since the 5DS-polyacrylamide gel electrophoresis method shows that the amount is approximately 60,000 to 65,000, this enzyme is an octamer consisting of eight identical subunits.
（６）阻 害：
各種代謝産物による百害を検討したところ・アミノ酸類
ではグリシン、Ｌ−トリプトファン、Ｄ−スレオニン等
で若干の医書が見られる程度であるが、ヌクレオチド、
ヌクレオシド類による阻害は大きく、アデノシン、 Ａ
ＭＰ。(6) Inhibition: After examining the damage caused by various metabolites, there are only a few medical records for amino acids such as glycine, L-tryptophan, and D-threonine, but for nucleotides,
Inhibition by nucleosides is large; adenosine, A
M.P.
ＡＤＰなどによって活性は強く■害される。Its activity is strongly impaired by ADP and the like.
（′ｒ）金属イオンの影響：
酵素反応には金属イオンとしてＭｇ １＋を要求しＳＭ
ｎ”十でも３４％の活性がある〇（８） Ｋｍ値：
各、基質に対するＫｍ値を求めたところ、Ｌ−グルタミ
ン酸に対して７．９ ｘ １０−”Ｍ％塩化アンモニウ
ムに対して５．０ ｘ １０−”Ｍ、 ＡＴＰに対して
１．２ Ｘ １０−’Ｍであった。('r) Effect of metal ions: Enzyme reactions require Mg 1+ as metal ions, and SM
34% activity even with n"0 (8) Km value: When the Km value for each substrate was determined, it was 7.9 x 10-" for L-glutamic acid and 5.9 for M% ammonium chloride. 0 x 10-'M and 1.2 x 10-'M for ATP.
（９）酵素活性測定法： 酵素活性の測定は次のようにして求めた。(9) Enzyme activity measurement method: Enzyme activity was measured as follows.
ｓｏｏｍＭグルタミン酸ナトリツナトリウム溶液０ｍｉ
ｘ２５０ｍＭ塩化アンモニウム溶液０．１ｆｆｉＡ！、
７５ｍＭ ＡＴＰ溶液０．１ ｍｌ ｓ ３００ ｍＭ
Ｍｇ０１ｇ溶液０．１尼、１Ｍイミダゾール−塩酸緩
裡液（ｐＨ７，０）Ｑ、 ｌ ｍｌ 、水０．４ ｒｎ
ｌおよび適当に希釈した酵素液Ｑ、 ｌ ｍｉ 、反応
液ｆｌ １． Ｏｒａｔで３７°Ｃ，１０分間反応させ
、生成する無機リン酸をフイスヶーサバロウの方法で測
定する方法、および生成するグルタミンをペーパークロ
マトダラフィーで分離し、ニンヒドリン発色法で測定す
る方法により求めた◇グルタミン合成活性の１単位は上
記反応系で１分間に１μＭの無機リン酸あるいはグルタ
ミンを生成する酵素量として表示した。soomM sodium glutamate solution 0mi
x250mM ammonium chloride solution 0.1ffiA! ,
75mM ATP solution 0.1ml s 300mM
Mg01g solution 0.1ml, 1M imidazole-hydrochloric acid solution (pH 7,0) Q, lml, water 0.4rn
l and appropriately diluted enzyme solution Q, l mi , reaction liquid fl 1. The reaction was carried out at 37°C for 10 minutes in Orat, and the produced inorganic phosphoric acid was measured using the method of Fuisuka-Sabarou, and the produced glutamine was separated using paper chromatography and measured using the ninhydrin color method. One unit of the determined ◇glutamine synthesis activity was expressed as the amount of enzyme that produces 1 μM of inorganic phosphoric acid or glutamine per minute in the above reaction system.
なお、グルタミン合成酵素の製造方法については特開昭
５７−３３５９４号に記載されている。The method for producing glutamine synthetase is described in JP-A-57-33594.
アンモニアおよびＡＴＰの定量法
本発明者らは、被検液中のアンモニアまたはＡＴＰの定
量方法について鋭意検討を重ねた結果・グルタミン合成
酵素を用いることにより高感度で簡便かつ安価なアンモ
ニアまたはＡＴＰの定量法を見出した。被検液中のアン
モニアはＬ−グルタミン酸とＡＴＰの存在下、グルタミ
ン合成酵素の作用により、Ｌ−グルタミン、ＡＤＰおよ
び無機リン酸を生成し、また被検液中のＡＴＰはＬ−ダ
ルタミン酸とアンモニアの存在下、本酵素作用により１
Ｌ−グルタミン、ＡＤＰおよび無機リン％を生成する。Quantification method of ammonia and ATP The present inventors have conducted extensive studies on a method for quantifying ammonia or ATP in a test solution. ・The quantification of ammonia or ATP is highly sensitive, simple, and inexpensive by using glutamine synthetase. I found the law. In the presence of L-glutamic acid and ATP, ammonia in the test solution produces L-glutamine, ADP, and inorganic phosphate by the action of glutamine synthetase, and ATP in the test solution produces L-daltamic acid and ammonia. 1 by the action of this enzyme in the presence of
Produces L-glutamine, ADP and % inorganic phosphorus.
酵素反応により生成したＡＤＰにキナーゼ基質用リン化
合物の存在下、キナーゼを作用させるとキナーゼ反応生
成物とＡＴＰが生成され、サイクリング反応が進行しＡ
ＴＰが増幅される。この反応の一例としてピルビン酸キ
ナーゼによるＡＤＰとホスホエノールピルビン酸からＡ
ＴＰとピルビン酸を生成する反応が挙げられる。生成し
たピルビン酸の定量には公知の方法を用いることができ
る。例えばピルビン酸にＮＡＤＨと乳酸脱水素酵素を作
用させ、ＮＡＤＨの３４０ｎｍの吸光度の減少より定量
するが、またはピルビン酸オキシダーゼを作用させ消費
される酸素を酸素電極で測定するか、生成する過酸化水
素をペルオキシダーゼ系による呈色反応、例えば４−ア
ミノアンチピリン−フェノール−ペルオキシダーゼ法を
用いた場合５００ｎｍで定量することかできる。When a kinase is allowed to act on ADP produced by the enzymatic reaction in the presence of a phosphorus compound for the kinase substrate, a kinase reaction product and ATP are produced, and the cycling reaction progresses.
TP is amplified. An example of this reaction is ADP and phosphoenolpyruvate by pyruvate kinase.
An example is a reaction that produces TP and pyruvic acid. A known method can be used to quantify the produced pyruvic acid. For example, by reacting pyruvate with NADH and lactate dehydrogenase, it is determined by the decrease in absorbance of NADH at 340 nm, or by reacting pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode, or by reacting with pyruvate oxidase and measuring the consumed oxygen with an oxygen electrode. can be quantified at 500 nm using a color reaction using a peroxidase system, for example, the 4-aminoantipyrine-phenol-peroxidase method.
反応に用いられる緩衝液は特に限定されず、リン酸緩衝
液、イミダゾール緩衝液、トリス緩衝液、グリシルグリ
シン緩衝液などが好適であり・ｐＨ６〜９Ｓ好ましくは
ｐＨ７の緩衝液が用ｐｂられるＯグルタミン合成酵素は
通常０．２〜２Ｑ単位、好ましくは１〜５単位・キナー
ゼ・例えハヒルビン増キナーゼは、１〜１Ｑ０単位、好
ましくは５単位以上用いられる。また・Ｌ−グルタミン
酸濃度は１〜５ＱｍＭ、好ましくは５〜ｌ ＱｍＭｓ
ＡＴＰやキナーゼ基質用リン化合物１例えばホスホエノ
ールピルビン酸およびＮＡＤＨは少なくとも被検液中の
アンモニアまたはＡＴＰのモル量以上用いればよい。反
応温度は２０〜４００Ｃ１反応時間は１〜２０分間で反
応は行なわれる。The buffer used in the reaction is not particularly limited, and phosphate buffer, imidazole buffer, Tris buffer, glycylglycine buffer, etc. are suitable.Buffers with pH 6 to 9S and preferably pH 7 are used. Glutamine synthetase is usually used in an amount of 0.2 to 2 Q units, preferably 1 to 5 units. Kinases such as hahirubin increase kinase are used in amounts of 1 to 1 Q0 units, preferably 5 or more units. Also, L-glutamic acid concentration is 1 to 5 QmM, preferably 5 to 1 QmMs
Phosphorus compounds 1 for ATP and kinase substrates, such as phosphoenolpyruvate and NADH, may be used in an amount at least equal to or greater than the molar amount of ammonia or ATP in the test solution. The reaction temperature is 20 to 400C and the reaction time is 1 to 20 minutes.
以下に本発明を、実施例をもって説明するが、本発明が
以下の実施例の範囲のみに限定されるものではない。The present invention will be explained below with reference to examples, but the present invention is not limited to the scope of the following examples.
実施例 １
アンモニアの定量 （ピルビン酸キナーゼ−乳酸脱水素
酵素反応系）
１ Ｍ イミダゾール−塩酸緩衝液（ｐＨ，７，０）
０．３ｍ１５０ｍＭＬ−グルタミン酸ナトリウム
Ｑ、ｌ ｍｉ３ Ｑ ｍＭ ＡＴＥ
Ｑ、１ｍｔ３ＱｍＭ
ホスホエノールピルビン酸 ０１１ｄ
７．５ ｍＭ ＮＡＤＨＱ、１ｍ１
１．５Ｍ ＭｇＯ１ｘ
０．１工！２．７Ｍ ＫＯＩ
０．１ｍ１３０単位７ｍｂ
グルタミン合成酵素（合成活性） ０．１１
ｒＬｌ水
１．３ ｍｌ上記混合溶液３．０罰に１．２，３．４お
よび５ｍＭの塩化アンモニウム溶液０．１ ｍｉをそれ
ぞれ添加し、３７℃で５分間反応した。第１図に示すよ
うに添加した塩化アンモニウム量と３４０ｎｍの吸光度
の減少量には良好な直線関係が得られた。Example 1 Quantification of ammonia (pyruvate kinase-lactate dehydrogenase reaction system) 1 M imidazole-hydrochloric acid buffer (pH, 7.0)
0.3ml 150mmL-sodium glutamate
Q, l mi3 Q mM ATE
Q, 1mt3QmM
Phosphoenolpyruvate 011d
7.5mM NADHQ, 1ml 1.5M MgO1x
0.1 engineering! 2.7M KOI
0.1m 130 units 7mb
Glutamine synthetase (synthetic activity) 0.11
rLl water
To 1.3 ml of the above mixed solution 0.1 ml of 1.2, 3.4 and 5 mM ammonium chloride solutions were added, respectively, and reacted at 37° C. for 5 minutes. As shown in FIG. 1, a good linear relationship was obtained between the amount of ammonium chloride added and the amount of decrease in absorbance at 340 nm.
実施例 ２
反応糸）
１ Ｍ イミダゾール−塩酸むＱ菌液（ｐＨ７，０）
０．３ｍ／１５０ｍＭ Ｌ−グルタミン酸ナトリ
ウム Ｑ、 ｌ ｍ１１３ Ｑ ｍＭ Ａ
ＴＰ ０．１ｍ
１３０ ｍＭ ホスホエノールピルビン酸０．１１２
４．６ｍＭ ４−７ミノアンチビリン
Ｑ、 ｌ ｍ１４２０ｍＭ フェノール
０．１ ｒＩＬｔｌ、５ Ｍ
ＭｇＯ１ｚ Ｑ、
１ｍＡ２．７Ｍ ＫＯＩ
Ｏ，１ｍＡ６ｍＭ チアミンピロホスフ
ェート ０．１ ｍＡｌｍＭ ＸＦＡ
Ｄ ０．１ｒｒｌ
Ａ３０単ｗｍｔ グルタミン合成酵素（合成活性）、
Ｑ、１ｍｊ水
１．４成上記混合溶液３． Ｏｎｌｅ；に
１，２，３．４および５ｍＭの塩化アンモニウム溶液Ｑ
、 ｌ ｍｌをそれぞれ添加し、３７°Ｃで５分間反応
した。＠２図に示すように添加した塩化アンモニウム量
と５００ｎｍの吸光度の増加量には良好な直線関係が得
られた〇
実施例 ３
ＡＴＰの定量
１ Ｍ イミダゾール−塩酸緩衝液（ｐＨ７，０）
０．３ｍ１１５ＱｍＭＬ−グルタミン酸ナトリウム
０．１ ｍｌ ５０ ｍＭ 塩化アンモニ
ウム Ｑ、 ｌ ｍｉ１３ Ｑ ｍ
Ｍ ホスホエノールピルビン酸 Ｑ、 ｌ
ｍｉ７．５ｍＭ ＮＡＤＨ０４１ｍ１ｊ
１．５ Ｍ Ｍｇ０１１
０．１ｍ１２．７Ｍ ＫＯＩ
ｏ、ｘｍｔ３０単Ｖ′ｍｔ
グルタミン合成酵素（合成活性） ｏ、 １
ｍｉ水 １
．３ｍｌ上記混合溶液３． Ｑ ｍｌに０．２５ 、０
．５０．０．７５およびｌ、ＱｍＭのＡＴＰ溶液０．１
蛯をそれぞれ添加。Example 2 Reaction thread) 1 M imidazole-hydrochloric acid Q bacterial solution (pH 7.0)
0.3m/150mM Sodium L-glutamate Q, l m113 Q mM A
TP 0.1m
130 mM Phosphoenolpyruvate 0.112
4.6mM 4-7 Minoantivirine
Q, lm1420mM phenol
0.1 rILtl, 5 M
MgO1z Q,
1mA2.7M KOI
O,1mA6mM Thiamine pyrophosphate 0.1 mAlmM XFA
D 0.1rrl
A30 monowmt glutamine synthetase (synthetic activity),
Q, 1mj water
1.4 Mixed solution 3. Onle; 1, 2, 3.4 and 5mM ammonium chloride solution Q
, 1 ml were added, and reacted at 37°C for 5 minutes. @2 As shown in the figure, a good linear relationship was obtained between the amount of ammonium chloride added and the increase in absorbance at 500 nm. Example 3 Quantification of ATP 1 M imidazole-hydrochloric acid buffer (pH 7,0)
0.3ml115QmML-Sodium Glutamate
0.1 ml 50 mM ammonium chloride Q, l mi13 Q m
M Phosphoenolpyruvate Q, l
mi7.5mM NADH041m1j 1.5M Mg011
0.1m12.7M KOI
o, xmt30 single V'mt
Glutamine synthetase (synthetic activity) o, 1
mi water 1
．． 3ml of the above mixed solution 3. Q 0.25 in ml, 0
．． 50.0.75 and l, QmM ATP solution 0.1
Add each elm.
し、３７°Ｃで反応させ、３分後と５分後のＮＡＤｌＫ
の減少の差を測定した。第３図に示すようにＡＴＰの添
加量とＮＡＤＨの減少量には良好な直線関係が得られた
。and reacted at 37°C, and NADlK after 3 and 5 minutes.
The difference in decrease was measured. As shown in FIG. 3, a good linear relationship was obtained between the amount of ATP added and the amount of decrease in NADH.
参考例
グルタミン合成酵素とグルタミン酸脱水素酵素を用いて
アンモニアを定量する場合の反応速度の比較
本発明で使用されるグルタミン合成酵素と酵母由来の従
来のグルタミン酸脱水素酵素を用いて、低濃度のアンモ
ニアを定量する場合、どちらか速やかに終点に達するか
比較試験を行なった。グルタミン合成酵素を用いる場合
の混合溶液組成は４５単位／ｒｎｌ（合成活性）のグル
タ・ミン介成酵素溶液Ｑ、 ｌ ｍｉを用いる以外は実
施例１と同様で、またグルタミン酸脱水素酵素を用いる
場合の混合溶液組成は３００ｍＭト’Ｊスー塩酸緩衝液
（ｐＨ８，０） １．０ｍ１ｓ ２２５ｍＭ ２−
’ｒ ）グルタＡ／藤溶液０．１ ｍε、５ ｍＭ
ＮＡＤＰＨ溶液０１づ、３００ｍＭ塩化アンモニウム溶
液Ｑ、 ｌ ｍｔ・４５単位／ ｍｅグルタミン酸脱水
素酵素溶液Ｑ、ｌ ｍＡおよび水１．５ ｍｉである０
上記両混合溶液３． Ｑ ｒｎｂに０．５゜１０および
２． Ｑ ｍＭの塩化アンモニウム溶液５０μｌをそれ
ぞれ添加し、３７°Ｃで反応させＮＡＤＨの減少に基づ
＜３４０ｎｍの吸光度を経時的に測定した。第４図に示
すようにグルタミン合成酵素を用いた場合（Ａ）・いず
れのアンモニア濃度でも釣３分間で反応が終了するのに
対し、グルタミン酸脱水素酵素を用いた場合（Ｂ）には
、６〜７分経過時でいずれのアンモニア濃度においても
反応は終了していなかった。Reference Example: Comparison of reaction rates when quantifying ammonia using glutamine synthetase and glutamate dehydrogenase. When quantifying, we conducted a comparative test to determine which method reaches the end point more quickly. When using glutamine synthetase, the mixed solution composition is the same as in Example 1 except for using 45 units/rnl (synthetic activity) of glutamine-mediated enzyme solution Q, lmi, and when using glutamate dehydrogenase. The mixed solution composition is 300mM To'J-HCl buffer (pH 8,0) 1.0mls 225mM 2-
'r) Gluta A/Wisteria solution 0.1 mε, 5 mM
NADPH solution 01, 300 mM ammonium chloride solution Q, l mt·45 units/me glutamic acid dehydrogenase solution Q, l mA and water 1.5 mi 0
Both of the above mixed solutions 3. 0.5°10 and 2. to Qrnb. 50 μl of Q mM ammonium chloride solution was added to each, reacted at 37° C., and the absorbance at <340 nm was measured over time based on the decrease in NADH. As shown in Figure 4, when glutamine synthetase is used (A), the reaction is completed in 3 minutes at any ammonia concentration, whereas when glutamate dehydrogenase is used (B), the reaction is completed within 3 minutes. After ~7 minutes had elapsed, the reaction had not been completed at any ammonia concentration.
以上詳細に説明したように、本発明の定量法によればア
ンモニアまたはＡＴＰを高感度かつ持具的に定量するこ
とができる。また本発明に用いるグルタミン合成酵素は
微生物より安価に調製することが可能であり臨床検査試
桑として優れた効果を有する。As explained in detail above, according to the quantitative method of the present invention, ammonia or ATP can be quantitatively determined with high sensitivity and in a convenient manner. Furthermore, the glutamine synthetase used in the present invention can be prepared at a lower cost than microorganisms, and has excellent effects as a clinical test sample.
第１図は本発明の定１号方法の実施例１における検１線
をアンモニア量と３４０ｎｍの吸光度差との関係で示し
たグラフである。第２図は本発明の定量方法の実施例２
における検量線をアンモニア量と５００ｎｍの吸光度差
との関係で示したグラフである。第３図は本発明の定量
方法の実施例３における検量線をＡＴＰ渣と３４０ｎｍ
の吸光度差との関係で示したグラフである。そして、第
４図は本発明に使用されるグルタミン合成酵素（Ａ）と
従来のグルタミン酸脱水素酵素（Ｂ）をそれぞれ用いて
、アンモニアを定量する場合の反応速度を反応時間（分
）と３４０ｎｍの吸光度差との関係で示したグーラフで
ある。FIG. 1 is a graph showing the relationship between the amount of ammonia and the absorbance difference at 340 nm for the first test line in Example 1 of Method No. 1 of the present invention. Figure 2 shows Example 2 of the quantitative method of the present invention.
2 is a graph showing the calibration curve in terms of the relationship between the amount of ammonia and the difference in absorbance at 500 nm. Figure 3 shows the calibration curve in Example 3 of the quantitative method of the present invention with ATP residue and 340 nm.
It is a graph shown in relation to the absorbance difference. Figure 4 shows the reaction time (minutes) and the reaction rate at 340 nm for quantifying ammonia using the glutamine synthetase (A) used in the present invention and the conventional glutamate dehydrogenase (B). This is a graph shown in relation to the absorbance difference.
Claims (1)
り、被定量成分ではないもう一方の物質およびＬ−グル
タミン酸の存在下で被検液にグルタミン、合成酵素を作
用させ、生成したＡＤＰとキナーゼ基質用リン化合物に
キナーゼを作用させて、生成したキナーゼ反応生成物を
定量することを特徴とするアンモニアまたはＡＴＰの定
量法。 ２、被検液中に、グルタミン合成酵素、キナーゼおよび
基質用リン化合物を同時に存在させる特許請求の範囲第
１項記載のアンモニアまたはＡＴＰの定量法。[Claims] 1. In quantifying ammonia or ATP in a test solution, glutamine and a synthetic enzyme are allowed to act on the test solution in the presence of another substance other than the component to be quantified and L-glutamic acid. A method for quantifying ammonia or ATP, which comprises causing a kinase to act on the generated ADP and a phosphorus compound for a kinase substrate, and quantifying the generated kinase reaction product. 2. The method for quantifying ammonia or ATP according to claim 1, wherein a glutamine synthetase, a kinase, and a phosphorus compound for substrate are present simultaneously in the test solution.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14124185A JPS623800A (en) | 1985-06-27 | 1985-06-27 | Determination of ammonia or atp |
| DE19863621448 DE3621448A1 (en) | 1985-06-27 | 1986-06-26 | Method for the determination of ammonia (or ATP) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14124185A JPS623800A (en) | 1985-06-27 | 1985-06-27 | Determination of ammonia or atp |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS623800A true JPS623800A (en) | 1987-01-09 |
Family
ID=15287374
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14124185A Pending JPS623800A (en) | 1985-06-27 | 1985-06-27 | Determination of ammonia or atp |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS623800A (en) |
| DE (1) | DE3621448A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03293467A (en) * | 1990-04-10 | 1991-12-25 | Toto Ltd | Formation of surface pattern of ceramic product |
| KR100433683B1 (en) * | 2001-06-18 | 2004-05-31 | 한국과학기술연구원 | A microassay method of glutamine synthetase |
| EP3315611A1 (en) | 2016-10-25 | 2018-05-02 | ARKRAY, Inc. | Quantification method for ammonia, quantification reagent kit, test piece, and ammonia quantification device |
| JP2018068278A (en) * | 2016-10-25 | 2018-05-10 | アークレイ株式会社 | Quantification method, quantification reagent kit, test piece, and quantification apparatus for ammonia |
| EP3441478A1 (en) | 2017-08-10 | 2019-02-13 | ARKRAY, Inc. | Improved glutamine synthetase reaction and method for quantifying ammonia utilizing the same |
| CN113075139A (en) * | 2021-03-29 | 2021-07-06 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2834704A1 (en) * | 1978-08-08 | 1980-02-21 | Boehringer Mannheim Gmbh | METHOD FOR THE QUANTITATIVE ENZYMATIC DETERMINATION OF ADP |
-
1985
- 1985-06-27 JP JP14124185A patent/JPS623800A/en active Pending
-
1986
- 1986-06-26 DE DE19863621448 patent/DE3621448A1/en not_active Withdrawn
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03293467A (en) * | 1990-04-10 | 1991-12-25 | Toto Ltd | Formation of surface pattern of ceramic product |
| KR100433683B1 (en) * | 2001-06-18 | 2004-05-31 | 한국과학기술연구원 | A microassay method of glutamine synthetase |
| EP3315611A1 (en) | 2016-10-25 | 2018-05-02 | ARKRAY, Inc. | Quantification method for ammonia, quantification reagent kit, test piece, and ammonia quantification device |
| JP2018068278A (en) * | 2016-10-25 | 2018-05-10 | アークレイ株式会社 | Quantification method, quantification reagent kit, test piece, and quantification apparatus for ammonia |
| US10731200B2 (en) | 2016-10-25 | 2020-08-04 | Arkray, Inc. | Quantification method for ammonia, quantification reagent kit, test piece, and ammonia quantification device |
| EP3441478A1 (en) | 2017-08-10 | 2019-02-13 | ARKRAY, Inc. | Improved glutamine synthetase reaction and method for quantifying ammonia utilizing the same |
| US11162123B2 (en) | 2017-08-10 | 2021-11-02 | Arkray, Inc. | Glutamine synthetase reaction and method for quantifying ammonia utilizing the same |
| CN113075139A (en) * | 2021-03-29 | 2021-07-06 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
| CN113075139B (en) * | 2021-03-29 | 2022-10-11 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
Also Published As
| Publication number | Publication date |
|---|---|
| DE3621448A1 (en) | 1987-01-08 |
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