JP2656065B2 - Enzyme activity measurement method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for measuring the enzyme activity of ω-aminoalkylaldehyde dehydrogenase in a subject.

Ω-Aminoalkylaldehyde dehydrogenase, an enzyme present in the body, is one of the enzymes that play an important role in the metabolic pathway of polyamines. Provides important clinical significance in knowing metabolism.

(Prior art and problems) Important metabolic pathways of polyamines include oxidative metabolic pathways by oxidase or transaminase. The ω-aminoalkyl aldehyde generated by the oxidative metabolism of these polyamines is usually converted to ω-aminoalkyl carboxylic acid by the action of ω-aminoalkyl aldehyde dehydrogenase. As the ω-aminoalkylaldehyde dehydrogenase, 4-aminobutanal dehydrogenase, 3-aminopropanal dehydrogenase and the like are known. However, the measurement of the activity of these enzymes is very complicated, and at present, there has been almost no detailed study on their distribution in living organisms. Also,
The presence of 5-aminopentanal dehydrogenase, which is considered to be involved in the metabolism of cadaverine, one of the polyamines, is also expected for ω-aminoalkylaldehyde logenase, but no research has yet been found.

The reason why studies on these ω-aminoalkylaldehyde dehydrogenases are difficult is that the method for measuring the activity of the enzyme is very complicated. For this, the adjustment of ω-aminoalkylaldehyde, which is a substrate used when measuring the activity of the enzyme, is very complicated,
The greatest cause is the lack of stability as a compound. That is, in the conventional method for measuring the enzyme activity of ω-aminoalkylaldehyde dehydrogenase,
Each time an enzyme activity is measured, an aminoalkylaldehyde is synthesized by a complicated organic chemical means, and a test substance is added to the obtained substrate in the presence of an oxidized nicotinamide coenzyme,
A method of measuring the production rate of the produced reduced nicotinamide coenzyme has been performed. For example, the journal of
Biological chemistry (J. Biol. Chem.) No. 234
Vol., Pp. 2145 to 2150 (1959), when measuring the enzymatic activity of 4-aminobutanal dehydrogenase, as the substrate 4-aminobutanal,
A reaction mixture obtained by reacting ornithine and N-bromosuccinimide under reduced pressure conditions in the presence of a debrominating agent and then heating in a boiling water bath is used as a substrate.

However, the 4-aminobutanal obtained by this preparation method contains unreacted raw materials and impurities such as bromine generated with the progress of the reaction, and has low purity as a substrate for measuring the enzyme activity. However, there is a problem that the enzyme activity value of ω-aminoalkylaldehyde dehydrogenase measured using such a substrate is not reliable. or,
It is very difficult to accurately adjust the substrate concentration due to differences in reaction yield due to differences in reaction conditions and instability of 4-aminobutanal. Furthermore, the enzymatic activity method itself requires two operations including a chemical synthesis operation of a substrate and an activity measurement operation by an enzymatic reaction, which is a very complicated enzyme activity measurement method.

(Means for Solving the Problems) The present inventors have studied a simple and highly accurate method for measuring the enzyme activity of ω-aminoalkylaldehyde dehydrogenase in order to solve the conventional drawbacks. As a result, the present inventors have found a simple and accurate method for measuring enzyme activity in which the target substrate can be easily adjusted and the operation of isolating the substrate in the middle is not required, and the present invention has been completed.

That is, in the present invention, when measuring the enzyme activity of ω-aminoalkylaldehyde dehydrogenase in a subject, putrescine oxidase and / or polyamine oxidase are allowed to act on polyamine to obtain ω-aminoalkylaldehyde, and then oxidized nicotine Measuring the enzyme activity of ω-aminoalkyl aldehyde dehydrogenase, comprising reacting the ω-aminoalkyl aldehyde with a test substance in the presence of an amide coenzyme and measuring the rate of the reduced nicotinamide coenzyme produced. Is the way.

In the present invention, the target ω-aminoalkylaldehyde dehydrogenase includes 4-aminobutanal dehydrogenase, 3-aminopropanal dehydrogenase, and 5-aminopentanal dehydrogenase.

The most important feature of the present invention is that ω-aminoalkylaldehyde obtained by reacting polyamine with putrescine oxidase and / or polyamine oxidase is used as a substrate for measuring the enzyme activity of ω-aminoalkylaldehyde dehydrogenase. Is to do. By using such an ω-aminoalkyl aldehyde,
The finding that the enzyme activity of ω-aminoalkylaldehyde dehydrogenase can be measured accurately and safely was found for the first time in the present invention.

The polyamine used in the present invention is not particularly limited as long as it generates a substrate of ω-aminoalkylaldehyde dehydrogenase, which is an enzyme to be measured.For example, the enzyme activity of 4-aminobutanal dehydrogenase is measured. In this case, putrescine is suitable, spermidine or spermine is suitable for measuring the enzyme activity of 3-aminopropanal dehydrogenase, and cadaverine is suitable for measuring the enzyme activity of 5-aminopentanal dehydrogenase.

The putrescine oxidase used in the present invention may be of any origin as long as it can produce ω-aminoalkylaldehyde from a polyamine such as putrescine or spermidine. And putrescine oxidase derived from microorganisms derived from the genera Aspergillus, Arthrobacter, and Pseudomonas, from plants such as germinated soybean, and derived from animals such as pig kidney. The polyamine oxidase may be of any origin as long as it can generate one kind of ω-aminoalkylaldehyde from polyamines such as spermine and spermidine.For example, barley, oats,
Examples thereof include polyamine oxidases of plant origin such as corn origin, animal origins such as bovine plasma and rat liver, and microbial origins such as Aspergillus, Penicillium and Streptomyces.

In the present invention, the ω-aminoalkyl aldehyde is
Putrescine oxidase and / or
Alternatively, it can be obtained by acting polyamine oxidase.

In the above reaction, the concentrations of putrescine oxidase and polyamine oxidase used as enzymes are not particularly limited, but it is preferable to select a concentration at which the polyamine is completely converted into the corresponding ω-aminoalkylaldehyde. Further, the pH condition and the temperature condition are different depending on the type of putrescine oxidase and polyamine oxidase used, and any condition may be used as long as the activity of these enzymes is expressed. Is 4.0 to 10.5 and the temperature condition is 15 to 4
A range of 0 ° C. is preferred.

In the present invention, for the above reaction, a reaction that generates ω-aminoalkylaldehyde as a substrate corresponding to ω-aminoalkylaldehyde dehydrogenase, which is an enzyme to be measured, may be selected.

For example, when the enzyme to be measured is 4-aminobutanal dehydrogenase, it is 4-aminobutanal (γ-aminobutyraldehyde), and when the enzyme to be measured is 5-aminopentanal dehydrogenase, it is 5-aminopentanal. When the enzyme to be measured is 3-aminopropanal dehydrogenase, the raw material polyamine and the enzyme to be used are selected so as to produce 3-aminopropanal.

As described above, in the method of the present invention, the substrate used for measuring the enzymatic activity acts only on polyamine and is reacted with putrescine oxidase and / or polyamine oxidase whose action mechanism has been clarified. Since it is produced, it has an advantage that its purity is very high and its concentration is obvious, and as a result, the enzyme activity of ω-aminoalkylaldehyde dehydrogenase can be measured accurately.

In the present invention, the measurement of the enzyme activity of ω-aminoalkylaldehyde dehydrogenase
The reaction is carried out by reacting an aminoalkylaldehyde with an analyte in the presence of an oxidized nicotinamide coenzyme, and measuring the production rate of a reduced nicotinamide coenzyme.

The oxidized nicotinamide adenine dinucleotide (hereinafter referred to as NAD) or oxidized nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP) is used as the oxidized nicotinamide adenine dinucleotide (NAD). Whether to use NAD or NADP is appropriately selected depending on the type or purpose of the enzyme to be measured.

In practicing the present invention, the oxidized nicotinamide coenzyme, which is a coenzyme of ω-aminoalkylaldehyde dehydrogenase, is caused to exist by either a method of adding it together with the analyte or a method of pre-existing in a polyamine solution. And may be selected according to the purpose of measuring the enzyme activity.

Among them, when the contact time between the oxidized nicotinamide coenzyme and the analyte is to be shortened as much as possible or when the method for measuring the enzyme activity is to be simplified, a method in which the enzyme is present in the polyamine in advance is selected. .

In the above reaction, the concentration of NAD or NADP, which is an oxidized nicotinamide coenzyme, was measured as ω-
The concentration may be a concentration that sufficiently expresses the enzyme activity of aminoalkylaldehyde dehydrogenase, that is, a concentration that is at least about twice the km value, and preferably a concentration in the range of 0.1 to 200 mM.

The reaction conditions, for example, pH conditions and temperature conditions vary depending on the type of the enzyme of ω-aminoalkylaldehyde dehydrogenase to be measured, and may be any condition as long as the activity of these enzymes is expressed.
Preferably, the pH condition is in the range of 4.0 to 10.5 and the temperature condition is in the range of 15 to 40 ° C.

Further, the production rate of reduced nicotinamide coenzyme produced by the above reaction can be determined by increasing the rate of direct ultraviolet absorption, which has been conventionally performed, or a visible rate that appears by changing to a formazan dye using a tetrazolium salt and an electron transfer system. What is necessary is just to measure by the well-known method, such as the method of measuring the increase rate of absorption, the luminescence amount obtained by leading to chemiluminescence, etc.

(Effect) The method for measuring the enzyme activity of ω-aminoalkylaldehyde dehydrogenase according to the present invention provides a simpler and more accurate method than the conventional method for measuring the activity of the enzyme. That is, according to the present invention, ω-aminoalkylaldehyde, which is a substrate required for measuring the activity of the enzyme, is produced by reacting polyamine with putrescine oxidase and / or polyamine oxidase. It can be easily adjusted at the time of activity measurement without going through the purification step. Therefore, the measurement of the enzyme activity was greatly simplified.

In addition, in the conventional measurement of the activity of the enzyme, ω-aminoalkylaldehyde, which is a substrate of the enzyme, was separately chemically synthesized and purified. According to this, the substrate of the enzyme is automatically adjusted enzymatically at the time of activity measurement, so that a fresh substrate free of degradation products can always be used, and the enzyme activity value can be obtained more accurately. became.

Further, the substrate used in the conventional method for measuring the activity of the enzyme contains compounds such as bromine and succinic acid, which are by-products of the enzyme because it is based on chemical synthesis. It had an adverse effect on the expression of activity. According to the method for measuring the activity of the enzyme according to the present invention, there is no compound as described above in the substrate of the enzyme, and it is possible to obtain an activity value of the enzyme with extremely high accuracy.

Therefore, according to the present invention, the routine measurement of the enzyme activity of ω-aminoalkylaldehyde dehydrogenase can be carried out simply, in a short time, and with high accuracy in the field of clinical examination or biochemical research.

(Examples) Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the scope described in these examples.

Example 1 A 0.1 M Tris-HCl buffer (pH 8.0) containing 10 mM putrescine dihydrochloride was placed in a cuvette having an optical path width of 1 cm.
2.5 ml was dispensed, and 20 μ (5 units) of Micrococcus putrescine oxidase was added and mixed.
After incubation at 30 ° C. for 10 minutes, 0.5 ml of a 20 mM NAD aqueous solution was added to the reaction solution, and the amount of γ-aminobutyric acid produced was measured in advance by an activity measurement method for quantifying by liquid chromatography. -50 subjects with an aminobutanal dehydrogenase activity of 4.5 units / ml
μ was added and mixed. Immediately at 30 ° C., the rate of increase in absorbance at 340 nm was measured. As a result, the amount of increase in absorbance per minute (A) was 0.455. Using the following conversion formula (1), the enzymatic activity value of 4-aminobutanal dehydrogenase per 1.0 ml of the subject was calculated to be 4.5 units (μm
ole / min).

Example 2 Putrescine was replaced with cadaverine and an additional 20μ (5
Unit) putrescine oxidase was replaced by 20 μ (20 units) putrescine oxidase, and the same operation as in Example 1 was carried out.
The increase (A) in the absorbance at 0 nm was 0.130. According to the above conversion formula (1), the enzyme activity value of 5-aminopentanal dehydrogenase per 1.0 ml of the specimen is 1.3 units (μm
ole / min). On the other hand, when the 5-aminopentanal dehydrogenase activity was measured by an activity measuring method for quantifying the amount of δ-aminovaleric acid produced by liquid chromatography, it was 1.2 units / ml.

Example 3 20 mM spermidine in a cuvette with an optical path width of 1.0 cm
1.0 ml of 10 mM phosphate buffer (pH 6.5) containing tetrahydrochloride
Was further added, 20 μ (2 units) of Aspergillus-derived polyamine oxidase was added thereto, and the mixture was incubated at 30 ° C. for 10 minutes. Then, 0.1 ml of 0.1 M Tris containing 2.0 ml of 10 mM NAD was added to the reaction solution. A hydrochloric acid buffer (pH 8.0) was added, and then a 3-aminopropanal dehydrogenase activity of 1.7 units / ml, which was previously determined by an activity measurement method for determining the amount of β-aminopropionic acid produced by liquid chromatography. 50 μl of the sample was added and mixed, and the rate of increase in absorbance at 340 nm was immediately measured at 30 ° C. As a result, the amount of increase in absorbance per minute (A) is
It was 0.186. From the above conversion formula (1), the enzyme activity value of 3-aminopropanal dehydrogenase per 1.0 ml of the specimen was 1.8 units (μmole / min).

Example 4 A 10 mM putrescine solution was placed in a cuvette having an optical path width of 1.0 cm.
3.0 ml of 0.1 M Tris-HCl buffer (pH 8.0) containing dihydrochloride and 20 mM NAD was dispensed, and 20 μl (2
Unit), and the mixture was incubated at 30 ° C for 10 minutes, and then the reaction solution was measured in advance by liquid chromatography to determine the 4-butanal dehydrogenase activity. A test sample (50 μ at 2.4 units / ml) was added and mixed, and the increase rate of the absorbance at 340 nm was immediately measured at 30 ° C. As a result, the amount of increase in absorbance per minute (A) is
It was 0.242. According to the above conversion formula (1), the enzyme activity value of 4-aminobutanal dehydrogenase per 1.0 ml of the specimen was 2.4 units (μmole / min).

COMPARATIVE EXAMPLE 10 ml of an aqueous solution of 200 mM N-bromosuccinimide was added to 10 ml of an aqueous solution containing 100 mM of L-ornithine dihydrochloride and mixed, and then the reaction mixture was placed in a desiccator under reduced pressure using an aspirator. In the desiccator, a beaker containing a 40% aqueous solution of potassium iodide was co-present for the purpose of trapping bromine generated as the reaction progressed. After reacting at 20 ° C. for 1 hour under reduced pressure, the reaction was heated in boiling water for 5 minutes. The obtained reaction product (dried product) was dissolved in 10 ml of a 10 mM aqueous hydrochloric acid solution. The solution obtained in this operation contained 80 mM 4-aminobutanal.

0.1M Tris-HCl buffer (p
H8.0) was dispensed in 2.2 ml and the 80 mM 4-
After dispensing 0.32 ml of aminobutanal aqueous solution and mixing, add another 20
0.5 ml of an NAD aqueous solution of mM was added, and then 50 μm of the same analyte as that shown in Example 1 was added and mixed. Immediately 30 ° C
As a result of measuring the rate of increase in absorbance at 340 nm below, the amount of increase in absorbance per minute (A) was 0.125. The enzymatic activity value of 4-aminobutanal dehydrogenase per 1.0 ml of the subject was calculated using the conversion formula (1) shown in Example 1 and was found to be 1.2 units (μmole / min).

Claims (1)

(57) [Claims] When measuring the enzyme activity of .omega.-aminoalkylaldehyde dehydrogenase in a test sample, a polyamine is reacted with putrescine oxidase and / or polyamine oxidase to obtain an .omega.-aminoalkylaldehyde. Reacting the test sample with the ω-aminoalkylaldehyde in the presence of the nicotinamide coenzyme, and measuring the rate of formation of the reduced nicotinamide coenzyme to be formed; measuring the enzyme activity of the ω-aminoalkylaldehyde dehydrogenase. Measuring method.