JP2017038569A - Activity measurement method of autotaxin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide measurement methods of autotaxin activity in a sample.SOLUTION: A measurement method of autotaxin activity comprises the steps of: (1) contacting lysophosphatidyl glycerol with a sample to produce glycerol and lysophosphatidic acid from lysophosphatidyl glycerol; (2) contacting a particular glycerol kinase, a first nucleotide coenzyme, and a second nucleotide coenzyme differed from the first nucleotide coenzyme in a nucleoside portion with a treated product obtained in the step (1); (3) detecting the variation of at least any one of the first nucleotide coenzyme, a dephosphorylated product of the first nucleotide coenzyme, the second nucleotide coenzyme, and a phosphorylated product of the second nucleotide coenzyme, in the step (2); and (4) calculating the autotaxin activity in a sample based on the variation detected in the step (3).SELECTED DRAWING: None

Description

  The present invention relates to a method for measuring autotaxin activity. More specifically, the present invention relates to a method for measuring autotaxin activity in a sample and a composition for measurement using a reaction of autotaxin using lysophosphatidylglycerol as a substrate.

  Autotaxin is a glycoprotein that was isolated and identified in 1992 from the culture supernatant of human malignant melanoma cell line A2058, and was purified and obtained from bovine serum and human plasma. Enzyme research has found that it is identical to lysophosphatidic acid-producing enzyme lysophospholipase D, and plays an important role in vivo as a lysophosphatidic acid (hereinafter sometimes referred to as LPA) producing enzyme ( Non-Patent Documents 1 and 2).

  As a method for measuring the activity of autotaxin in serum, an enzyme method is known in which choline released using lysophosphatidylcholine (hereinafter sometimes abbreviated as LPC) as a substrate is measured using choline oxidase (non-patent document). 3). In this method, LPC and a sample are heated in the presence of magnesium chloride, calcium chloride, and sodium chloride at 37 ° C. for a fixed time, for example, 3 hours, and then a coloring solution containing choline oxidase, peroxidase, and a coloring dye is added. It is added to quantify the choline produced by the autotaxin reaction.

  A measurement method using a synthetic substrate has also been proposed (Patent Documents 1 and 2). In this method, lysophosphatidyl-p-nitrophenol is synthesized in place of the natural substrate LPC, and this is used as a substrate, and p-nitrophenol released by the reaction of lysophospholipase D is developed under alkaline conditions. A compound in which the oxygen atom on the side opposite to the glycerol skeleton of the lipid phosphate diester is substituted with a sulfur atom, and a thiol compound released from this compound by the reaction of lysophospholipase D is synthesized in the presence of a disulfide compound. This is a method of measuring from the absorbance derived from the thiol compound produced by the exchange reaction.

  On the other hand, a measurement method using an antibody has also been reported (Patent Document 3). In this method, two types of antibodies against autotaxin are used instead of enzyme activity, one antibody is immobilized on an insoluble carrier, autotaxin is allowed to act, and another antibody labeled with an enzyme or the like is used. Furthermore, autotaxin is recognized and measured as mass.

WO2002 / 053569 JP2009-149582 JP2013-178286A

J Cell Biol. 158: 227-233,2002 JBiol Chem. 277: 39436-39442,2002 Clin Biochem 40: 274-277, 2007

Several methods for measuring autotaxin activity are known as described above.
However, the method for enzymatically detecting choline released from LPC may be influenced by endogenous LPC or free choline. In addition, autotaxin in blood is stable, but it has been reported that endogenous LPC increases in the serum after blood collection according to the heating time (Non-patent Document 3). For this reason, when a biological sample is used, there is a problem that the measured value of autotaxin activity is affected by the storage state after blood collection.

  Furthermore, a method for measuring autotaxin activity using a synthetic substrate has been reported, but it is difficult to reduce the cost. Furthermore, although an antibody method has been reported, there is a problem that the operation is complicated and a special apparatus is required.

  In order to solve these problems, the present invention is not affected by endogenous autotaxin substrates such as LPC and free choline or products thereof, and is not affected by autotaxin substrates such as LPC that increase after blood collection. In addition, a method for measuring the activity of autotaxin and a composition for measurement are provided.

  The present inventors have intensively studied to solve the above-mentioned problems, and surprisingly, lysophosphatidylglycerol, which has not been known so far, is used as a substrate for autotaxin. Confirmed for the first time. Then, by reacting glycerol, which is a product of this enzyme reaction, and / or its phosphoric acid with a novel enzyme cycling method using glycerol kinase, endogenous components such as LPC and free choline are compared with conventional methods. The present inventors have found a method and composition for measuring autotaxin activity that is inexpensive and simple to measure the activity of autotaxin without being affected by an autotaxin substrate or a product thereof and without being affected by an autotaxin substrate such as LPC that increases after blood collection. That is, the present invention relates to the following.

[0]
A method for measuring the activity of autotaxin in a sample, comprising the steps of (1) contacting a sample with lysophosphatidylglycerol and generating glycerol and lysophosphatidic acid from lysophosphatidylglycerol,
Measuring method including
[1]
A method for measuring autotaxin activity in a sample comprising:
(1) A step of bringing a sample into contact with lysophosphatidylglycerol, and a process for producing glycerol and lysophosphatidic acid from lysophosphatidylglycerol;
(2) Glycerol kinase, which catalyzes a forward reaction and its reverse reaction from glycerol in the presence of a nucleotide coenzyme, and at least different nucleoside moieties in the forward reaction and the reverse reaction, respectively. Glycerol kinase using a nucleotide coenzyme having a first nucleotide coenzyme, and a second nucleotide coenzyme having a nucleoside moiety different from that of the first nucleotide coenzyme is obtained in the above step (1). Contacting with the treated sample, and causing a cycling reaction of the following formula (I):
Carrying out the cycling reaction of
(3) Any of the first nucleotide coenzyme, the dephosphorylation oxide of the first nucleotide coenzyme, the second nucleotide coenzyme, and the phosphorylation of the second nucleotide coenzyme in step (2) Detecting one or more changes; and (4) calculating the autotaxin activity of the sample based on the changes detected in step (3);
Measuring method including
[1-1]
Simultaneously with step (2) or prior to step (2), the following steps are further performed:
(1a) A step of bringing lysophospholipase into contact with the treated product obtained in step (1) and performing a treatment for producing glycerol-3-phosphate from lysophosphatidic acid;
The measurement method according to [1], wherein step (2) is a step of bringing glycerol kinase into contact with the treated product obtained in steps (1) and (1a).
[1-1-2]
The step (2) is a step of detecting a signal change amount corresponding to an increased amount of the dephosphorylation oxide of the first nucleotide coenzyme or the second nucleotide coenzyme [1] Or the measuring method as described in [1-1].
[1-2]
The nucleoside part of the first nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine. The measurement method according to any one of [1] to [1-1-2].
[1-3]
The measurement method according to any one of [1] to [1-2], wherein the nucleoside portion of the first nucleotide coenzyme is any one of adenosine, inosine, guanosine, and deoxyadenosine.
[1-4]
The nucleoside part of the second nucleotide coenzyme is any one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine, and deoxyinosine. The measurement method according to any one of [1] to [1-3].
[1-5]
The measurement method according to any one of [1] to [1-4], wherein the nucleoside portion of the second nucleotide coenzyme is any one of adenosine, inosine, guanosine, and deoxyadenosine.
[1-6]
The nucleoside portion of the first nucleotide coenzyme is any of adenosine, inosine, guanosine, and deoxyadenosine, and the nucleoside portion of the second nucleotide coenzyme is any of adenosine, inosine, guanosine, and deoxyguanosine The measuring method according to any one of [1] to [1-5].
[1-7]
The combination of the nucleotide portion of the first nucleotide coenzyme and the nucleoside complement portion of the second nucleotide coenzyme comprises adenosine and inosine, guanosine and adenosine, deoxyadenosine and guanosine, deoxyadenosine and deoxyguanosine, deoxyadenosine and inosine, and The measurement method according to any one of [1] to [1-6], which is either inosine or adenosine.
[1-8]
The measurement method according to any one of [1] to [1-7], wherein the nucleoside portion of the first nucleotide coenzyme is adenosine, and the nucleoside portion of the second nucleotide coenzyme is inosine.
[1-9]
The measurement according to any one of [1] to [1-8], wherein the first nucleotide coenzyme is adenosine triphosphate (ATP) and the second nucleotide coenzyme is inosine diphosphate (IDP). Method.
[1-10]
Step (3) is: a detection enzyme that uses the dephosphorylation oxide of the first nucleotide coenzyme as a substrate but does not use the second nucleotide coenzyme as a substrate; or does not use the first nucleoside coenzyme as a substrate, An amount of change of the first nucleotide coenzyme due to the forward reaction or an amount of increase of the second nucleotide coenzyme due to the reverse reaction The measurement method according to any one of [1] to [1-9], which is a step of detecting.
[1-11]
The dephosphorylation oxide of the first nucleotide coenzyme or the phosphate of the second nucleotide coenzyme is adenosine diphosphate (ADP);
Step (3) is a step of detecting a change in signal that changes in response to an increase in ADP in the presence of glucose, using ADP-dependent glucokinase (EC 2.7.1.147). [1] The measurement method according to any one of [1-10].
[1-12]
Step (3) uses adenosine diphosphate (ADP) -dependent glucokinase (EC 2.7.1.147) to thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (Thio-NAD), nicotinamide adenine dinucleotide phosphate (NADP), and any one of nicotinamide adenine dinucleotide (NAD) coenzymes and glucose 6-phosphate dehydrogenase in the presence of ADP The measurement method according to [1-11], which is a step of detecting a change amount of a changing signal.
[1-13]
A method for measuring autotaxin in a sample comprising:
(1) a step of contacting a sample with lysophosphatidylglycerol, and a step of producing glycerol and lysophosphatidic acid from lysophosphatidylglycerol; (1a) contacting a lysophospholipase with the treatment product obtained in step (1); Treating lysophosphatidic acid to produce glycerol-3-phosphate;
(2) a glycerol kinase that catalyzes a forward reaction of glycerol-3-phosphate from glycerol and its reverse reaction in the presence of adenosine triphosphate (ATP), and uses ATP in the forward reaction; Glycerol kinase, ATP, and IDP using inosine diphosphate (IDP) in the reverse reaction are brought into contact with the treated product obtained in steps (1) and (1a), and the following formula (II)

Carrying out the cycling reaction of
(3) Using adenosine diphosphate (ADP) -dependent glucokinase (EC 2.7.1.147), thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (thioNAD) ), Nicotinamide adenine dinucleotide phosphate (NADP), and nicotinamide adenine dinucleotide (NAD) coenzyme, glucose and glucose 6-phosphate dehydrogenase in the presence of glucose 6-phosphate dehydrogenase increase in step (2) Detecting the amount; and (4) calculating the autotaxin activity of the sample based on the increased amount of ADP detected in step (3);
Measuring method including
[1-14]
[1] A clinical specimen test method comprising the measurement method according to any one of [1-13].

[2]
A composition for measuring autotaxin activity in a sample,
(A) lysophosphatidylglycerol,
(B) Glycerol kinase, which catalyzes a forward reaction and its reverse reaction to produce its phosphate from glycerol in the presence of a nucleotide coenzyme. In the forward reaction and the reverse reaction, different nucleoside moieties are used. Glycerol kinase using a nucleotide coenzyme having,
(C) a first nucleotide coenzyme;
(D) a second nucleotide coenzyme that differs in nucleoside moiety from the first nucleotide coenzyme;
A composition comprising:
[2-1]
(A1) The composition according to [2], further comprising lysophospholipase.
[2-2]
The nucleoside portion of the first nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine. The composition according to any one of [2] and [2-1].
[2-3]
The composition according to any one of [2] to [2-2], wherein the nucleoside portion of the first nucleotide coenzyme is any one of adenosine, inosine, guanosine, and deoxyadenosine.
[2-4]
The nucleoside portion of the second nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine. The composition according to any one of [2] to [2-3].
[2-5]
The composition according to any one of [2] to [2-4], wherein the nucleoside portion of the second nucleotide coenzyme is any one of adenosine, inosine, guanosine, and deoxyguanosine.
[2-6]
The nucleoside portion of the first nucleotide coenzyme is any of adenosine, inosine, guanosine, and deoxyadenosine, and the nucleoside portion of the second nucleotide coenzyme is any of adenosine, inosine, guanosine, and deoxyguanosine The composition according to any one of [2] to [2-5].
[2-7]
The combination of the nucleoside portion of the first nucleotide coenzyme and the nucleoside complement portion of the second nucleotide coenzyme comprises adenosine and inosine, guanosine and adenosine, deoxyadenosine and guanosine, deoxyadenosine and deoxyguanosine, deoxyadenosine and inosine, and The composition according to any one of [2] to [2-6], which is either inosine or adenosine.
[2-8]
The composition according to any one of [2] to [2-7], wherein the nucleoside moiety of the first nucleotide coenzyme is adenosine and the nucleoside moiety of the second nucleotide coenzyme is inosine.
[2-9]
The first nucleotide coenzyme is adenosine triphosphate (ATP), and the second nucleotide coenzyme is inosinic acid diphosphate (IDP), according to any one of [2] to [2-8] Composition.
[2-10]
Detection enzyme that uses dephosphorylate of first nucleotide coenzyme as substrate but does not use second nucleotide coenzyme as substrate, or second nucleotide coenzyme that does not use first nucleoside coenzyme as substrate Enzyme for detection using phosphorous oxide as a substrate,
The composition according to any one of [2] to [2-9], further comprising:
[2-11]
The composition according to [2-10], wherein the detection enzyme is adenosine diphosphate (ADP) -dependent glucokinase (EC 1.7.1.147) and further contains glucose.
[2-12]
The composition according to any one of [2] to [2-10], further comprising glucose.
[2-13]
Any of thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (thioNAD), nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide (NAD);
Glucose 6-phosphate dehydrogenase;
The composition according to any one of [2] to [2-12], further comprising:
[2-14]
A composition for measuring autotaxin activity in a sample,
(A) lysophosphatidylglycerol,
(A1) lysophospholipase;
(B) A glycerol kinase that catalyzes the forward reaction and the reverse reaction of producing glycerol-3-phosphate from glycerol in the presence of adenosine triphosphate (ATP), and in the forward reaction, adenosine triphosphate ( Glycerol kinase utilizing ATP) and utilizing inosine diphosphate (IDP) in the reverse reaction;
(C) ATP;
(D) IDP;
(E) adenosine diphosphate (ADP) dependent glucokinase (EC 1.7.1.147);
(F) glucose;
(G) any one of thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (thioNAD), nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide (NAD);
(H) glucose 6-phosphate dehydrogenase;
A composition comprising:
[3]
A reagent kit for measuring autotaxin activity, comprising the composition according to any one of [2] to [2-14].
[4]
A clinical specimen measuring device for measuring autotaxin activity, comprising the composition according to any one of [2] to [2-14].

  According to the present invention, endogenous autotaxin such as LPC and free choline can be obtained by combining lysophosphatidylglycerol as a substrate for autotaxin and combining glycerol and lysophosphatidic acid with an enzyme cycling reaction by glycerol kinase. The activity of autotaxin can be measured easily and inexpensively without being affected by the substrate or its product, and without being affected by an autotaxin substrate such as LPC that increases after blood collection.

FIG. 3 is a graph showing the relationship between the substrate lysophosphatidylglycerol concentration and the absorbance corresponding to the amount of glycerol produced in a reaction using recombinant autotaxin (Example 2). 10 is a graph showing the relationship between the amount of recombinant autotaxin and the absorbance corresponding to the amount of glycerol produced in a reaction using lysophosphatidylglycerol as a substrate (Example 3). It is the graph which showed the lysophospholipase addition effect in the autotaxin activity measuring method of this invention (Example 4). FIG. 6 is a correlation diagram of autotaxin activity measurement between the conventional method using LPC as a substrate and the method of the present invention (Example 5).

  Hereinafter, an embodiment of the present invention (hereinafter also referred to as “the present embodiment”) will be specifically described. However, it should not be understood that the following embodiments limit the present invention. It should be understood that various alternative embodiments will be apparent to those skilled in the art from this disclosure, and that the present invention includes various embodiments and the like not specifically described herein. is there. The EC number (Enzyme Commission numbers) in this specification is a number confirmed as of August 18, 2015.

This embodiment is a method for measuring the activity of autotaxin in a sample: (1) A step of bringing a sample into contact with lysophosphatidylglycerol, and a process for producing glycerol and lysophosphatidic acid from lysophosphatidylglycerol;
(2) Glycerol kinase, which catalyzes a forward reaction and a reverse reaction of glycerol from its glycerol in the presence of a nucleotide coenzyme. In the forward reaction and the reverse reaction, different nucleoside moieties are used. A treatment obtained in step (1) comprising a glycerol kinase using a nucleotide coenzyme having, a first nucleotide coenzyme, and a second nucleotide coenzyme having a nucleoside moiety different from that of the first nucleotide coenzyme The following formula (I):
Carrying out the cycling reaction of
(3) Any of the first nucleotide coenzyme, the dephosphorylation oxide of the first nucleotide coenzyme, the second nucleotide coenzyme, and the phosphorylation of the second nucleotide coenzyme in step (2) Detecting one or more changes; and (4) calculating the autotaxin activity of the sample based on the changes detected in step (3);
It is the measuring method containing.

  The measurement target in the method according to the present embodiment is autotaxin activity. Autotaxin plays an important role as an LPA-producing enzyme in vivo, but the measurement target in the method according to the present embodiment is not limited to that existing in vivo. Examples thereof include those produced by genetic manipulation and those synthesized by chemical synthesis. Further, as described above, autotaxin may be called lysophospholipase D (Non-patent Document 3), and this activity is also included in the measurement target in the method according to the present embodiment.

  The substrate for autotaxin in the method according to the present embodiment is lysophosphatidylglycerol, which may be derived from a living body, chemically synthesized, or enzymatically synthesized, and its origin is It doesn't matter.

  The sample that can be used in the method according to the present embodiment is not particularly limited, but a human or animal biological sample may be used, preferably human blood, more preferably human serum. The sample may contain autotaxin but may not contain it as a result. Moreover, the sample adjusted in the laboratory may be sufficient.

  “Contacting” in the method according to the present embodiment refers to an act of bringing two or more objects into contact with each other, and does not indicate whether or not they are in contact with each other. For example, operations such as mixing, addition, pouring, and suitable conditions are included, but the present invention is not limited thereto.

  In step (1) of the method according to the present embodiment, the sample is brought into contact with lysophosphatidylglycerol, and glycerol and lysophosphatidic acid are produced from lysophosphatidylglycerol as a substrate by the action of autotaxin in the sample. For example, the following conditions may be used.

Lysophosphatidylglycerol (LPG) can be dissolved using a surfactant such as Triton X-100, but is not limited thereto. The LPG concentration during the reaction is preferably 2 to 40 mmol / L, more preferably 4 to 20 mmol / L. In order to maintain the pH during the reaction, for example, a pH buffer is added at 20 mmol / L or more and 200 mmol / L or less to obtain a reaction solution. As the pH buffering agent, for example, phosphate buffer, Tris-HCl buffer, and Good buffers such as PIPES, MES, and HEPES can be used.
The pH at the time of the reaction may be selected appropriately under conditions that allow the reaction to proceed efficiently. Usually, the pH is adjusted from neutral to weakly alkaline at pH 7.0 or more and 10.0 or less, and magnesium chloride is used in the same manner as in the previously reported activity measurement method. And a metal salt such as calcium chloride is appropriately added in an amount of 0.5 mmol / L to 20 mmol / L. Similarly, a salt such as sodium chloride can be added at 1 mol / L or less.
The reaction solution is preheated to 25 ° C. or higher and 42 ° C. or lower, more preferably around 37 ° C., and the autotaxin reaction is performed. As will be described later, the autotaxin reaction and the enzyme cycling reaction in the step (2) can be carried out simultaneously.

  In the method according to the present embodiment, “Generate” refers to an action to be generated, regardless of whether it is generated.

In one aspect, the method according to the present embodiment further includes the following steps:
(1a) A step of bringing lysophospholipase into contact with the treated product obtained in step (1) and performing a treatment for producing glycerol-3-phosphate from lysophosphatidic acid;
In this case, step (2) is a step of bringing glycerol kinase into contact with the processed product obtained in steps (1) and (1a). This step (1a) can be performed simultaneously with the step (2) or prior to the step (2).

  In the step (1a) of the present embodiment, the lysophospholipase is not particularly limited as long as it produces glycerol-3-phosphate using lysophosphatidic acid as a substrate. For example, a commercially available product (LYPL; T-32, Asahi Kasei Pharma Co., Ltd.) can be used.

  In the step (1a) of the present embodiment, a method for bringing lysophospholipase into contact with the treated product obtained in step (1) to produce glycerol-3-phosphate from lysophosphatidic acid is referred to a known method. (Clin Chim Acta 333, 59-67, 2003, etc.). For example, specifically, lysophospholipase is further added at 2.5 u / mL to 100 u / mL, more preferably at 5 u / mL to 50 u / mL under the conditions that cause the enzyme cycling reaction by glycerol kinase in the step (2) shown below. What is necessary is just to add.

Since lysophospholipase in step (1a) uses lysophospholipid as a substrate, if it is added at the same time as step (1), it also acts on lysophosphatidylglycerol, which is a substrate for autotaxin in step (1). May hinder measurement of autotaxin activity. In this case, the reaction may be carried out in multiple steps, such as adding lysophospholipase after stopping the autotaxin reaction in step (1). Examples of the method for stopping the reaction include addition of a chelating agent such as EDTA, change of pH to the acidic side, or addition of an antibody that binds to the active site. Moreover, since the search of an autotaxin inhibitor is actively advanced now as a pharmaceutical use, these inhibitors can also be used. By adding lysophospholipase in this way, both the glycerol produced in step (1) and the glycerol-3-phosphate produced in step (1a) are involved in the enzyme cycling reaction of step (2). Therefore, the autotaxin activity measurement sensitivity can be substantially doubled. Whether or not lysophospholipase is added may be appropriately selected according to the amount of autotaxin activity in the sample.
The step (1a) can be performed prior to the step (2) or can be performed simultaneously with the step (2). For example, after stopping the autotaxin reaction in step (1), glycerol kinase and lysophospholipase are added to the treated product obtained in step (1), and in parallel with the execution of step (2), step (1a ) Can also be performed.

  Next, step (2) of the method according to the present embodiment will be described. Glycerol kinase that can be used in step (2) is an enzyme that exhibits reversible reactivity for at least dephosphorylation and phosphorylation, and acts on two or more nucleotide coenzymes with different nucleoside moieties. It can be used regardless of its origin. Since the enzyme is widely used for measuring triglycerides, many microorganism-derived enzymes are commercially available and are easily available. For example, enzymes derived from the genus Cellulomonas are commercially available from Toyobo Co., Ltd., and enzymes derived from Flabobacterium meningosepticum are commercially available from Asahi Kasei Pharma Corporation. These derived cells and tissues may be cultured, and the enzyme may be isolated and purified and obtained by a conventional method. For example, after culturing a microorganism, an enzyme having a purity increased to a practical level can be obtained by combining known purification methods such as column chromatography. Alternatively, the enzyme can be obtained by culturing transformed cells such as E. coli into which the gene has been incorporated. Furthermore, the glycerol kinase according to the present embodiment may be genetically modified for the purpose of improving enzyme properties. For example, a gene encoding the modified enzyme protein can be isolated by a conventional method, and the modified enzyme can be obtained by a method of expressing in a suitable host using a gene recombination technique.

  As a method for confirming the specificity to the nucleotide coenzyme (nucleoside phosphate) in the forward reaction and reverse reaction of glycerol kinase, for example, for nucleoside phosphate, a known detection method by high performance liquid chromatography (HPLC) method is used. This is possible by checking the reaction product.

  Further, the specificity to the positive reaction nucleotide coenzyme is that glycerol-3-phosphate, which is a product of the positive reaction, is combined with glycerol-3-phosphate oxidase and peroxidase, and a coupler such as 4-aminoantipyrine and phenol. In the presence of a hydrogen donor such as

  Although there are not as many reports on the reverse reaction of glycerol kinase as the normal reaction, whether the reverse reaction proceeds can be easily confirmed by experiments by detecting the product. For example, Eur J Biochem 267, 2323-2333, 2000 exemplifies a method of detecting ATP produced by a reverse reaction as a chemiluminescence amount using luciferase in the presence of luciferin. When the reverse reaction is confirmed in this way, the pH at the time of the reaction is further changed, and the optimum pH for the reverse reaction is investigated, so that the condition for the step (2) in the present embodiment is set. It can be used as a reference. The degree of the reverse reaction is not particularly limited, but the relative activity is preferably 1.5% or more of the normal reaction.

  Glycerol kinase using the nucleotide coenzyme having a different nucleoside moiety in step (2) as a reactant in the forward reaction or the reverse reaction is a nucleotide coenzyme having a different nucleoside moiety that works best for each of the forward reaction and the reverse reaction. For example, it is desirable to have a specificity of, for example, 4% or more, preferably 8% or more, but is not limited thereto.

  In the method according to the present embodiment, “nucleotide coenzyme” refers to a nucleoside phosphate. The first nucleotide coenzyme and the second nucleotide coenzyme used in the method according to the present embodiment are nucleotide coenzymes having different nucleoside moieties.

  In the method according to the present embodiment, “dephosphorylation / dephosphorylation of the first nucleotide coenzyme by the positive reaction” means that in the kinase positive reaction, the phosphate from the first nucleotide coenzyme is the positive of the kinase. This refers to the dephosphorylating oxide of the first nucleotide coenzyme after transfer to the reaction substrate. In addition, “dephosphorylation of the second nucleotide coenzyme by reverse reaction” means that after phosphate transfer from the kinase reverse reaction substrate to the second nucleotide coenzyme in the kinase reverse reaction. Of the second nucleotide coenzyme. Here, the number of transferred phosphoric acid may be one, two, or three. Preferably one.

  The nucleoside part of the nucleotide coenzyme in the method according to the present embodiment means not only a naturally occurring substance, but also a derivative substance chemically or enzymatically synthesized and a similar substance.

  Note that step (2) of the method according to the present embodiment can be performed with one type of glycerol kinase. For example, two or more different glycerol kinases that catalyze the same cycling reaction are used. It can also be implemented.

  The type of nucleoside moiety of the nucleotide coenzyme used in the method according to the present embodiment is not particularly limited. Examples include uridine, deoxycytidine, deoxyxanthosine, and deoxyinosine. In addition, these phosphorus oxides may be monophosphorus oxide, 2-phosphorus oxide, or 3-phosphorus oxide.

  Examples of nucleotide coenzymes include ribonucleotides and deoxyribonucleotides.

  Specific examples of ribonucleotides as nucleotide coenzymes include ATP, ADP, adenylate (AMP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanylate (GMP), 5-methyluridine 3 Phosphoric acid (TTP), 5-methyluridine diphosphate (TDP), 5-methyluridine monophosphate (TMP), uridine triphosphate (UTP), uridine diphosphate (UDP), uridine monophosphate (UMP) ), Cytidine triphosphate (CTP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), xanthosine triphosphate (XTP), xanthosine diphosphate (XDP), xanthosine monophosphate (XMP), Examples include inosine triphosphate (ITP), inosine diphosphate (IDP), and inosine monophosphate (IMP). But it is not limited to.

  Specific examples of deoxyribonucleotides as nucleotide coenzymes include deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), deoxyguanosine triphosphate (dGTP) , Deoxyguanosine diphosphate (dGDP), deoxyguanosine monophosphate (dGMP), thymidine triphosphate (dTTP), thymidine diphosphate (dTDP), thymidine monophosphate (dTMP), deoxyuridine triphosphate (dUTP) ), Deoxyuridine diphosphate (dUDP), deoxyuridine monophosphate (dUMP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP), deoxycytidine monophosphate (dCMP), deoxyxanthosine Triphosphate (dX P), deoxyxanthosine diphosphate (dXDP), deoxyxanthosine monophosphate (dXMP), deoxyinosine triphosphate (dITP), deoxyinosine diphosphate (dIDP), and deoxyinosine monophosphate (dIMP) However, it is not limited to these.

  Preferred nucleotide coenzymes are, for example, ATP, ADP, GTP, GDP, TTP, TDP, UTP, UDP, CTP, CDP, XTP, XDP, ITP, IDP, dATP, dADP, dGTP, dGDP, dTTP, dTDP, dUTP, Examples include, but are not limited to, dUDP, dCTP, dCDP, dXTP, dXDP, dITP, and dIDP.

  In some cases, for example, a substituent having a coloring ability may be bound to these nucleotide enzymes so that the amount of change in step (3) described later can be easily detected.

Examples of the nucleoside portion of the first nucleotide coenzyme in the positive reaction of step (2) of the method according to the present embodiment include those described above for the nucleotide coenzyme, and preferably adenosine, inosine, guanosine, and deoxy Adenosine etc. are mentioned, More preferably, adenosine is mentioned.
In addition, the first nucleotide coenzyme which is a phosphoric acid thereof may be a monophosphate, a diphosphate, or a triphosphate. In one embodiment, the first nucleotide coenzyme may be dephosphorylated by glycerol kinase. From the viewpoint of being oxidized, triphosphorus oxide is preferable. Specific examples of the first nucleotide coenzyme include ATP, GTP, TTP, UTP, CTP, XTP, ITP, dATP, dGTP, dTTP, dUTP, dCTP, dXTP, and dITP in addition to the specific examples described above. Is mentioned.

Examples of the nucleoside moiety of the second nucleotide coenzyme in the reverse reaction of the step (2) of the method according to the present embodiment include those described above for the nucleotide coenzyme, preferably inosine, guanosine, adenosine, and deoxy Guanosine etc. are mentioned, More preferably, inosine is mentioned.
Further, the second nucleotide coenzyme which is a phosphoric acid thereof may be a monophosphoric acid oxide or a diphosphoric acid oxide. In one embodiment, the phosphoric acid is phosphorylated by glycerol kinase. Is preferred. Specific examples of the second nucleotide coenzyme include ADP, GDP, TDP, UDP, CDP, XDP, IDP, dADP, dGDP, dTDP, dUDP, dCDP, dXDP, and dIDP in addition to the specific examples described above. Can be mentioned.

The preferred nucleoside moieties of the first nucleotid coenzyme and the second nucleotide coenzyme used in the method according to the present embodiment are not particularly limited as long as they are different from each other. For example, adenosine, inosine, guanosine, or deoxyadenosine Etc. For example, the combination of the nucleoside part of the first nucleotide coenzyme and the nucleoside part of the second nucleotide coenzyme includes adenosine and inosine, guanosine and adenosine, deoxyadenosine and guanosine, deoxyadenosine and deoxyguanosine, deoxyadenosine and inosine, Or inosine and adenosine are mentioned. More preferably, the nucleoside portion of the first nucleotide coenzyme is adenosine and the nucleoside portion of the second nucleotide coenzyme is inosine.
In one embodiment of the present invention, preferably, the first nucleotide coenzyme is ATP and the second nucleotide coenzyme is IDP.

  The cycling reaction in step (2) of the method according to the present embodiment is performed, for example, as follows.

For example, in the presence of glycerol kinase, an excessive amount of nucleoside triphosphate as the first nucleotide coenzyme and nucleoside diphosphate as the second nucleotide coenzyme, specifically 0.1 mmol / L or more and 12 mmol / L or less More preferably, 0.2 mmol / L or more and 6 mmol / L or less is prepared, and in order to maintain the pH during the reaction, a pH buffering agent is added at 20 mmol / L or more and 200 mmol / L or less to obtain a reaction solution. Examples of the pH buffer include phosphate buffer, Tris-HCl buffer, and Good buffers such as PIPES, MES, and HEPES.
The pH at the time of the reaction may be appropriately selected under conditions that allow the reaction to proceed efficiently. Usually, the pH is adjusted to around neutrality between pH 5.0 and pH 9.0, such as magnesium chloride, which is an activator of glycerol kinase. For example, a metal salt of 0.5 mmol / L or more and 20 mmol / L or less is added. Then, the reaction solution is preheated to 25 ° C. or more and 42 ° C. or less, more preferably around 37 ° C., and the nucleoside triphosphate and nucleoside diphosphate produced in step (1) and optionally step (1a) Compared to glycerol (product of step (1)) in a trace amount (for example, 1/10 or less molar amount of nucleotide coenzyme, which is the lesser of nucleoside triphosphate and nucleoside diphosphate) and optionally glycerol-3 -The reaction may be initiated in the presence of phosphoric acid (product of step (1a)).

In a general enzyme cycling reaction, the cycling rate depends on the amount of enzyme, and it is known that the smaller the Michaelis constant (Km value) for the substrate, the higher the sensitivity can be obtained with a small amount of enzyme. Therefore, as a measure of the amount of glycerol kinase to be added, an enzyme unit (unit of 1 unit or more, preferably 2 times or more of the larger Km value (mmol / L) of glycerol and glycerol-3-phosphate per 1 mL of the reaction solution) : U), may be added. The upper limit is not particularly limited as long as it can be substantially added. The Km value of glycerol kinase with respect to glycerol and glycerol-3-phosphate can be determined by a known method. In addition, said addition amount is an example and is not limited to these.
For example, Candida mycoderma-derived glycerol kinase is reported to have a Km value of 0.015 mmol / L for glycerol and 0.08 mmol / L for glycerol-3-phosphate (J Biol Chem 249, 2562-2566, 1974), the enzyme is preferably added in an amount of 0.08 U / mL or more according to the above-mentioned standard for the amount of enzyme added, but is not limited thereto.
Step (2) can be carried out with one kind of glycerol kinase, but it can also be carried out with two or more kinds of glycerol kinases having different origins.

Step (3) of the method according to the present embodiment will be described below.
In the step (3), any one or more of the first nucleotide coenzyme, the dephosphorylation oxide of the first nucleotide coenzyme, the second nucleotide coenzyme, and the phosphorylation of the second nucleotide coenzyme The change amount can be appropriately detected using a method capable of measuring each detection target. For example, the change amount can be detected by measuring a signal change amount corresponding to the change amount of each detection target.
The amount of change to be detected may be a decrease in the first nucleotide coenzyme or the second nucleotide coenzyme, or an increase in the dephosphorylation oxide of the first nucleotide coenzyme or the phosphorylation of the second nucleotide coenzyme. Often, from the viewpoint of simplicity of detection, it is preferably an increased amount of the dephosphorylation oxide of the first nucleotide coenzyme or the phosphate of the second nucleotide coenzyme.
As a method for detecting the amount of change of each detection target or the amount of change of a signal corresponding thereto, a known HPLC method or the like can be used. In the case of using the HPLC method, a chelating agent or the like is added to terminate the enzyme reaction, and then a reverse phase column or the like is appropriately selected to quantitate the detection target before and after the reaction. Information on HPLC analysis is readily available from resin manufacturers.

  In addition, step (3) of the method according to the present embodiment includes: a detection enzyme that uses the dephosphorylation oxide of the first nucleotide coenzyme by a positive reaction as a substrate but does not use the second nucleotide coenzyme as a substrate. It may be used to detect a signal change amount that changes with an increase in the dephosphorylation amount of the first nucleotide coenzyme due to a positive reaction. Alternatively, phosphorylation of the second nucleotide coenzyme by the reverse reaction using a detection enzyme that does not use the first nucleotide coenzyme as a substrate but uses the phosphate of the second nucleotide coenzyme by the reverse reaction as a substrate. You may detect the signal variation | change_quantity which changes with respect to the increase amount of a thing.

  For example, when ITP, GTP or CTP is used for the first nucleotide coenzyme and ADP is used for the second nucleotide coenzyme, it is a phosphate of the second nucleotide coenzyme by an enzyme that can specifically detect only ATP. Only ATP can be detected. The specificity of the nucleotide coenzyme may be appropriately selected with reference to literature information. For example, among the synthases belonging to the enzyme number EC6, many enzymes that specifically detect only ATP have been reported. For example, NAD synthase derived from E. coli (EC 6.3.1.5) is ATP. Since it is reported that it does not act on nucleotide coenzymes other than those, these enzymes can be used as detection enzymes.

  Thus, in step (3) of the method according to the present embodiment, detection is appropriately performed while paying attention to the combination of the first nucleotide coenzyme and the second nucleotide coenzyme in consideration of the specificity of the glycerol kinase to be used. What is necessary is just to select the enzyme for use. The specificity for coenzyme may be affected not only by the origin of the enzyme but also by conditions such as the amount of coenzyme and the amount of enzyme added, and the same specificity as the results reported in the literature is obtained. In some cases, it is desirable to actually confirm the specificity when selecting an enzyme for detection.

  In one embodiment of the present invention, when the amount of change to be detected is ADP, adenosine diphosphate-dependent glucokinase (EC 2.7.1.147) can be preferably used as the detection enzyme, and more preferably Pyrococcus furiosus-derived adenosine diphosphate-dependent glucokinase (EC 2.7.1.147) can be used. When ADP-dependent glucokinase is used, the product AMP or glucose-6-phosphate can be quantified by a known method.

In one embodiment, the amount of glucose-6-phosphate is changed in the presence of thio-NADP, thio-NAD, NADP and NAD coenzyme, glucose, and glucose-6-phosphate dehydrogenase (G6PDH) in the presence of ADP. For example, it can be measured as an absorbance change amount at 340 nm based on reduced NAD (P). Further, a coenzyme analog such as thio-NAD (P) can be used instead of NAD (P).
As an example, the measurement of the amount of change in absorbance based on reduced NAD (P) is, for example, nitrotetrazolium blue (Nitro-NB), 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2 , 4-Disulfophenyl) -2H-tetrazolium, sodium salt (WST-1), and 2- (4-iodophenyl) -3- (2,4-dinitrophenyl) -5-disulfophenyl) -2H- In the presence of a hydrogen acceptor such as tetrazolium, sodium salt (WST-3): using 1-methoxy PMS as an electron carrier; or forming a formazan dye using the enzyme diaphorase; by measuring visible absorbance Can also be quantified. These compounds are commercially available and can be easily obtained. Moreover, it can also detect in combination with a fluorescent reagent or a luminescent reagent.

  Alternatively, the amount of glucose-6-phosphate change can be quantified with high sensitivity by an enzyme cycling method using G6PDH (Japanese Patent No. 3036708). The step of detecting the amount of change of these changing signals may be performed simultaneously with the cycling reaction in the method according to the present embodiment, or may be performed separately.

Step (4) of the method according to the present embodiment will be described below.
In step (4), a known method can be used as a method for calculating the activity of autotaxin in the sample based on the amount of change detected in step (3). For example, in the enzyme cycling reaction in step (2), the dephosphorylation of the first nucleotide coenzyme produced by the forward reaction and the phosphate of the second nucleotide coenzyme produced by the reverse reaction are proportional to time. Increase. Therefore, in order to calculate the amount of the test substance in the sample, the reaction time of the enzyme cycling reaction in step (2) is specified (for example, 5 minutes to 7 minutes after the start of the reaction), and the concentration and activity are known. The amount of the test substance in the sample can be calculated by measuring the amount of change in the signal corresponding to the change in the calibrator using the reference substance (calibrator) as a control.

  To calculate the amount of autotaxin activity in the sample in step (4) of the method according to the present embodiment, specifically, for example, the following procedure is performed.

  Prior to the enzyme cycling reaction in the step (2), the autotaxin reaction in the step (1) is performed for a certain period of time, the autotaxin activity is stopped by an operation such as heat treatment, and a part or all of the autotaxin reaction is subjected to the enzyme cycling reaction. For the calibrator, glycerol kinase or glycerol-3-phosphate, which is a substrate of glycerol kinase or its phosphate, can be used. Enzyme activity is a quantity that represents the catalytic capacity per unit time, that is, the amount of substance change. In this example, glycerol and / or glycerol-3-phosphate produced by the autotaxin reaction increases in proportion to time. . Therefore, autotaxin activity can be calculated by dividing the amount of glycerol and / or glycerol-3-phosphate produced by the reaction time of autotaxin.

When only glycerol, which is a product of the autotaxin reaction, is detected, the enzyme cycling reaction in step (2) and the autotaxin reaction in step (1) can be performed simultaneously. In such a case, since the amount of glycerol produced in the step (1) increases in proportion to time, the phosphorylation and desorption of the nucleotide coenzyme produced in the enzyme cycling reaction of the step (2) using this glycerol. It is known that the amount of phosphorus oxide increases in a quadratic function without being proportional to time. Therefore, if autotaxin with known enzyme activity is used for the calibrator, the autotaxin activity in the sample can be easily calculated. Autotaxin used for calibrator use may be obtained by purifying serum or by a known genetic recombination method. In addition, even when autotaxin cannot be used as a calibrator, if a substance having a known concentration such as glycerol or glycerol-3-phosphate is used and the cycling rate k _c in the enzyme cycling reaction alone is measured in advance, The enzyme activity can also be determined by calculation as follows:
Enzyme activity (glycerol production per unit time) = 2p / (k _c · t ² )
(Where t = reaction time, p = product concentration from reaction start to time t (calculated from absorbance change))

In one aspect, the measurement method of the present embodiment is:
(1) a step of contacting a sample with lysophosphatidylglycerol, and a step of producing glycerol and lysophosphatidic acid from lysophosphatidylglycerol; (1a) contacting a lysophospholipase with the treatment product obtained in step (1); Treating lysophosphatidic acid to produce glycerol-3-phosphate;
(2) a glycerol kinase that catalyzes a forward reaction of glycerol-3-phosphate from glycerol and its reverse reaction in the presence of adenosine triphosphate (ATP), and uses ATP in the forward reaction; Glycerol kinase, ATP, and IDP using inosine diphosphate (IDP) in the reverse reaction are brought into contact with the treated product obtained in steps (1) and (1a), and the following formula (II)
Carrying out the cycling reaction of
(3) Presence of thio-NADP, thio-NAD, NADP, and NAD coenzyme, glucose, and glucose 6-phosphate dehydrogenase using ADP-dependent glucokinase (EC 2.7.1.147) A step of detecting an increased amount of ADP in step (2); and (4) a step of calculating autotaxin activity of the sample based on the increased amount of ADP detected in step (3);
including. Each step can be performed as described above.

  The present embodiment also relates to a clinical specimen testing method including the above-described method for measuring autotaxin activity. As such a method, for example, a test method using a body fluid such as blood, cerebrospinal fluid, pleural effusion, ascites, urine or the like as a clinical sample can be cited. Even a test method implemented in a point-of-care test (POCT) apparatus equipped with a system for detecting autotaxin activity by a method for measuring autotaxin activity and optionally equipped with a sample collection system and / or a sample processing system Good.

A further embodiment is a composition for measuring autotaxin activity in a sample,
(A) lysophosphatidylglycerol,
(B) Glycerol kinase, which catalyzes a forward reaction and its reverse reaction to produce its phosphate from glycerol in the presence of a nucleotide coenzyme. In the forward reaction and the reverse reaction, different nucleoside moieties are used. Glycerol kinase using a nucleotide coenzyme having,
(C) a first nucleotide coenzyme;
(D) a second nucleotide coenzyme that differs in nucleoside moiety from the first nucleotide coenzyme;
A composition comprising

The composition according to this embodiment further includes:
Lysophospholipase;
Detection enzyme that uses dephosphorylate of first nucleotide coenzyme as substrate, but does not use second nucleotide coenzyme as substrate, or second nucleotide coenzyme that does not use first nucleoside coenzyme as substrate A detection enzyme using a phosphorous oxide as a substrate; and any one of thioNADP, thioNAD, NADP and NAD coenzymes, glucose, and glucose 6-phosphate dehydrogenase;
Any one or more of these may be included.

  About each component in said composition, it is as having demonstrated with the method which concerns on this Embodiment.

  If the composition which concerns on this Embodiment is used, the measuring method of the autotaxin activity which concerns on said this Embodiment, for example can be implemented.

The composition according to the present embodiment may be a reagent kit for measuring autotaxin activity. The “reagent kit” in the present embodiment may be divided into a plurality of reagents, or may be combined into one. For example, the composition according to the present embodiment may be appropriately divided into two or three or more reagents to form a reagent kit. For example, each of the components involved in the above steps (1) to (4) can be made into a reagent kit appropriately divided into one or two or more reagents.
Specifically, for example: a reagent in which ATP, glucose, NADP, and ADP-dependent glucokinase are assigned to one reagent, and lysophosphatidylglycerol, IDP, glucose-6-phosphate dehydrogenase, and glycerol kinase are assigned to the other reagent A reagent kit in which lysophosphatidylglycerol, ATP, glucose-6-phosphate dehydrogenase and ADP-dependent glucokinase are assigned to one reagent, and IDP, NADP, glucose and glycerol kinase are assigned to the other reagent It can also be.
Further, for example, a detection enzyme that uses thio-NADP, thio-NAD, NADP, or NAD, glucose, and a dephosphorylated oxide of the first nucleotide coenzyme as a substrate, but does not use the second nucleotide coenzyme as a substrate; One nucleotide coenzyme is not used as a substrate, but a composition containing a detection enzyme that uses a phosphate of the second nucleotide coenzyme as a substrate can be appropriately distributed to the above two or more reagent kits. .
In one embodiment, preferably, the composition containing ATP, glycerol kinase and IDP can be divided into two reagent kits as appropriate. Furthermore, NADP, ADP-dependent glucokinase, and glucose-6-phosphate dehydrogenase may be appropriately distributed into two reagent kits.
Each component may be included redundantly in a plurality of divided reagents.

  The present embodiment also relates to a clinical specimen measuring apparatus for measuring autotaxin activity, which includes the composition for measuring autotaxin activity. As such a device, for example, a composition for measuring autotaxin activity as described above, such as a device represented by a self-blood glucose measuring device, using body fluid such as blood, cerebrospinal fluid, pleural effusion, ascites, urine, etc. as a clinical sample. And a point-of-care test (POCT) apparatus including a sample collection system and / or a sample processing system.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.

Example 1 Measurement of autotaxin activity in serum 1

(1) Measurement of glycerol of known concentration by glycerol kinase cycling [Reaction solution 1]
50 mM PIPES buffer (pH 7.0)
10 mM glucose 1 mM NAD
10 mM magnesium chloride 1 u / mL ADP-dependent glucokinase (Asahi Kasei Pharma: ADP-HKPII)
1 u / mL glucose-6-phosphate dehydrogenase 0.5 mmol / L deoxy ATP
20u / mL Glycerol kinase (Asahi Kasei Pharma: GKZ)

The reaction solution 1 was pre-warmed at 1 mL and 37 ° C. 0.025 mL of 0.2 mmol / L glycerol solution was added, and 0.02 mL of IDP solution prepared to 20 mmol / L was further added to start the enzyme cycling reaction. A reagent blank was prepared by adding physiological saline instead of the glycerol solution and performing the same operation. For each of the glycerol solution and physiological saline, the absorbance at 340 nm accompanying the production of reduced NAD 3 minutes and 5 minutes after the start of the reaction was read, and the amount of change in absorbance per 2 minutes was determined. 0.0151.

(2) Autotaxin reaction Lysophosphatidylglycerol (LPG (manufactured by Sigma)) was weighed so as to be 10 mmol / L, and dissolved in 0.5% Triton X-100 Normal serum (purchased from Bizcom Japan) 1 volume To the solution, 0.2 mol / L Tris-HCl buffer solution 0.2 solution, 5 mmol / L magnesium chloride 0.072 solution, and LPG solution 0.4 solution were added, and the reaction was started at 37 ° C. (total) Immediately after the reaction, 30 minutes and 60 minutes later, 0.025 mL was sampled and added to the reaction solution 1 used in (1), and the same operation as in (1) was performed to start the reaction. The absorbance after 3 minutes and 5 minutes was read out, and the amount of change in absorbance per 2 minutes was determined, and the amount of change in absorbance was 0.0400, 0.0442 and 0.0475, respectively. It increased in accordance with the heating time of the Qing.

(3) Calculation of autotaxin activity The calculation method will be described below, taking as an example the case where the heating time of serum is 30 minutes. Since the amount of change in absorbance for 30 minutes is determined by (0.0442-0.0400), the glycerol concentration in the liquid containing serum and LPG is 0.2 (mmol / L) × (0.0442-0.400). ) / (0.0713-0.0151) = 0.0149947 (mmol / L). Since this corresponds to the amount of change in glycerol concentration per 30 minutes, in order to convert to the enzyme activity in serum, it is sufficient to divide this amount of change by time and apply a coefficient for liquid volume correction. Calculated by the formula. Enzyme activity in serum = (14.947 (μmol / L) / 30 (min.)) × 1.672
= 0.833U / L
Similarly, the enzyme activity in the serum when the serum was heated for 60 minutes was calculated to be 0.744 U / L. The amount by which 1 μmol of substrate changes per minute was defined as 1 U (unit).

Example 2 Activity measurement of recombinant autotaxin

(1) Acquisition of recombinant autotaxin cDNA is purchased from ORIGEN, and a human autotaxin gene is acquired according to a conventional method (see, for example, J. Biol. Chem. 283,7776-7789, 2008), CHO cells (ATCC (Obtained from American type culture collection).

(2) Activity measurement (non-cycling)
[Reaction solution 2-1]
100 mmol / L Tris-HCl (pH 8.0)
5mmol / L magnesium chloride

[Reaction solution 2-2]
50 mmol / L PIPES buffer (pH 7.0)
1mmol / L ATP
0.03% 4-aminoantipyrine 0.02% N, N-bis (4-sulfobutyl) -3-methylaniline, disodium salt (TODB)
5 U / mL peroxidase (derived from horseradish, manufactured by Sigma)
2U / mL Glycerol kinase (Asahi Kasei Pharma: GKZ)
10 U / mL glycerol-3-phosphate oxidase (Asahi Kasei Pharma: GPOSP)

It was examined whether the recombinant autotaxin can use lysophosphatidylglycerol (LPG) as a substrate. The principle of activity measurement is as follows.
Glycerol produced by the action of autotaxin is converted to hydrogen peroxide by the action of GKZ and GPOSP, and peroxidase is added to this in the presence of a chromogen and quantified as absorbance at 550 nm.
First, 20 mmol / L LPG was prepared using 0.2% Triton X-100.
The LPG concentration in the reaction solution 2-1 was adjusted to 0.5, 1, 2, 5 and 10 mmol / L, respectively. This was dispensed into a 0.25 mL test tube, pre-warmed at 37 ° C., 0.025 mL of 0.1 mg / mL recombinant autotaxin was added, and the mixture was reacted at 37 ° C. for 20 minutes.
Subsequently, 0.75 mL of the reaction liquid 2-2 previously heated to 37 ° C. was added, and the absorbance at 550 nm was read 5 minutes after the addition.
The relationship between the substrate concentration and the absorbance is shown in FIG. It was shown that the recombinant autotaxin also acts on LPG and its enzyme activity is dependent on the substrate concentration. Further, assuming that the molecular extinction coefficient of TODB was 3,8000 L / mol · cm, the activity of 0.1 mg / mL recombinant autotaxin when the LPG concentration was 10 mmol / L was calculated to be 0.0195 U / mL. Therefore, when converted to activity per weight, it was 0.195 U / mg.

Example 3 Quantitativeness of recombinant autotaxin [Reaction solution 3-1]
100 mmol / L Tris-HCl (pH 8.0)
5 mmol / L Magnesium chloride 15 mmol / L LPG (0.15% Triton X-100)

[Reaction solution 3-2]
Same as reaction liquid 2-2 in Example 2

Example 2 confirmed that recombinant autotaxin acts on lysophosphatidylglycerol (LPG). Therefore, reaction solution 3-1 was prepared so that LPG was 15 mmol / L, and the quantitative property of the recombinant autotaxin was examined. That is, reaction solution 1 was dispensed into a 0.25 mL test tube, pre-warmed at 37 ° C., and recombinant autotaxin at concentrations of 0.01, 0.02, 0.05, 0.1, and 0.2 mg / mL 0.025 mL of each was added and reacted at 37 ° C. for 20 minutes. Subsequently, 0.75 mL of the reaction solution 3-2 previously heated to 37 ° C. was added, and the absorbance at 550 nm after 5 minutes from the addition was read. The relationship between the substrate concentration and the absorbance is shown in FIG. Absorbance increased approximately in proportion to enzyme concentration.

Example 4 Combined Use of Lysophospholipase [Reaction Solution 4-1]
100 mmol / L Tris-HCl (pH 8.0)
5 mmol / L Magnesium chloride 0.25 mol / L Sodium chloride 10 mmol / L LPG (0.1% Triton X-100)
2mmol / L deoxy ATP

[Reaction solution 4-2]
100 mM PIPES buffer (pH 6.5)
10 mM glucose 1.3 mM thio-NAD (manufactured by Oriental Yeast Co., Ltd.)
12 mM magnesium chloride 1.5 u / mL ADP-dependent glucokinase (Asahi Kasei Pharma: ADP-HKPII)
1.5 u / mL glucose-6-phosphate dehydrogenase 5.5 mmol / L IDP
27 u / mL Glycerol kinase (Asahi Kasei Pharma: GKZ)
1u / mL lysophospholipase (Asahi Kasei Pharma: LYPL)

The above reaction solutions were prepared. 0.005, 0.01, 0.015, and 0.025 mL of serum with respect to 0.25 mL of the reaction solution 4-1 (the amount of solution using physiological saline so that the amount of sample added becomes 0.025 mL) Was added) and reacted at 37 ° C. for 10 minutes. At this time, a sample blank to which no LPG was added was used for each serum amount. Subsequently, 0.75 mL of reaction solution 4-2 was added, and the absorbance at 400 nm was monitored. The amount of change in absorbance after 2 to 7 minutes from the addition of the reaction solution was calculated, and the same amount of change in each sample blank was subtracted.
Separately from this, a reagent was prepared by removing lysophospholipase (LYPL) from the reaction solution 4-2, and the same operation was performed.
The results are shown in FIG. By adding LYPL, glycerol-3-phosphate is produced from phosphatidic acid, which is a product of the autotaxin reaction, and this becomes a substrate for the glycerol kinase cycling reaction. Therefore, compared with the case where LYPL is not added, approximately 2 Double sensitivity was obtained.

Example 5 Measurement of autotaxin activity in serum 2
As a conventional method, LPC was used as a substrate, and the method of measuring released choline was compared with the method of the present invention using five types of human serum (purchased from Bizcom Japan).

(Conventional method)
[Comparative reaction solution 1]
100 mM Tris-HCl (pH 9.0)
500 mM NaCl
5 mM magnesium chloride 5 mM calcium chloride 2 mM lysophosphatidylcholine (derived from egg yolk lecithin: manufactured by Sigma)
0.05% Triton X-100

[Comparative reaction solution 2]
50 mmol / L PIPES buffer (pH 7.0)
0.03% 4-aminoantipyrine 0.02% N-ethyl-N (2-hydroxy-3-sulfopropyl) -3-methylaniline, sodium salt (TOOS)
5 U / mL peroxidase (derived from horseradish, manufactured by Sigma)
10 U / mL choline oxidase (manufactured by Asahi Kasei Pharma: COD)

0.02 mL of each serum was dispensed into 0.1 mL of the above comparative example reaction solution 1, and an enzyme reaction was performed at 37 ° C. for 3 hours. Thereafter, 2 mL of Comparative Example Reaction Solution 2 was added, and after heating at 37 ° C. for 5 minutes, the absorbance at 555 nm was measured. For each sample, a sample blank that was not subjected to an enzyme reaction at 37 ° C. for 3 hours was used as a sample blank, and the absorbance was subtracted.

(Method of the present invention)
[Reaction solution 5-1]
100 mmol / L Tris-HCl (pH 8.0)
5 mmol / L Magnesium chloride 0.25 mol / L Sodium chloride 10 mmol / L LPG (0.1% Triton X-100)

[Reaction solution 5-2]
100 mM PIPES buffer (pH 6.5)
10 mM glucose 1 mM NADP (manufactured by Oriental Yeast Co., Ltd.)
12 mM magnesium chloride 1.5 u / mL ADP-dependent glucokinase (Asahi Kasei Pharma: ADP-HKPII)
1.5u / mL glucose-6-phosphate dehydrogenase 4mmol / L IDP
100 u / mL Glycerol kinase (manufactured by Asahi Kasei Pharma: GKZ)

[Reaction solution 5-3]
10 mM deoxy ATP

0.02 mL of serum was added to 0.1 mL of the reaction solution 5-1, and the mixture was heated at 37 ° C. for 15 minutes. In addition, a sample blank was not heated. Thereafter, 1 mL of the reaction solution 5-2 was added, and 0.05 mL of the reaction solution 5-3 was further added to start the enzyme cycling reaction. After the addition of the reaction solution 5-3, the change in absorbance at 340 nm after 3 to 5 minutes was measured, and the measured value of each specimen blank was subtracted. The measurement results of both methods are shown in FIG. The correlation with the conventional method was good, and the autotaxin reaction of this method was 15 minutes compared to 3 hours of the conventional method, indicating that autotaxin can be measured in a shorter time.

  According to the present invention, autotaxin is not affected by endogenous autotaxin substrates such as LPC and free choline or products thereof, and is not affected by autotaxin substrates such as LPC which increase after blood collection. It has the industrial applicability that it can provide a method and a composition for measurement of measuring activity. The present invention is particularly useful in the fields of medicine, medicine, disease diagnosis, specimen testing, reagents and the like.

Claims (22)

A method for measuring autotaxin activity in a sample comprising:
(1) A step of bringing a sample into contact with lysophosphatidylglycerol, and a process for producing glycerol and lysophosphatidic acid from lysophosphatidylglycerol;
(2) Glycerol kinase, which catalyzes a forward reaction and a reverse reaction of glycerol from its glycerol in the presence of a nucleotide coenzyme. In the forward reaction and the reverse reaction, different nucleoside moieties are used. A treatment obtained in step (1) comprising a glycerol kinase using a nucleotide coenzyme having, a first nucleotide coenzyme, and a second nucleotide coenzyme having a nucleoside moiety different from that of the first nucleotide coenzyme The following formula (I):
Carrying out the cycling reaction of
(3) Any of the first nucleotide coenzyme, the dephosphorylation oxide of the first nucleotide coenzyme, the second nucleotide coenzyme, and the phosphorylation of the second nucleotide coenzyme in step (2) Detecting one or more changes; and (4) calculating the autotaxin activity of the sample based on the changes detected in step (3);
Measuring method including Simultaneously with step (2) or prior to step (2), the following steps are further performed:
(1a) A step of bringing lysophospholipase into contact with the treated product obtained in step (1) and performing a treatment for producing glycerol-3-phosphate from lysophosphatidic acid;
The measurement method according to claim 1, wherein step (2) is a step of bringing glycerol kinase into contact with the treated product obtained in steps (1) and (1a).   The step (3) is a step of detecting a dephosphorylated oxide of the first nucleotide coenzyme or an increased amount of a phosphorylated oxide of the second nucleotide coenzyme. Measuring method.   The nucleoside portion of the first nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine. The measuring method according to any one of claims 1 to 3.   The nucleoside portion of the second nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine. The measuring method according to any one of claims 1 to 4.   The measurement method according to claim 1, wherein the first nucleotide coenzyme is adenosine triphosphate (ATP) and the second nucleotide coenzyme is inosine diphosphate (IDP).   Step (3) is: a detection enzyme that uses the dephosphorylation oxide of the first nucleotide coenzyme as a substrate but does not use the second nucleotide coenzyme as a substrate; or does not use the first nucleoside coenzyme as a substrate, An enzyme for detection using a phosphate of the second nucleotide coenzyme as a substrate; an increase in dephosphorylation of the first nucleotide coenzyme by a forward reaction or a second nucleotide coenzyme by a reverse reaction The measuring method according to claim 1, which is a step of detecting an increase amount. The dephosphorylation oxide of the first nucleotide coenzyme or the phosphate of the second nucleotide coenzyme is adenosine diphosphate (ADP);
Step (3) is a step of detecting a change in signal that changes in response to an increase in ADP in the presence of glucose, using ADP-dependent glucokinase (EC 2.7.1.147). The measuring method in any one of Claims 1-7.   Step (3) further comprises thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (thioNAD), nicotinamide adenine dinucleotide phosphate (NADP), and nicotinamide adenine dinucleotide ( The measurement method according to claim 8, which is a step of detecting a change amount of a signal that changes in response to an increase amount of ADP in the presence of any coenzyme of NAD) and glucose 6-phosphate dehydrogenase. A method for measuring autotaxin in a sample comprising:
(1) a step of contacting a sample with lysophosphatidylglycerol, and a step of producing glycerol and lysophosphatidic acid from lysophosphatidylglycerol; (1a) contacting a lysophospholipase with the treatment product obtained in step (1); Treating lysophosphatidic acid to produce glycerol-3-phosphate;
(2) a glycerol kinase that catalyzes a forward reaction of glycerol-3-phosphate from glycerol and its reverse reaction in the presence of adenosine triphosphate (ATP), and uses ATP in the forward reaction; Glycerol kinase, ATP, and IDP using inosine diphosphate (IDP) in the reverse reaction are brought into contact with the treated product obtained in steps (1) and (1a), and the following formula (II)
Carrying out the cycling reaction of
(3) Using adenosine diphosphate (ADP) -dependent glucokinase (EC 2.7.1.147), thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (thioNAD) ), Nicotinamide adenine dinucleotide phosphate (NADP), and nicotinamide adenine dinucleotide (NAD) coenzyme, glucose and glucose 6-phosphate dehydrogenase in the presence of glucose 6-phosphate dehydrogenase increase in step (2) Detecting the amount; and (4) calculating the autotaxin activity of the sample based on the increased amount of ADP detected in step (3);
Measuring method including   A clinical specimen testing method comprising the measurement method according to claim 1. A composition for measuring autotaxin activity in a sample,
(A) lysophosphatidylglycerol,
(B) Glycerol kinase, which catalyzes a forward reaction and its reverse reaction to produce its phosphate from glycerol in the presence of a nucleotide coenzyme. In the forward reaction and the reverse reaction, different nucleoside moieties are used. Glycerol kinase using a nucleotide coenzyme having,
(C) a first nucleotide coenzyme;
(D) a second nucleotide coenzyme that differs in nucleoside moiety from the first nucleotide coenzyme;
A composition comprising:   The composition according to claim 12, further comprising (a1) lysophospholipase.   The nucleoside portion of the first nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine. 14. The composition according to claim 12 or 13, wherein   The nucleoside portion of the second nucleotide coenzyme is one of adenosine, guanosine, thymidine, uridine, cytidine, xanthosine, inosine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, deoxycytidine, deoxyxanthosine and deoxyinosine. The composition according to any one of claims 12 to 14.   The composition according to any one of claims 12 to 15, wherein the first nucleotide coenzyme is adenosine triphosphate (ATP) and the second nucleotide coenzyme is inosine diphosphate (IDP). Detection enzyme that uses dephosphorylate of first nucleotide coenzyme as substrate but does not use second nucleotide coenzyme as substrate, or second nucleotide coenzyme that does not use first nucleoside coenzyme as substrate Enzyme for detection using phosphorous oxide as a substrate,
The composition according to any one of claims 12 to 16, further comprising:   18. The composition of claim 17, wherein the detection enzyme is adenosine diphosphate (ADP) dependent glucokinase (EC 2.7.1.147) and further comprises glucose. Any of thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (thioNAD), nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide (NAD);
Glucose 6-phosphate dehydrogenase;
The composition according to any one of claims 12 to 18, further comprising: A composition for measuring autotaxin activity in a sample,
(A) lysophosphatidylglycerol,
(A1) lysophospholipase;
(B) A glycerol kinase that catalyzes the forward reaction and the reverse reaction of producing glycerol-3-phosphate from glycerol in the presence of adenosine triphosphate (ATP), and in the forward reaction, adenosine triphosphate ( Glycerol kinase utilizing ATP) and utilizing inosine diphosphate (IDP) in the reverse reaction;
(C) ATP;
(D) IDP;
(E) adenosine diphosphate (ADP) dependent glucokinase (EC 2.7.1.147);
(F) glucose;
(G) any one of thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide adenine dinucleotide (thioNAD), nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide (NAD);
(H) glucose 6-phosphate dehydrogenase;
A composition comprising:   A reagent kit for measuring autotaxin activity, comprising the composition according to any one of claims 12 to 20.   A clinical specimen measuring device for measuring autotaxin activity, comprising the composition according to any one of claims 12 to 20.