JPS6034200A - Measurement of activity of gamma-glutamyl transpeptidase - Google Patents

Abstract

PURPOSE:To measure activity of gamma-glutamyl transpeptidase in high accuracy, by treating gamma-L-glutamyl-L-glutamic acid with gamma-glutamyl transpeptidase, treating liberated L-glutamic acid with L-glutamate oxidase. CONSTITUTION:gamma-L-Glutamyl-L-glutamic acid is used as a substrate to provide gamma- glutamyl group, it is treated with gamma-glutamyl transpeptidase so that L-glutamic acid is liberated. Liberated L-glutamic acid is treated with L-glutamate oxidase, an amount of consumption of oxygen caused by the reaction, or an amount of formation of hydrogen peroxide, ammonia, or alpha-ketoglutaric acid is detected, to measure activity of gamma-glutamyl transpeptidase. L-Glutamate oxidase used has high substrate specificity ot L-glutamic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to γ-glutamyl transpeptidase (EC
2, 8, 2, 2: Hereinafter abbreviated as "γ-GTPJ". Concerning a method for measuring activity. Specifically, L-glutamate oxidase (hereinafter sometimes abbreviated as rGLXDJ).
The present invention relates to a method for measuring γ-GTP activity using γ-GTP activity.

f-GTP is an enzyme involved in the metabolism of γ-glutamyl peptide in vivo, and has the effect of hydrolyzing r-glutamyl bebutide with glutathione and simultaneously transferring the r-glutamyl group to other peptides or amino acids. It is an enzyme. γ-GTP is an enzyme that is widely distributed in various tissues in the body, and measurement of r-GTP activity in body fluids, especially serum, is useful as a means of diagnosing liver diseases such as cirrhosis, alcoholic hepatitis, and liver cancer, and understanding their pathological conditions. It is important and widely implemented.

Conventional technology There are known methods for measuring r-GTP activity that use peptides such as glutathione as a substrate and use complicated methods such as chromatography and gas analysis. A commonly used method is to use a certain γ-glutamylamide as a substrate and quantify the amines liberated by the action of γ-GTP. Typical methods include (1) γ-glutamyl p
A method using °-nitroanilide as a substrate, and (2) γ
- A method using glutamyl-α-naphthylamide or r-glutamyl-β-naphthylamide as a substrate is known.

In method (1), p-nitroaniline liberated from the substrate by the action of γ-GTP can be quantified by direct colorimetric determination of yellow p-nitroaniline (1-1) or (1-2 ) I) - A method is adopted in which the nitroaniline is colored red with an aldehyde compound and then the color is compared. These methods have the disadvantage that the substrate concentration during the preparation of the reagent is limited because the substrate γ-glutamyl-p-nitroanilide is poorly soluble in water and has poor dissolution/dissolution stability. Furthermore, in the determination of liberated p-nitroaniline, method (1-1) is susceptible to the influence of serum components in colorimetric determination because this substance is yellow, and method (1-2) is It lacks practicality as a daily testing method because it requires complicated operations such as the need to strictly control the temperature during the color reaction.

In method (2), a- or β-naphthylamine released from the substrate by the action of γ-GTP can also be determined by colorimetrically fixing these amines as (2-1) diazonium salts. method, or (2-2)8
-Methyl-2-benzothiazolinone hydrazone and an oxidizing agent are used to develop a color and quantify the amount. These methods are not preferred as testing methods, not only because the operations are complicated, but also because the amines, which are the raw material for the substrate compound and the standard substance, have cancerous properties.

In addition to the above methods, as a method for measuring γ-GTP activity (8)
A method using amine oxidase is also known (JP-A-58-9698). That is, this method uses a γ-glutamyl-substituted methylamide compound as a substrate,
This is a method in which a substituted methylamine compound liberated by the action of γ-GTP is acted on with a corresponding amine oxidase, and the amount of oxygen consumed or the amount of hydrogen peroxide or ammonia produced accompanying the enzymatic reaction is quantified. This method is difficult to obtain a substrate, and the substrate specificity and stability of the amine oxidase used are not necessarily high (it is difficult to say that it is practical).

Note that, as a conventional method for measuring f-GTP activity, a method using a dipeptide as a substrate (r-glutamyl group donor) is not known, nor is a method using L-amino acid oxidase.

SUMMARY OF THE INVENTION The present inventor has disclosed that f-
As a result of intensive research on methods for measuring GTP activity, γ-L
-Glutamyl- monoglutamic acid and an r-glutamyl group receptor are used as substrates, and an extremely highly specific monoamino acid oxidase is applied to L-glutamic acid, which is liberated by the action of γ-GTP on this substrate. discovered that it is possible to measure γ-GTP activity by detecting the balance of substances associated with this reaction, and completed the present invention.

The present invention provides γ-
L-glutamyl-monoglutamic acid is used, and r
- Applying glutamyl transpeptidase, then releasing monoglutamic acid, and applying L-glutamate oxidase to detect the amount of oxygen consumed or the amount of hydrogen peroxide, ammonia, or α-ketoglutarate produced during the reaction. The present invention provides a method for measuring r-glutamyl transpeptidase activity, which is characterized by the following.

The enzymatic reaction in the method of the present invention is schematically shown below.

Detection treatment of r-L-glutamyl-L-glutamic acid + receptor enzyme reaction ■ The substrate of γ-GTP used in the present invention basically exists in nature, and the measured γ-GTP
The activity reflects γ-GTP activity in the true sense.

■The substrate is a safe substance and is easily available.

■The reaction using GLXD of the present invention is an oxidase reaction that is most widely used in clinical tests, etc.
For the measurement of enzymatic reactions, commonly used methods can be applied as is.

■ Since the above GLXD has extremely high substrate specificity for L-glutamic acid, is it possible that other amino acids coexist? However, only monoglutamic acid produced by the action of γ-GTP can be specifically measured.

(2) The above GLXD has high thermal stability, pH stability and storage stability, and is convenient for use as a reagent for measuring γ-GTP activity.

■The above GLXD has a wide action pH range, with an optimum pH of 7~
-8.5 and works efficiently near neutrality, making it easy to couple with the reaction system of γ-GTP whose optimum pH is in the same range.

Furthermore, the above-mentioned G L Therefore, γ−
It is also possible to perform two consecutive reactions or all the reactions simultaneously among the enzymatic reaction using GTP, the enzymatic reaction using G L XD, and the reaction for detecting hydrogen peroxide.

The substrates in the γ-glutamyl group transfer reaction of γ-GTP are an r-glutamyl group donor and a γ-glutamyl group acceptor.

The method of the present invention is based on γ-1. -Glutamyl-One feature lies in the use of L-glutamic acid as a substrate that donates an r-glutamyl group (γ-glutamyl group donor).

Various peptides or amino acids can be used as the γ-glutamyl group receptor. Usually glycylalanine is used, but in some cases other dipeptides such as glycylalanine, tripeptides such as glycylglycylglycine, and amino acids such as glycine, alanine, valine, leucine, and phenylalanine can also be used. .

Another feature of the method of the present invention is that monoglutamate oxidase, which is a monoamino acid oxidase with high substrate specificity, is used to quantify monoglutamic acid, which is liberated by the action of f-GTP.

The monoglutamate oxidase used in the present invention basically exhibits the action shown by the following reaction formula, and L-
Any enzyme that has high substrate specificity for glutamic acid (and has little effect on other amino acids) is sufficient.

A specific example of L-glutamate +02 + H20□α-ketoglutarate +NHB -1-H2O2L-glutamate oxidase is an enzyme discovered by the present inventor IC, and Streptomyces sp.
19X-119-6 (Strepto SP, X
-119-6; Kaikoken Bacteria No. 6560, ATCC89
848), Culture 1323-1828 (1988), Japanese Patent Application No. 57-112271, etc.; this enzyme is hereinafter referred to as "
It is also called "this enzyme". ) can be exemplified.

The characteristic properties of this enzyme are shown below.

This enzyme has extremely high substrate specificity for L-glutamic acid and has no substantial effect on other amino acids. That is, at pH 7.4, L-aspartic acid and L-asparagine show slight activity (L = 0.6-0.7% of the activity against glutamic acid), but other amino acids including L-glutamine and It has no effect on D-amino acids. Further, at pH 6.0, it does not substantially show activity against L-aspartic acid and L-asparagine.

Effects of this enzyme 1) H range is wide (pH 5-10), optimal 1) H is pH 7-8.5. In other words, it is an enzyme that acts efficiently near neutrality.

This enzyme is stable over a wide pH range. That is,
Under the condition of holding at 37°C for 160 minutes, pH is 5.5-10.
．． In the range of 5, under the conditions of holding at 45 °C for 15 minutes, I) In the range of 5 to 9.5, 60 °C1
Under the condition of holding for 15 minutes, each was stable in the pH range of 5.5 to 7.5.

The optimal temperature range for this enzyme's action is wide, from 30 to 60'C.

The optimum temperature for action is around 50°C.

The thermostability of this enzyme is very high. Sunawaji, pH5.5
It is stable up to 65°C, and even at 85°C it is stable at about 5°C.
Shows 0% residual activity. I) 50° for H7,5
It is stable up to 75°C and shows about 60% residual activity even at 75°C. Stable up to 45°C at pH 9.5,
It shows about 50% residual activity at 70°C.

The activity of this enzyme is inhibited by approximately 45% by parachloromercuribenzoate, but not by other common inhibitors.

An example of the method for producing this enzyme is detailed in Reference Examples.

(2) The assay method of the present invention basically consists of about one step of the two-step enzymatic reaction as described above. Specifically, (1) r-GTP to be measured is applied to a solution containing γ-L-glutamyl monoglutamic acid and an r-glutamyl group receptor, and γ-
A reaction in which a glutamyl group is transferred to the above-mentioned receptor and L-glutamic acid is released;
This is a reaction in which hydrogen peroxide, ammonia and α-ketoglutaric acid are produced by the action of D.

The conditions for each reaction can be adjusted so that each enzyme reacts appropriately, but if the reaction conditions for (1) and (2) are in the same range, they can be continued in the same reaction system without changing the reaction conditions. This is convenient because the reaction can be carried out with

Since r-GTP is optimal 1) H is 7.5-9.0, it is compatible with the reaction conditions of GL X D, and (1) and (2)
These reactions can be carried out continuously in the same reaction system.

Specifically, the reaction solution (1) contains receptors such as γ-L-glutamyl monoglutamic acid and glycylglycine, and may further contain a buffer and magnesium chloride as necessary. Trishydroxymethylaminomethane, barbiturates, triethanolamine, glycine, phosphates, phosphates, carbonates can be used as buffers. The concentration of each substrate in the reaction solution is not limited, but usually 2 to 50 mM of γ-L-glutamyl monoglutamic acid and 50 to 250 mM of γ-glutamyl group acceptor.
Used at a concentration of mM. The reaction is carried out under conditions suitable for the γ-GTP reaction, that is, usually at pH 7.5-9.0.15-
It is carried out under conditions in the range of 40°C.

The reaction (2) is carried out by bringing the reaction solution of (1) into contact with GLXD. As for the reaction conditions, it is not necessary to particularly change the conditions in (1), but the reaction pH, reaction temperature, etc. may be changed as necessary. The reaction is usually carried out at a pH of 5 to 9.
It is carried out under arbitrary conditions in the range of 15 to 60°C.

In addition, reactions (1) and (2) can be performed using a dry system (chemistry area, 35f4
1.278-280 (1981)).

In the reaction, GLXD is used as a soluble or immobilized enzyme.

Immobilized enzymes can be prepared using general methods such as entrapment methods (lattice type, microcapsule type, etc.), carrier bonding methods (covalent bonding, ionic bonding, physical adsorption, etc.), and cross-linking methods (``immobilized enzyme'', etc.).
Partly edited by Chibata, March 20, 1975, published by Kodansha)
It can be manufactured by

For example, immobilization carriers include cellulose (cellophane, filter paper, etc.), acetyl cellulose derivatives, various ion-exchanged celluloses, polysaccharides such as carrageenan; proteins such as collagen and gelatin; polystyrene, polyaminostyrene, photocurable resins (ethylene Hydrophilic photocurable resins having sexually unsaturated groups (Special Publication No. 55-40, Japanese Patent Publication No. 1983)
-20676), synthetic organic polymer substances such as ion exchange resins, glass (porous glass beads), and inorganic substances such as silica. When immobilizing enzymes, these carriers are used as they are, or after being subjected to pretreatment such as introduction of appropriate functional groups, introduction of spacers, and activation of functional groups. The carrier and the enzyme can be bonded by a diazo method, a peptide method, an alkylation method, or a method using a crosslinking reagent (glutaraldehyde, hexamethylene diisocyanate, etc.).

Immobilization can also be achieved by cross-linking enzymes using a cross-linking reagent such as the one described above.

Immobilized enzymes can be in membrane, gel, granule, chip, powder, or
It is prepared in a form depending on the mode of use, such as microcapsule form, tube form, container form, fiber form, and holofiber form. For example, it can be prepared in the form of a membrane to serve as an enzyme electrode, prepared in granules and packed with column 1 to form a reactor-type biosensor, or prepared as an enzyme reagent layer of a multilayer analysis film together with a coloring reagent, a binder such as gelatin, etc. can be used.

Detection The γ-GTP activity according to the present invention is measured by detecting the amount of oxygen consumed or the amount of hydrogen peroxide, ammonia, or α-ketoglutaric acid produced in the two-step reaction. Note that the detection method may be a rate method or an endpoint method.

(]) Oxygen consumption The amount of oxygen consumed is usually detected using the Warburg pressure method (Biochemistry Experiment Course 5. Enzyme Research Methods, pp. 35-41, Tokyo Kagaku Doujin, August 20, 1975) or Oxygen electrode method (Oxygen measurement using electrode method.

Edited by Bunji Hagiwara, ■Kodansha, published November 20, 1977)
carried out by.

As the oxygen electrode, for example, a Clark type oxygen electrode is used, which has a platinum electrode as a cathode, a lead electrode as an anode, uses a potassium hydroxide solution as an internal liquid, and has an oxygen-permeable Teflon membrane attached to the surface of the platinum cathode. be able to.

Oxygen electrodes can also be used as enzyme electrodes. t
For example, it can be used as an electrode-attached enzyme electrode in which the immobilized enzyme of GLXD or the soluble enzyme covered with a semipermeable membrane is attached closely to the electrode membrane of a Clark type electrode, and the immobilized enzyme is filled. It can also be used as a reactor-type enzyme electrode consisting of one reactor such as a column and an oxygen electrode attached separately ([Region of Chemistry, Special Issue TA 4, Biomaterial Science m 1
fil+)I), 69-79. April 20, 1982,
■Published by Nankodo; [Chemical Industry J June 1982 issue, pl
), 491-496; "Ion electrode and enzyme electrode",
Edited by Shu Suzuki.

pp, 65-106. Published by Kodansha, November 1, 1981; "Region of Chemistry" Vol. 36, No. 5, pp.

343-849. (1982)).

(2) Amount of hydrogen peroxide produced The amount of hydrogen peroxide produced can be detected using known methods such as electrochemical methods, spectroscopic methods, chemiluminescence methods, and chemistries (Japanese Patent Publication No. 56-82919, etc.)1. It can be carried out.

The most common electrochemical method is to use a hydrogen peroxide electrode, but hydrogen peroxide can also be produced by reacting hydrogen peroxide with iodine ions and measuring the decrease in the activity of the iodine ions using an iodine ion electrode. can be detected.

As the hydrogen peroxide electrode, for example, a Clark type hydrogen peroxide electrode using a platinum electrode can be used. It can also be used as an enzyme electrode similar to an oxygen electrode.

Spectroscopic methods include ■ peroxidase method, ■ catalase method, ■ chemical methods using Ti (1% ll / 4- (2-pyridylazo) resorcinol system, V (Vl / xylenol orange system, etc.) Organic Synthetic Chemistry - Color 9F71. 659-666 (1981)),
■Method using glutathione/glutathione peroxidase system (Anal, 13iochem 76).

184-191 (1976)), etc. can be used.

The peroxidase method (2) is a method in which an oxidizable coloring agent is reacted with hydrogen peroxide in the presence of peroxidase or a substance exhibiting similar activity, and the reaction product, the dye, is quantified by absorbance measurement. As peroxidase, an enzyme derived from horseradish is usually used.

As a coloring agent, for example, 0-dianisidine, 4-methoxy-1-naphthol or 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) alone; 4-aminoantipyrine and phenol, p-chloro Combinations of phenolic compounds such as phenol, 2,4°6-dribromophenol; 4-aminoantipyrine and N.

Combinations with aniline compounds such as N-dimethylaniline and N,N-diethylaniline; combinations of 4-aminoantipyrine and toluidine compounds such as N,N-diethyl-m-)luidine; or 3-methyl- A combination of 2-benzothiazolinone hydrazone and N,N-dimethylaniline, etc. can be used.

Note that the coloring reaction by the peroxidase method is often carried out at a pH around neutrality. For example, in the case of 4-aminoantipyrine/phenol, the color reaction is I) H7,
It will be held at 5. Therefore, since the reaction by GLXD is usually carried out near neutrality, the reaction can be carried out continuously in the same reaction system without changing the pH during the coloring reaction. Furthermore, enzymatic reactions using γ-GTP can also be carried out in the same pH range. That is, r-
The three reactions of the enzymatic reaction using GTP, the enzymatic reaction using GLXD, and the coloring reaction can be performed continuously in the same reaction system.

For the catalase method (2), hydrogen peroxide is reacted with alcohol (such as methanol) in the presence of catalase, and the aldehyde (such as formaldehyde) produced is introduced into a coloring system and the absorbance of the produced dye is measured. A method of quantifying using dehydrogenase, etc. can be adopted. Formaldehyde can be colored by reacting it with ammonia or by reacting it with hydrazone (4-amino-8-hydrazino-5-mercapto-1) in the presence of an oxidizing agent (sodium periodate, red blood salt (potassium ferricyanide), etc.). ,,2,4-
triazole, etc.) (Japanese Patent Publication No. 19288/1983).

As the chemiluminescence method, a method (Japanese Unexamined Patent Publication No. 58-47484) in which hydrogen peroxide is reacted with luminol in the presence of red blood salt and the amount of chemiluminescence is measured with a photon counter is suitable. In the same manner, peroxidase may be used instead of red blood salt (JP-A-57-71899, JP-A-57-71400), and isoluminol or pyrogallol may be used instead of luminol. .

A method for determining this is to react homovanillic acid with hydrogen peroxide in the presence of peroxidase and measure the fluorescence intensity. As the fluorescent reagent, p-hydroxyphenylacetic acid or diacetylfluorescin (Japanese Patent Application Laid-open No. 48656/1983) may be used.

(3) Ammonia production amount Ammonia can be analyzed by methods such as the microkelpool method, the Nessler method, the indophenol method, the ninhydrin method, and the phenosafranine method. In addition, analysis can be performed using electrochemical analysis methods, such as a method in which ammonium ions are analyzed using a cation-selective electrode, or a method in which ammonia gas is analyzed using an ammonia gas electrode consisting of a glass electrode equipped with a hydrophobic gas-permeable membrane. Yotl
. Furthermore, ammonia may be detected using an enzyme electrode that is a combination of these electrodes and C, L x D.

(4) Amount of α-ketoglutaric acid produced.

a-Ketoglutaric acid is reacted with 8-methyl-2-benzothiazolinone hydrazone (MBTH) and the absorbance of the product is measured, and 2.4-dinitrophenylhydrazine is reacted and the absorbance of the product is measured. How to do, 0-
A method of reacting with phenylenediamine and measuring the absorbance of the product, and other known methods can be used.

EXAMPLE Hereinafter, an example and L
- The present invention will be explained more specifically using reference examples showing a method for producing glutamate oxidase. The present invention is not limited to these examples. In Examples and Reference Examples, the enzyme unit ([Jnit) in ps is expressed as rUJ.

Example 1 (1) Preparation of reagents Substrate solution 42 IRg of glycylglycine, 7 mfl of γ-L-glutamyl-monoglutamic acid and 1 OIIIg of magnesium chloride hexahydrate were mixed with 5 ml of 02M Tris-HCl buffer (
pH 8.5).

■γ-GTP standard solution γ-GTP (manufactured by Sigma, derived from bovine kidney, 6.2U/#+
g) 6.2 ml of 0.2 M l-lis-
It was dissolved in a hydrochloric acid buffer (pH 8.5) and diluted with the same buffer to prepare an enzyme solution of 50 to 1000 mU/*/.

■ Color reagent phenol 40Il 1g, 4-aminoantipinone 4
0mg and peroxidase (manufactured by Toyobo ■, derived from horseradish, 100 U711g) 6ay in 0.2M potassium phosphate buffer (p)l 7>4) 80ttt
Dissolved in l.

■GLXD solution Partially purified GLXD (12U/zy) 5q was dissolved in 50*t of 0.1M phosphate buffer (pl ('7.4) (
Approximately 1.2 U/gt).

(2) Procedure Take 0.1 ml of the substrate solution in a test tube, add 0.2a+t (10-200 mU) of γ-GTP standard solution,
After incubation for 20 minutes, the reaction was stopped by adding 20 μl of 25% IJ chloroacetic acid.

IM potassium phosphate buffer (p) 17.4 as reaction stop solution
) 01 layer t, 0.1 ml of GLXD solution and 0.5 wt of coloring reagent were added and incubated aerobically for 37 °C with shaking at 500 °C as a control.
The absorbance at nm was measured and a calibration curve shown in FIG. 1 was created.

Example 2 The same substrate solution as in Example 1 containing GLXD2U was poured into a polarographic oxygen electrode cuvette by circulating constant temperature water at 25°C, and 20 μl of γ-G was added to the cuvette.
A TP standard solution (12.5 to 200 mtJ) was injected, the oxygen consumption rate was measured, and the calibration curve shown in FIG. 2 was prepared.

Reference example 500g/ Erlenmeyer flask with 20' of wheat bran/and 1 part of water
Streptomyces sp.
119-6 (Feikoken Bacteria No. 6560) was inoculated and the temperature was 28°.
C. Inoculum was prepared by culturing for 7 days.

200 q of bran and 160*t of water were placed in each of 25 51-volume Erlenmeyer flasks, autoclaved at 120'C for 30 minutes, and then aseptically inoculated with the above-mentioned inoculum. After culturing at 28°C for 2 days, the temperature was lowered. The temperature was lowered to 208C and cultured for another two weeks.

After soaking the obtained culture in 87.51 water for 1 hour,
Filter and pass through diatomaceous earth to obtain crude enzyme liquid 84
I got 1. Add 5096 ammonium sulfate to this crude enzyme solution.
Add to saturation, collect precipitate by centrifugation, and reduce to 0.02
Dissolved in M acetate buffer (pH 5,5) 3.9e, 57°
Heated at C for 30 minutes. This heat-treated enzyme solution was heated to 56C.
After cooling, 2x fil of pre-chilled ethanol was added, and the resulting precipitate was collected by centrifugation, dissolved in 0.02M phosphate buffer (pH 7,4) 2e, and dialyzed overnight against the same buffer. . DEAE (diethylaminoethyl) was obtained by centrifuging and removing the precipitate generated during dialysis, and equilibrating the supernatant with the same new buffer solution.
- Passed through a cellulose column (8,5x50α) and eluted the adsorbed enzyme using the same buffer containing 035M NaCl. The eluted active fraction was collected and dialyzed overnight against 005M acetate buffer (p) (55) containing 005M saline. This dialysate was treated with a DEAE-Sepharose CL-6B (manufactured by Pharmanoa Fine Chemicals 71) column (2X
10z) and soak the adsorbed enzyme with salt 0.05-075
It was eluted by the linear granoent method of M. The eluted active fraction was collected, dialyzed and concentrated, and then separated using Sephadex G-200 (
Pharmacia Fine Chemicals Co.) Column (2,
Gel filtration was performed using 002M potassium phosphate buffer (pH 7) to collect and concentrate the active fraction.
, 4). This dialyzed fluid was centrifuged, the supernatant was microfiltered, and then freeze-dried to obtain a purified specimen of L-glutamate oxidase (specific activity: 55.IU/Elf protein).

Yield: 18.4%) 80 scraps 2 were obtained.

[Brief explanation of drawings]

FIG. 1 shows a calibration curve for f-GTP activity in Example 1. FIG. 2 shows a calibration curve for γ-GTP activity in Example 2. Patent Applicant (677) Yamasa Soy Sauce Co., Ltd.≦Kasakai 2 Written amendment to this procedure (voluntary) July 1, 1980 Manabu Shiga, Commissioner of the Patent Office ◆ ■ Indication of the case 1988 Patent Application No. 14'8484 No. 2 Name of the invention Method for measuring γ-glutamyl transpeptidase activity 3
Relationship with the person making the amendment Patent applicant address (zip code 288) 2-10 Shinseicho, Choshi City, Chiba Prefecture Telephone: 0479
(22)0095 Column 5 of detailed explanation of the invention in the specification, contents of amendment and R47,11 2) rGLXD on page 13, line 15 of the specification, 1 after J
Add this "(this enzyme)". 3) After rGL and XDJ on page 21, line 1 of the specification, “
(this enzyme)” will be added. 4) In the first line of page 25 of the specification, [(12U 1 mg per month) is corrected to ``(this enzyme; t2U, /y)j.'' 5) j' in the 16th line of page 25 of the specification -Add 1 (this enzyme) after GLXDJ. 6) 1 after “kindase” on page 28, line 1 of the specification
Add C-enzyme).

Claims (1)

[Claims] 1) γ-■5 as a substrate donating γ-glutamyl group
-Glutamyl-monoglutamic acid is used, and γ-
Glutamyl transpeptidase is allowed to act, and then L-glutamate oxidase is made to act on the released monoglutamic acid, and the amount of oxygen consumed or the amount of hydrogen peroxide, ammonia, or a-ketoglutarate generated during the reaction is measured. 1. A method for measuring γ-glutamyl transpeptidase activity, which comprises detecting γ-glutamyl transpeptidase activity. 2) L-glutamate oxidase has extremely high substrate specificity for L-glutamic acid, does not substantially act on amino acids other than L-glutamic acid, and is a highly stable enzyme according to claim 1. Measurement method. 8) L-glutamate oxidase has an optimum pH of 7~
The measuring method according to claim 1 or 2, wherein the enzyme has a concentration of around 8.5. 4) L-glutamate oxidase is an enzyme having stability such that its activity does not decrease until 65°C under conditions of holding at pH 5.5° for 15 minutes. Described measurement method.