JPH06135997A - Purification and activity measurement of inositol triphosphate receptor - Google Patents

Abstract

PURPOSE:To provide a method for purifying the inositol triphosphate receptor existing on microsomes in cells and acting as a calcium ion-releasing channel, and to provide the method for measuring the activity of the inositol triphosphate receptor with the same. CONSTITUTION:The method for purifying the inositol triphosphate receptor comprises purifying by subjecting a specimen containing the inositol triphosphate receptor to an affinity chromatography using a carrier bound to an antibody recognizing the receptor. The activity measuring method comprises measuring the activity of the inositol triphosphate receptor on the basis of the amount of calcium ions passed through a lipid double membrane containing the inositol triphosphate receptor purified by the above method, thereby permitting to measure the activity of the receptor in high accuracy.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for purifying inositol triphosphate receptor and a method for measuring activity. More specifically,
The present invention relates to a method for purifying an inositol triphosphate receptor that acts as a calcium ion release channel and a method for measuring the activity of the receptor.

[0002]

2. Description of the Related Art The inositol triphosphate receptor is a calcium ion channel which exists on the endoplasmic reticulum and releases calcium stored in its lumen into the cytoplasm in an inositol triphosphate-dependent manner. This activity is regulated by various factors in cells, for example Supattapone.
Et al. Reported that phosphorylation of the inositol triphosphate receptor contained in the membrane fraction of rat cerebellum by the phosphorylating enzyme phosphokinase A suppresses the receptor channel activity. (Proc. Natn. Acad. Sc
i. USA. Vol.85. 8747-8750, 1988). Regarding the separation and purification of the inositol triphosphate receptor, in addition to the above documents, the present inventors have vigorously studied, and the details have been clarified by the present inventors (Metabolism Vol. 28. Extra number p141-148, 1991). That is, a method for purifying the inositol triphosphate receptor by a method such as gel filtration has been reported using a microsomal fraction of mouse cerebellum, which is considered to be rich in receptors, and solubilizing it. On the other hand, the physiological activity of the inositol triphosphate receptor is measured by an antagonist or activator for the inositol triphosphate binding site of the inositol triphosphate receptor (that is, an enhancer or inhibitor of Ca ²⁺ release). Is the measurement required when developing
As such an activity measuring method, many methods have heretofore been proposed in which microsomes, which are biological membranes, are mainly used and the release of calcium ions in the microsomes is used as an index. Further, as reported by Supattapone et al., A method of reconstructing an inositol triphosphate receptor by the lipid membrane method and measuring its receptor activity is also known.

[0003]

However, there are some problems in the purification method and activity measurement of the inositol triphosphate receptor in the above-mentioned prior art. That is, in the purification method, in the in vivo fraction rich in receptors,
It is known that there is diversity, that several purification steps are required, and that denaturation of the receptor protein occurs during the purification process. On the other hand, in the method for measuring the activity of the receptor, since the partially purified diverse receptor proteins are used as described above, there is a problem that the measurement sensitivity is poor and the measurement values are not reliable.
There has been a demand for an inositol triphosphate receptor activity measuring method having excellent sensitivity and accuracy. The present invention has been made to solve the above problems, and the present inventors have proposed a method for easily obtaining a highly purified inositol triphosphate receptor, and highly accurate inositol triphosphate receptor activity. We have conducted intensive research to develop a method that can measure. As a result, it was found that the receptor can be separated and purified from the sample containing the inositol triphosphate receptor in one step, and by using it to reconstitute the liposome, the inositol triphosphate receptor activity can be increased. The present invention has been completed by finding that the measurement can be performed with high accuracy. That is, an object of the present invention is to provide a method for purifying the inositol triphosphate receptor and a method for measuring the activity of the receptor.

[0004]

Means for Solving the Problems The purification method of the present invention, which was made to solve the above-mentioned problems, comprises the steps of treating a sample containing an inositol triphosphate receptor with a carrier to which an antibody recognizing the receptor is bound. It consists of separating and purifying by affinity chromatography used. In particular, as the antibody, an antibody that recognizes the C-terminal portion of the inositol triphosphate receptor is preferably used. Further, the method for measuring the activity of the inositol triphosphate receptor of the present invention uses a lipid bilayer membrane containing the inositol triphosphate receptor purified by the above-mentioned method, and measures the amount of calcium ion passing through the membrane. It consists of measuring phosphate receptor activity.
In particular, it is preferable to use liposomes as the lipid bilayer membrane.

The present invention has the above-mentioned constitution, and in the purification method of the present invention, a carrier to which an antibody recognizing the inositol triphosphate receptor is bound is used. The antibody used here may be either a polyclonal antibody or a monoclonal antibody, and such an antibody can be prepared according to a conventional method. Further, the antibody may recognize any site of the inositol triphosphate receptor, but preferably an antibody that recognizes the terminal site of the inositol triphosphate receptor, particularly the C-terminal site. used.
That is, the C-terminal portion of the inositol triphosphate receptor easily binds to an affinity carrier to which an antibody that recognizes the terminal portion is bound, and is easily released from the carrier in the eluate due to structural factors. Therefore, the inositol triphosphate acceptor with a high degree of purification can be obtained while simplifying the purification step. As an example of the method for preparing the antibody used in the present invention, C of inositol triphosphate receptor is used.
Explaining how to prepare an antibody that recognizes the terminal site,
First, a peptide corresponding to the C-terminal portion of the inositol triphosphate receptor is synthesized. This peptide is based on the amino acid sequence of the inositol triphosphate receptor which has already been clarified by the present inventors (Nature, Vol. 342, 32, 1989), and the peptide corresponding to the C-terminal site is commonly used. It can be synthesized using the peptide synthesis method. The peptide thus obtained is combined with a carrier for imparting antigenicity (eg, serum albumin, serum globulin, cytoglobulin, hemoglobin, etc.) to prepare an immune antigen. The obtained immunizing antigen is administered to an experimental animal with an adjuvant such as Freund's complete adjuvant to immunize, and blood is collected from the immunized experimental animal, and serum is separated using an appropriate means such as salting out or centrifugation. By doing
A polyclonal antibody that recognizes the C-terminal portion of the inositol triphosphate receptor can be obtained. A monoclonal antibody that recognizes the C-terminal portion of the inositol triphosphate receptor can also be prepared according to a conventional method. For example, a mouse spleen cell and a myeloma cell immunized with the above-mentioned immunizing antigen can be prepared using polyethylene glycol or the like. Cell fusion is performed using a cell fusion promoter. The fused cells are selected for hybridomas using an appropriate selection medium, then antibody production is checked and positive hybridomas are cloned. By culturing the antibody-producing hybridoma strain thus obtained in an appropriate medium, a monoclonal antibody that recognizes the C-terminal portion of the inositol triphosphate receptor can be obtained. More specifically, the method reported by the present inventors (J. Neurochem., Vol. 51, 1724-1730, 198).
8; Nature, Vol. 342, 32-38, 1989) and the like.

Next, a conjugate of an antibody that recognizes the inositol triphosphate receptor and a carrier is prepared. As a carrier,
Any carrier conventionally used as a carrier for affinity chromatography can be used, and sepharose gel is preferably used. The binding between the antibody and the carrier can be carried out by a conventionally known conventional method for affinity chromatography, and examples thereof include a chemical binding method using BrCN and a physical binding method.

The thus obtained antibody-bound carrier is brought into contact with a sample containing the inositol triphosphate receptor to bind the inositol triphosphate receptor to the carrier, and then the carrier is brought into contact with an appropriate eluent to give the inositol triphosphate receptor. The purified inositol triphosphate receptor can be obtained by eluting the phosphate receptor. This operation can be performed by either a column method or a batch method, but the column method is preferable from the viewpoint of purification efficiency. In order to show the above steps more specifically, a method for purifying an inositol triphosphate receptor using a carrier to which an antibody that recognizes the C-terminal portion of the inositol triphosphate receptor is bound will be described.
A suitable buffer solution (eg, Tris-hydrochloric acid) containing a stabilizer or the like is used for organs / tissues (eg, cerebellum, cerebrum, pancreas, gastric mucosa, endocrine cells, etc.) that are thought to be rich in inositol triphosphate receptors. Buffer solution) and then centrifuged to separate the supernatant containing the inositol triphosphate receptor. This supernatant is brought into contact with the carrier to which the above-mentioned antibody is bound to bind the inositol triphosphate receptor to the carrier. Next, the obtained carrier is packed in a column, and the peptide solution corresponding to the C-terminal portion of the above-mentioned inositol triphosphate receptor is flown through the column as an eluent to remove the inositol triphosphate receptor bound to the carrier. Is removed to give a highly purified inositol triphosphate receptor. According to the above method
The desired inositol triphosphate receptor can be purified in one step without causing protein denaturation.

Further, the method for measuring the activity of the inositol triphosphate receptor of the present invention uses the inositol triphosphate receptor highly purified by the above method, and uses the inositol triphosphate receptor in the lipid bilayer membrane. The amount of Ca ²⁺ that passes through the membrane is reconstructed, and the activity of the receptor is measured from the value. It is preferable to use a liposome as the lipid bilayer membrane. The above lipid bilayer membrane can be prepared according to a conventional method.For example, in order to reconstitute an inositol triphosphate receptor into a liposome, phospholipids such as phosphatidylcholine and phosphatidylserine, A lipid film is prepared by dissolving it in an organic solvent (eg, chloroform) and evaporating the solvent from the resulting solution under an inert gas stream or under reduced pressure. The obtained lipid film was suspended in an appropriate buffer solution, and the inositol triphosphate receptor was added thereto, incubated sufficiently, and then purified using a means such as dialysis to give the inositol triphosphate receptor. Can be obtained.

The amount of calcium ions passing through the membrane is measured, for example, by adding radioactive calcium ion and inositol triphosphate to one of the membranes and measuring the amount of calcium ions transferred to the opposite side of the membrane. . In the case of liposome, it is carried out by adding radioactive calcium ion and inositol triphosphate to the outer liquid of the membrane and measuring the amount of calcium ion transferred to the inside of the liposome. Note that calcium ions labeled with an appropriate label may be used instead of the radioactive calcium ions. The activity of the inositol triphosphate receptor is determined based on the thus measured amount of calcium ion passing through the membrane. At this time, if there is an antagonist or activator for the inositol triphosphate binding site of the inositol triphosphate receptor together with the above calcium ions in the measurement system, the amount of calcium ions passing through the membrane decreases or increases, Screening for antagonists or activators for the inositol triphosphate receptor can be performed. In addition, by using this system, inositol triphosphate can be quantified.

In the above-described method for measuring the activity of inositol triphosphate receptor, a lipid bilayer membrane in which purified inositol triphosphate receptor is reconstructed is used, and the activity of inositol triphosphate receptor is measured. It is possible to measure easily and accurately. That is, publicly known literature (The Journal of
Biological Chemistry Vol.257, No. 16, 9372-9378,
1982 and Nature Vol. 342, 87-89, 1989), the method using a microsome or a planar lipid bilayer membrane uses calcium ion-incorporated samples to measure the release of calcium ions. Although the preparation of such a reagent is complicated, according to the method of the present invention, it suffices to add calcium ions or the like to the external liquid, so that the measurement can be performed by an extremely simple operation.

[0011]

Industrial Applicability According to the purification method of the present invention, a pure receptor protein can be obtained in one step from a sample containing inositol triphosphate receptor (particularly in vivo fraction rich in the receptor). Further, according to the measuring method of the present invention, the inositol triphosphate receptor can be easily measured, and it is useful for analysis and research on various physiological phenomena related to the receptor.

[0012]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples. Example 1 Inositol triphosphate receptor C-terminal peptide
Obtaining of polyclonal antibody The amino acid sequence of the inositol triphosphate receptor is known from the already known cDNA. This time, part 2
736th to 2747th amino acid sequence, that is, G
The HPPHMNVNPQQ peptide was prepared by conventional methods. 10 mg of the peptide was added to 1-ethyl-3-
(3-Dimethylaminopropyl) carbodiimide (ED
C) Combined with 10 mg BSA in the presence. The resulting mixture was stirred overnight and unreacted EDC or peptide was separated by gel filtration using a Sephadex G-50 column. The column was equilibrated with 50 mM ammonium acetate. The purified BSA-bound peptides were collected and dissolved in phosphate buffer. Next, 1 ml of BSA conjugate containing 1 ml of Freund's complete adjuvant and 200 μg of peptide was added to New Zealand white rabbits.
1 was subcutaneously administered for primary immunization. 3 weeks later 1 ml
Sensitize with Freund's incomplete adjuvant and 1 ml of BSA conjugate corresponding to 100 μg of peptide. This operation was repeated every two weeks until the antibody titer of the antiserum was saturated.
A polyclonal antibody against the inositol triphosphate receptor could be prepared by the above method.

Example 2 Preparation of Microsomal Fraction A mature ddY mouse was anesthetized and then decapitated to fragment the cerebellum. Tissue is 0.32M sucrose, 1mM ED
A mixture of TA, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin, 10 μM pepstatin A, 1 mM 2-mercaptoethanol, and 5 mM Tris-hydrochloric acid was mixed to a pH of 7.4. Next, homogenize with a glass-Teflon potter homogenizer at 850 rpm for 10 strokes. The supernatant was centrifuged at 12,000g for 15 minutes at 2 ° C. The residue was washed again under the same conditions. The resulting supernatant was then centrifuged at 105,000 g for 60 minutes at 2 ° C. to obtain the desired microsome fraction. The resulting microsomal fraction was 0.1 M sucrose, 1
pH 7.3 consisting of a mixture of mM EDTA, 0.2 mM 2-mercaptoethanol, 10 mM Tris-HCl.
Mix with Buffer A adjusted to 4% to give 1% CH
It was solubilized with APS.

Example 3 Inositol triphosphate receptor by immunoaffinity
Purification of the inositol triphosphate receptor polyclonal antibody obtained in Example 1 was performed by ammonium sulfate precipitation to obtain C
It was bound to NBr-activated Sepharose 4B beads. Next, for the purpose of isolating inositol triphosphate from the microsomal fraction obtained in Example 2, 1% CHAPS was used.
The buffer A containing solubilized microsomal fraction was incubated with the beads for 2 hours at 4 ° C. with agitation. Next, add the obtained beads to the column,
It was washed with 50 ml of buffer A and 1 mg of phosphatidylcholine / ml. Then, as the eluent, 1 mg
Buffer A containing phosphatidylcholine / ml with a peptide (peptide described in Example 1) concentration of 5
Using a solution adjusted to have a concentration of μM, this solution was passed through a column to separate a fraction containing an inositol triphosphate receptor, thereby obtaining a purified inositol triphosphate receptor.

Example 4 Reconstitution of Inositol Triphosphate Receptor into Liposomes A phospholipid solution (prepared to have phosphatidylcholine and phosphatidylserine in chloroform at a ratio of 3: 1) was prepared and obtained. The resulting mixture was dried on a thin film under a stream of nitrogen gas. Further, the obtained lipid film was treated with 1 mg / ml of buffer B (50 mM NaC).
Suspended to pH 7.4) and 1% CHAPS with a mixture of 1, 50 mM KCl, 20 mM Tris-HCl. Here, the inositol triphosphate receptor obtained in Example 3 was mixed with the lipid-containing solution prepared above to adjust the inositol triphosphate receptor to 5 μg / ml.
Next, after incubating the mixture of the protein, lipid and surfactant obtained above on ice for at least 20 minutes, 2
-Mercaptoethanol, 1 mM EDTA, 1 mM
It was dialyzed against 1000 times volume of buffer B containing EGTA. The buffer used for dialysis was replaced every 8 hours and dialyzed for 48 hours. Finally, the dialysate was changed to 1000 times the volume of buffer B containing 2 mM 2-mercaptoethanol, and the EDTA and EGTA used were removed, and 8
Dialysis was performed for an hour. The liposomes obtained as described above are centrifuged at 100,000 g for 20 minutes, and the supernatant is collected.
The target inositol triphosphate receptor could be reconstituted into liposomes.

[0016] Measurement of ⁴⁵ Ca ²⁺ influx of Example 5 ⁴⁵ Ca ²⁺ influx amount was measured by the method known Ferris et al and Nunoki et al. That is, the amount of ⁴⁵ Ca ²⁺ inflow was measured by using 25 μl of the liposome particle solution obtained in Example 4 as 2 mM 2-mercaptoethanol and 2 μCi of Ca.
^It can be measured by incubating a solution diluted with 75 μl of buffer B containing ²⁺ . For example, 3 for 1 minute
After incubation at 0 ° C., calcium ion conductance is measured under conditions where ⁴⁵ Ca ²⁺ enters the liposome when the calcium channel is opened with inositol triphosphate. Next, after further incubating at 30 ° C. for 1 minute, 0.3 mM CaCl ₂ , 5 mM MgS
After inhibiting the influx with 300 μl buffer B containing O ₄ , 1 mM CdCl ₂ and 100 μg / ml heparin,
400 μl of the mixture was added to 3 ml of a chlex100 column (Bio-Rad) adjusted with 200 mM sucrose, 20 mM Tris-HCl (pH 7.4, 25 ° C.) in advance, and the liposome particles were further washed with 5 ml of the same buffer, This can be done by measuring ⁴⁵ Ca ²⁺ contained in the liposome particles. When the inositol triphosphate receptor purified by the method of the present invention was used, excellent measurement accuracy and reproducibility were obtained.

Example 6 Inositol triphosphate receptor by immunoaffinity
In Purification Example 3 of Example 1, instead of the polyclonal antibody of inositol triphosphate receptor, a monoclonal antibody of inositol triphosphate receptor (J. Neurochem., Vol. 51, 1724-173).
The inositol triphosphate receptor was purified by the same method except that the monoclonal antibody prepared by the method described in 0, 1988 was used).

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Claims (4)

[Claims] 1. An inositol triphosphate receptor, which comprises purifying a sample containing the inositol triphosphate receptor by affinity chromatography using a carrier to which an antibody that recognizes the receptor is bound. Purification method. 2. The method for purifying an inositol triphosphate receptor according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody that recognizes the C-terminal portion of the inositol triphosphate receptor. 3. A lipid bilayer membrane containing an inositol triphosphate receptor purified by the method according to claim 1,
A method for measuring inositol triphosphate receptor activity, which comprises measuring inositol triphosphate receptor activity based on the amount of calcium ions passing through a membrane. 4. The method for measuring inositol triphosphate receptor activity according to claim 3, wherein the lipid bilayer membrane is a liposome.