JPH09262085A - New enzyme and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new enzyme effective for specifically hydrolyzing neuro- peptide and useful as a reagent for the investigation of the metabolism of neuro- peptide and peptide hormone and an agent for the treatment of nervous system diseases such as Alzheimer's disease. SOLUTION: This enzyme is separated from the cytoplasm of ray liver and has the following characteristics. (1) It is inert to polymer protein and specifically hydrolyzes a peptide containing 6-17 amino acid residues; (2) it is inactivated in the presence of a cheating agent; (3) the molecular weight is 76kDa (SDS- PAGE; under reducing condition); (4) PAS staining is negative; (5) isoelectric point is 4.9-5.0; (6) optimum pH is 7.6; (7) it has the N-terminal amino acid sequence of the formula. The enzyme can be purified by ammonium sulfate fractionation, ion-exchange chromatography and affinity chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel enzyme and a method for producing the same. Specifically, S under reducing conditions
3. Molecular weight by DS-PAGE is 76 kDa, isoelectric point 4.
9-5.0, optimum pH of 7.6, a new enzyme which does not act on a high molecular weight protein and has an activity of specifically hydrolyzing a neuropeptide having 6 to 17 amino acid residues, and It relates to a method for producing a novel enzyme, which comprises purifying rat liver cytoplasm by ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography. Due to its activity, the enzyme of the present invention is useful as a reagent for studying metabolism of neuropeptides and peptide hormones, or as a therapeutic drug for nervous system diseases such as Alzheimer's disease.

[0002]

2. Description of the Related Art Various physiologically active peptides have been revealed to act as intercellular signal transduction substances, and as peptide hormones, those involved in endocrine type signal transduction and local chemical Some function as mediators, and some function as neurotransmitters as mediators between nerve cells. Since both types of peptides are closely related to the functional expression of target cells, it is considered that the peptide turnover is also strictly controlled. Particularly in the case of local transmission such as in the nervous system, the turnover of transmitters is rapid, and it is an important subject to sort out the functions of degrading enzymes involved in it. As a degrading enzyme for peptide hormones and neuropeptides, prolyl oligopeptidase (EC 3.4.21.
26), neprilysin (EC 3.4.24.11), thimet oligopeptidase (EC
3.4.24.15), neurolysin (EC 3.4.24.
16) etc. are known, but there are some that are widely distributed in the body and some are localized in cells, and no clear conclusion has been reached.

With the increase of the elderly population, dementia is attracting attention as a nervous system disease. Among them, Alzheimer's disease is one of the most analyzed diseases, and it is suggested that peptidase may be involved in the production of β-amyloid protein, which is considered to be a substance that induces this disease. Thimet oligopeptidase is one of the candidates, and it was discovered as a metalloendopeptidase that is widely distributed in the body and exists mainly in the cytoplasm.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have conducted an earnest search for a new enzyme having the same activity as the above-mentioned enzyme, and as a result, found a new enzyme having the same activity in rat liver cytoplasm. It was That is, an object of the present invention is to provide a novel enzyme and a method for producing the same. Specifically, the molecular weight by SDS-PAGE under reducing conditions was 76 k.
Da, isoelectric point 4.9-5.0, optimum pH 7.6,
Does not act on high molecular weight proteins and has 6 or more amino acid residues 1
It is an object to provide a novel enzyme having an activity of specifically hydrolyzing a neuropeptide having 7 residues or less, and a method for producing the same. Due to its activity, the enzyme of the present invention is useful as a reagent for studying metabolism of neuropeptides and peptide hormones, or as a therapeutic drug for nervous system diseases such as Alzheimer's disease.

[0005]

[Means for Solving the Problems] The novel enzyme of the present invention has a molecular weight of 76 by SDS-PAGE under reducing conditions.
It has an isoelectric point of 4.9-5.0 and an optimum pH of 7.6.
It does not act on a high molecular weight protein and has an activity of specifically hydrolyzing a neuropeptide having 6 to 17 amino acid residues.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The novel enzyme of the present invention can be obtained from rat liver cytoplasm. That is, the liver is removed from the rat and the homogenized product is centrifuged. The obtained supernatant is further subjected to ultracentrifugation to obtain a cytoplasmic solution. Ammonium sulphate fractionation of cytosolic solution, ammonium sulphate saturation 35-6
A 0% precipitated fraction is obtained, which is dialyzed. The crude fraction after dialysis is fractionated with DEAE-cellulose to obtain an active fraction. Next, this is purified with a hydroxyapatite column, a sephacryl column, a blue sepharose column, and a MonoQ column, and a purified enzyme can be obtained by collecting fractions.

[0007]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

[0008]

Example 1 Purification of rat liver cytoplasm-derived metalloendopeptidase (1) Enzyme activity measurement The hydrolysis activity of the target enzyme was determined by the method of Serizawa et al. (J. Bi
ol. Chem., Vol.270, p2029, 1995).
That is, 0.01 mM 7-methoxycoumarin-3-carboxyl-3-phe
7-methoxycouma produced by decomposition using nylpropyl-Pro-Leu-Gly-Pro-Lys (DNP) NH ₂ (NovaBiochem) as a substrate
The time change of rin-3-carboxyl-3-phenylpropyl-Pro-Leu-OH was measured. The amount of enzyme capable of producing 1 μmol of the product per minute was defined as 1U.

(2) Cell Fraction and Ammonium Sulfate Fractionation Liver (410 g) was extracted from 28 female Wistar rats aged 8 to 12 weeks, which had been fasted overnight, and the liver (410 g) was removed and rinsed with physiological saline.
Double amount of 10 mM Tris / HCl (pH 7.8), 250 mM sucrose, 5 mM 2
-Mercaptoethanol, 0.1 mM PMSF (Phenyl methyl
Sulfonyl fluoride (Wako Pure Chemical Industries, Ltd.) was added and the mixture was homogenized with a potter-type homogenizer. Then 10,000 ×
g, centrifugation was performed for 10 minutes, and the supernatant was recovered. The collected supernatant was further subjected to ultracentrifugation at 100,000 xg for 1 hour, and the supernatant was collected to give a cytoplasmic solution. The cytosolic solution was subjected to ammonium sulfate fractionation, and an ammonium sulfate saturation degree 35-60% precipitation fraction was collected. Then a small amount of 10 mM Tris-HCl (pH 7.8), 0.1
mM ZnCl ₂ , 5 mM 2-mercaptoethanol, 0.1 mM P
It was dissolved in MSF and dialyzed against the same buffer for 24 hours.

(3) DEAE cellulose chromatography
ー Pre - dialyze the crude fraction with 10 mM Tris-HCl (pH 7.8), 5 mM
2-Mercaptoethanol, 0.1 mM ZnCl ₂ , 0.05% Br
The mixture was mixed with 120 g of DEAE-cellulose (Whatman) equilibrated with ij-35 (Wako Pure Chemical Industries) and stirred at 4 ° C for 2 hours. Then, DEAE-cellulose was separated and collected, and washed with the buffer solution used for equilibration. Then the column (inner diameter
50 mm). Flow rate 3.0 ml / min, NaCl concentration 0-0.
Fractionation was performed with a gradient elution of 3/6000 ml, and the active fraction was collected. The active fraction is 10 mM sodium phosphate (pH
6.8), 5 mM 2-mercaptoethanol, 0.05% Brij
It was diafiltered against -35, 0.1 mM ZnCl ₂ and finally concentrated to 100 ml to make a DE pool.

(4) Hydroxyapatite chromatograph
Fee DE pool with hydroxyapatite column (26mm x 2
0 cm, Bio-Rad), flow rate 1.0 ml / min
, Fractionated amount 15ml, phosphoric acid concentration 0.01-0.3M / 1
Fractionation was performed with a gradient elution of 500 ml. The hydrolytic activity of the fractions was measured, and the active fractions were combined and used as the HA pool.

(5) Sephacryl S-200 gel fill tray
The cation HA pool was concentrated to 9 ml by ultrafiltration and preliminarily adjusted to 10 mM Tri
s-HCl (pH 7.8), 5 mM 2-mercaptoethanol, 0.1 mM
It was applied to a Sephacryl S-200 column (Pharmacia) equilibrated with NaCl ₂ , 0.15M NaCl and 0.05% Brij-35, and separated and fractionated at a flow rate of 0.4 ml / min and a fractionation amount of 6 ml. The degradation activity of each fraction was measured, and the active fractions were combined and G
The pool was F.

(6) Blue Sepharose chromatography
-The GF pool was mixed with 10 mM Tris / HCl (pH 7.8), 5 mM 2-mercaptoethanol, 0.1 mM ZnCl ₂ , 0.05% Brij-35,
It was applied to a HiTrap Blue column (5 ml, Pharmacia) equilibrated with 0.1 M NaCl, and a flow-through fraction was collected.

(7) The flow- through fraction of the MonoQ FPLC blue column was further fractionated by the MonoQ column (Pharmacia). Separation conditions: flow rate 0.5 ml / min
Gradient elution with a NaCl concentration of 0.1-0.2 M / 30 minutes was performed to obtain a fraction volume of 0.5 ml. Combine the active fractions,
It was a purified enzyme. The yield of the purification process is shown in Table 1. Finally, 0.8 mg of purified enzyme with a specific activity of 4.7 U / mg was recovered.

[0015]

[Table 1]

[0016]

Example 2 Characteristics of the enzyme of the present invention (1) Molecular weight and isoelectric point The results of SDS-PAGE (reducing conditions) of the final purified product of the enzyme of the present invention are shown in FIG. Finally, the fraction collected by fractionation with MonoQ showed a single band, confirming that it could be isolated by this method. The molecular weight of the isolated enzyme is 76 kDa.
Met. In addition, SDS-PAGE under non-reducing conditions,
Since the purified enzyme showed a single band with almost the same mobility, it was confirmed that this enzyme was a single peptide chain. Furthermore, as a result of analyzing the isoelectric point using the Fast System (Pharmacia), the isoelectric point of this enzyme was 4.9.
It was -5.0.

(2) Optimal pH 10 mM Tris-hydrochloric acid buffer and 10 mM phosphate buffer
The activity was adjusted from 6.6 to 8.0 in 0.1 steps and the activity at each pH was measured. The results are shown in FIG. As a result, the optimum pH was 7.6 in both buffer solutions.

(3) Inhibitors for this enzyme The effects of various protease inhibitors on this enzyme were investigated. E-64 (Peptide Institute), PMSF, Pepstain, O-Phenanthrolin, ED as protease inhibitors
TA, N-ethylmaleimide (all Wako Pure Chemical Industries, Ltd.) was used. The measurement was carried out after adding EDTA alone and incubating for 1 hour. Table 2 shows the results.

[0019]

[Table 2]

As a result, the isolated enzyme of the present invention was found to be PMS.
Since it was not inhibited by F and pepstatin at all,
It was confirmed that it was not a serine protease or an aspartic protease. On the other hand, it was confirmed that the enzyme of the present invention is a metalloprotease because it was inhibited by a chelating reagent such as EDTA or O-phenanthroline. However, since it was not inhibited by E-64 but was inhibited by N-ethylmaleimide, it was suggested that the cysteine residue contained in the present enzyme is involved in some activity expression.

(4) Km Value and Ki Value for Synthetic Peptide The characteristic of the isolated enzyme of the present invention is that 7-m which is a synthetic substrate is used.
ethoxycoumarin-3-carboxyl-3-phenylpropyl-Pro-Leu-
K for Gly-Pro-Lys (DNP) NH ₂ (Nova Biochem)
The m value was measured. As a result, the Km value was 24.3 μM. In addition, using the same synthetic peptide as a substrate, dynorphin A (1-13) and N- [1- (RS) -carboxyl-3-phenylprop
The Ki value for [yl] -Ala-Ala-Phe-pAb was measured. As a result, Ki values were 0.327 μM and 4.07 μM, respectively.

(5) Substrate specificity The substrate specificity of the present enzyme for synthetic or natural peptides was measured. That is, using a synthetic or natural peptide as a substrate,
The decomposed product was analyzed by HPLC. As a substrate, QF
01 (Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys, Nova Biochem), QF02 (7-methoxycoumarin-3-carboxyl-3-p)
henylpropyl-Pro-Leu-Gly-Pro-Lys (DNP) NH ₂ , NovaBiochem, Bradykinin, Neurotensin, Substance P, Luteinizing hormone-releasing hormone (LHR)
H), angiotensin I, angiotensin II, dynorphin A, dynorphin A 1-13, dynorphin A 1-8, atrial natriuretic peptide (ANP)
(All Sigma) were used. After separation and fractionation, the cleavage position was identified by amino acid analysis. The results are shown in Table 3.

[0023]

[Table 3]

As a result, the enzyme of the present invention did not decompose high molecular proteins such as collagen, bovine serum albumin, casein, sheep IgG, myoglobin, transferrin and ovalbumin. On the other hand, synthetic low molecular weight peptides and neuropeptides were decomposed by the enzyme of the present invention. As a result of analysis, it was confirmed that the peptide chain that can serve as a substrate has 6 to 17 residues, and therefore this enzyme has an oligoendopeptidase activity that specifically decomposes neuropeptides.

(6) N-terminal amino acid sequence N-terminal amino acid sequence analysis of the isolated metalloendopeptidase was carried out using an automatic amino acid sequence analyzer (A477 type, Applied Biosystems) according to its protocol. The obtained N-terminal amino acid sequence is
It is shown in SEQ ID NO: 1 in the sequence listing.

[0026]

From the above results, the present invention provides a novel enzyme. The enzyme of the present invention has a molecular weight of 76 kDa by SDS-PAGE under reducing conditions, an isoelectric point of 4.9-5.0, an optimum pH of 7.6, does not act on a high molecular weight protein, and has 6 or more amino acid residues. It has the activity of specifically hydrolyzing neuropeptides of 17 residues or less. Due to its activity, the enzyme of the present invention is useful as a reagent for studying metabolism of neuropeptides and peptide hormones, or as a therapeutic drug for nervous system diseases such as Alzheimer's disease.

[0027]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence: Thr Leu Gly Lys Glu Leu Ala Ser Pro Leu Gln Ala Met Ser Ser Try 1 5 10 15

[Brief description of drawings]

FIG. 1 SDS-P under reducing conditions of the enzyme of the present invention
The migration pattern by AGE is shown.

[Explanation of symbols]

 Molecular weight marker protein 97kD: phosphorylase 78kD: transferrin 68kD: bovine serum albumin 55kD: glutamate dehydrogenase 35kD: glyceraldehyde dehydrogenase 29kD: carbonic anhydrase 22kD: soybean trypsin inhibitor 12.5kD:

FIG. 2 shows the optimum pH of the enzyme of the present invention in each buffer solution.

[Explanation of symbols]

 ●: 10 mM phosphate buffer ○: 10 mM Tris-hydrochloric acid buffer

Claims (2)

[Claims] 1. An enzyme having the following characteristics. (1) Activity 1: It does not act on high molecular weight proteins and specifically hydrolyzes peptides having 6 to 17 amino acid residues. (2) Activity 2: deactivates in the presence of a chelating agent. (3) Molecular weight: 76 kDa (SDS-PAGE; under reducing conditions) (4) PAS staining: negative (5) Isoelectric point: 4.9-5.0 (6) Optimum pH: 7.6 (7) N Terminal amino acid sequence: It has the sequence shown in SEQ ID NO: 1 in the sequence listing. 2. The method for producing an enzyme according to claim 1, wherein the rat liver cytoplasm is purified by ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography.