JP2714430B2 - Novel peptide having Na, K-ATPase inhibitory action - Google Patents

Description

The present invention relates to a novel peptide having a Na, K-ATPase inhibitory action, and more specifically, it has been found from pig duodenum.
The present invention relates to a novel peptide effectively used in the field of pharmaceuticals.

[Problems to be solved by conventional technology and invention]

Many researchers have approached biological materials with such factors since it was reported in the 1960s that new factors regulating Na diuresis were present in body fluids.

In the urine and blood of animals that received salt loading in subsequent studies
Na, suppresses K-ATPase activity, shows a cross-reaction with an antibody against digoxin, a specific inhibitor of Na, K-ATPase,
It was reported that substances with diuretic activity were present.

Na, K-ATPase is considered to be a receptor for digitalis in vivo, and this substance is an endogenous digitalis-like facto.
r, EDF).

Na, K-ATPase is a kind of enzyme that regulates the balance between Na ⁺ and K ⁺ , has an important function on renal function, heart function, and also has an important function in the brain, It is deeply involved in maintaining homeostasis.

For example, in the urine and blood of hypertensive patients, such substances are detected more frequently than normal persons, so that they have a strong correlation with hypertension, and furthermore, digitalis has a strong relationship with various diseases due to its effects on the heart and kidney. It has also attracted attention in terms of pathology.

It has been reported in the following literatures that EDF is present in human urine, human plasma, pig plasma, guinea pig heart and the like. However, this substance has not yet been isolated and its structure has been determined, and its substance is still unknown.

-Kramer HJ, Renal Physiol., 8 , 80 (1985)-Kelly RA, J. Biol. Chem., 260, 11396 (1985)-Hamlyn JM, Mepertension, 7 , 847 (1985)-Tamura M., J. Biol. Chem., 260 , 9672 (1985) ・ Fagoo M., Biochem. Biophys. Res. Commum., 129 , 553 (19
85) ・ Halperin JA, J. Biol. Chem., 260 , 646 (1988) ・ Goto A., Biophem. Biophys. Res. Commun., 154 , 847 (198)
8) [Means for Solving the Problems] The present inventors have discovered that a substance having a Na, K-ATPase inhibitory action is present in a porcine duodenal extract. Then, the substance was further purified and isolated, and the chemical structure of the obtained substance was clarified. As a result, the substance was found to be a novel peptide, and the present invention was completed.

That is, the present invention provides a primary structural formula (Where X is Arg or Gly, and when X is Arg, Y is Se
When r and X are Gly, Y is Gly. N) as indicated by
a, It provides a novel peptide having a K-ATPase inhibitory action.

 Hereinafter, the present invention will be described in detail.

The peptide of the present invention is extracted and purified from pig duodenum. The extraction and purification may be carried out by applying a conventionally known method, and for example, the following outline method can be shown.

First, a VIP fraction is prepared from pig duodenum. In order to obtain VIP fractions from pig duodenum, for example, Suzuki,
Araki, Tachibana; The method described in Pharmaceutical Magazine, 99 , 172 (1979) can be used. Next, the VIP fraction was subjected to various purification treatments such as CM-Sephadex column chromatography, SP-Sephadex column chromatography, and CM-cellulose column chromatography to change the pH and salt concentration of the eluate. And elute. The tracking of the active fraction during this period is performed by measuring the Na, K-ATPase inhibitory action. Active ingredients having Na, K-ATPase inhibitory action are collected.
Next, desirably, gel filtration is performed using Sephadex G or Biogel P, and then reverse phase chromatography is performed using a silica gel carrier modified with a hydrophobic group such as nucleosyl C ₁₈ or nucleosyl CN to obtain a hydrophilic polymer having a concentration gradient. Elute with an aqueous solution of an organic solvent, measure the activity, and collect the target fraction. Examples of the hydrophilic organic solvent include lower aliphatic alcohols such as ethanol, methanol, and propyl alcohol, and lower aliphatic nitriles such as acetonitrile, with acetonitrile being particularly preferred. In the extraction and purification described herein, the order of the operations may be appropriately changed.

The peptide of the present invention can be produced by extracting and purifying pig duodenum as a raw material, but can also be produced by synthesis using a solid phase method or a liquid phase method. Moreover, you may manufacture by a gene recombination technique.

The structural analysis of the peptide of the present invention may be performed as follows, as shown in Examples below.

 First, the amino acid composition is confirmed by amino acid analysis.

The peptide of the present invention has eight cysteines and S-
It is known that there are four S bonds.

That is, from the results shown in the examples of the present invention, in the primary structural formula, Cys at position 20 and Cys at position 49, and Cys at position 27.
And 53rd Cys, 36th Cys and 48th Cys, 42nd C
This is an SS bond between ys and Cys at position 57. Next, a reduction treatment is performed with dithiothreitol to cleave the SS bond, and then carboxymethyl (CM) to obtain a CM-peptide. Further, a part thereof is treated with chymotrypsin or lysyl endopeptidase to obtain respective fragments. The amino acid sequences of CM-peptide and chymotrypsin or lysyl endopeptidase fragment are analyzed using a sequencer. The C-end is analyzed with carboxypeptidase Y.

 As described above, the entire amino acid sequence is determined.

〔Example〕

The present invention will be described more specifically with reference to the following examples.

Example 1 20 kg of fresh pig duodenum is treated in boiling water for 5 minutes, then homogenized with 0.1 M acetic acid and extracted. CM the supernatant
-After adsorption on cellulose, elute with 0.1N hydrochloric acid solution,
A precipitate is obtained by salting out. This is extracted with 95% isopropyl alcohol and then with methanol, and the isopropyl alcohol layer and the ethanol layer are concentrated to dryness. 0.1M
CM well buffered with citrate phosphate buffer (pH 6.5)
-Flush the cellulose, adsorb it, elute secretin with 0.01 M citrate phosphate buffer (pH 6.5) containing 0.06 M salt, then elute adsorbed substances that do not elute with 0.1 N hydrochloric acid solution, The material obtained by the precipitation is used as a raw material.

30 g of the obtained raw material was dissolved in 500 ml of water and adjusted to pH 6.0. This is passed through a CM-Sephadex G-25 column equilibrated with a 0.05 M ammonium formate buffer (pH 6.5) to be adsorbed. Next, 0.5 M ammonium formate buffer (pH 6.5)
After that, the adsorbed substance is eluted with 2M ammonium formate (pH 8.0). This fraction contains Na, K-ATPase
Inhibitory active substances are included. The obtained eluate was freeze-dried. 1.8 g of the obtained substance was dissolved in 30 ml of 0.1 M acetic acid, and the solution was applied to a Sephadex G-25 column equilibrated with 0.1 M acetic acid. A Na, K-ATPase inhibitory activity fraction was collected and lyophilized.

Dissolve 950 mg of the obtained substance in 0.1 M acetic acid, and add 10%
Nucleosil _C18 column (diameter 10 mm x length 300) equilibrated with acetonitrile solution (containing 0.1% trifluoroacetic acid)
mm), and a gradient elution was performed with the same solution and a 60% acetonitrile solution (containing 0.1% trifluoroacetic acid). Active fractions having Na, K-ATPase inhibitory activity were collected and lyophilized.

Dissolve 105 mg of the obtained substance in 0.1 M acetic acid, and add 10%
The solution is passed through nucleosyl CN (4.6 mm in diameter x 250 mm in length) equilibrated with an acetonitrile solution (containing 0.1% trifluoroacetic acid), and a concentration gradient elution is performed between the same solution and an 80% acetonitrile solution (containing 0.1% trifluoroacetic acid). Was. N
a, K-ATPase inhibitory fractions were collected and freeze-dried.

The obtained substance was dissolved in 0.1 M acetic acid, and the solution was passed through a nucleosil _C18 column (4.6 mm in diameter x 250 mm in length) equilibrated with 25% acetonitrile (containing 0.1% trifluoroacetic acid). To elute the active substance.

 The elution curve is shown in FIG.

The horizontal axis in the figure represents the elapsed time (minutes) since the sample was injected, and the vertical axis represents the absorbance (280 nm).

In the figure, the solid line shows an elution curve, and the active fraction having Na, K-ATPase inhibitory activity (the fraction (A) corresponding to the hatched portion in FIG. 1,
Fractions (B) and (C)) were collected and lyophilized.

Nucleosyl _C18 reverse phase chromatography was performed on the obtained substance, and all showed a single peak. The active substance (B) was subjected to amino acid analysis, and the composition ratio was as shown in Table 1.

Further, structural analysis was performed in the following manner, and it was confirmed that the peptide of the present invention was obtained.

Structural analysis 20 nmol of the active substance (B) was reduced with a 1% dithiothreitol solution, and the SS bond was cleaved to form carboxymethyl (CM).

CM-peptide 1n purified by reverse phase chromatography
The mol was taken, and the amino acid sequence was analyzed using a gas phase sequencer (Applied Biosystem).

Next, 2 nmol of CM-peptide was decomposed with chymotrypsin, and a nucleosil _C18 column (4.6 mm in diameter, rare 250 m
m), using a 1% to 60% acetonitrile solution (containing 0.1% trifluoroacetic acid) to perform concentration gradient elution,
Fragments were collected.

In addition, 2 nmol of CM-peptide was digested with lysyl endopeptidase, and six fragments were separated by the same operation.

An amino acid analysis was performed for a part of each, and the remaining amino acids were analyzed for amino acid sequence by a gas-phase sequencer.

The amino acid sequence at the C-terminal was confirmed by decomposing with carboxypeptidase Y using 0.5 nmol of CM-peptide. After 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 12 hours after the reaction, a part of the reaction solution was sampled, and the released amino acids were analyzed with Pitacog System (Nippon Water Limited).
(Cohen SA, Nature, 320 , 769 (1986)).

FIG. 2 shows the entire structure and the procedure of the amino acid sequence.

In the figure, L is lysyl endopeptidase and a fragment,
KP indicates a chymotrypsin fragment, Marks indicate sequencing with the gas phase sequencer and marks indicate sequencing with carboxypeptidase Y (CR-Y).

In addition, as a result of amino acid sequence analysis using a gas-phase sequencer, the active substance (A) starts from leucine, which is the amino acid at position 13 of the active substance (B), and then confirms that the active substance (B) is a similar peptide having the same sequence. Confirmed by the same method as described above.

After the carboxymethylation of the active substance (C), amino acid sequence analysis using a gas-phase sequencer confirmed that the active substance (C) was the same as the active substance (B) up to the N-terminal 18th position. Next, the carboxylmethylated peptide was decomposed with lysyl endopeptidase, and the resulting fragment was analyzed and fractionated by HPLC. As a result, only one new fragment which was not found in the active substance (B) was confirmed, and the other fragments were The retention time by HPLC and the amino acid analysis value of the active substance (B) were in agreement. Analysis of the amino acid sequence of the fragment newly identified as the active substance (C) using a gas-phase sequencer revealed that the fragment corresponds to the peptide from position 17 to position 35, and arginine at position 22 and serine at position 30 of active substance (B). Has been replaced with glycine, respectively, and the other amino acid sequences have the same novel structure.

The novel peptides having Na, K-ATPase inhibitory activity (active substances (B) and (C)) were all monospots under the following conditions for thin layer chromatography.

Thin plate; Silica gel 60 F-254 plate Developing solvent; n-butanol: acetic acid: water = 1: 1: 1 Active substance (B) R _f = 0.05 Active substance (C) R _f = 0.02 n-butanol: pyridine : Acetic acid: water = 15: 10: 3: 12 Active substance (B) _Rf = 0.32 Active substance (C) _Rf = 0.30 Detection; ninhydrin reagent Next, a novel peptide of the present invention having Na, K-ATPase inhibitory action (Hereafter, SPAI (Sodium Potassium ATPase Inhibi
(abbreviated as tor)).

10 nmol of the active substance (A) shown in FIG.
After dissolving in a phosphate buffer (pH 6.5), adding 10 μg of trypsin, reacting at 37 ° C. for 22 hours, and then equilibrating with a 1% acetonitrile solution (containing 0.1% trifluoroacetic acid), a nucleosyl C ₁₈ column ( Using a column with a diameter of 4.6 mm x 250 mm), a concentration gradient elution was performed using a 1% to 80% acetonitrile solution (containing 0.1% trifluoroacetic acid).
(Peaks T-1 to 4) were collected.

After fractionation of peak T-2 in FIG. 3, it was lyophilized and degraded with protease V8. That is, the peak T-2 is set to 400
Dissolve in μl phosphate buffer (pH 6.5) and add protease V
82 μg is added and reacted at 25 ° C. for 20 hours.

After the reaction, the fragments were collected by using a nucleosyl _C18 column under the same conditions as in the above-described trypsin decomposition, and the four fragments (V8-1 to V8-4) shown in FIG. 4 were obtained.

A part of each fragment was subjected to amino acid analysis, and the rest was analyzed for amino acid sequence using a gas phase sequencer. Table 2 shows the results of the amino acid analysis. Table 3 shows the results of the gas-phase sequencer.

From the above results, FIG. 5 shows the position of the SS bond of the active substance (A). In FIG. 5, the amino acids are represented by one-letter abbreviations, which correspond to the three-letter abbreviations as follows.

Since the N-terminal and 12 amino acids of the active substance (B) of the active substance (A) correspond to the peptide having a small number, the active substance (B) is considered to have a similar SS bond.

Similarly, the active substance (C) differs in two amino acids, but since the position of Cys is the same as that of the active substances (A) and (B), it may have the same coordination of the SS bond. Conceivable.

Therefore, the coordination of the SS bond of the SPAI of the present invention is shown in FIG. In FIG. 6, amino acids are represented by single letter abbreviations.

〔The invention's effect〕

The following experimental examples show that the peptides of the present invention have Na, K-ATPase inhibitory activity.

Experimental Example The following composition was used for the reaction solution.

<Reaction solution composition> 0.05 M Tris-HCl buffer (pH 7.4), 0.13 M sodium chloride, 3 mM magnesium chloride, 1 mM ethylenediaminetetraacetic acid, 3 mM adenosine triphosphate, 20 mM potassium chloride.

After adding 0.01 unit of Na, K-ATPase to 0.2 ml of the solution of this composition and reacting at 37 ° C. for 30 minutes, 0.04 ml of 60% perchloric acid was added.
To stop the reaction.

The Na, K-ATPase activity can be measured by coloring the released inorganic phosphoric acid with arachid green and ammonium molybdate and measuring the absorbance at 660 nm.

Results of Measurement About 50% Na, K- at a concentration of 2.5 nmol / ml of the peptide of the present invention
It was found to have ATPase inhibitory action.

[Brief description of the drawings]

FIG. 1 shows an elution curve of the Na, K-ATPase inhibitory activity fraction obtained in Example 1 on a nucleosil _C18 column, and FIG. 2 shows the entire structure and amino acid sequence of the active substance (B). 3 Nukureojiru C ₁₈ column elution curve, Figure 4 is the elution curve of Nukureojiru C ₁₈ column but the peak T-2 in FIG. 3 was decomposed with protease V8 tryptic of the active substance (A), FIG. 5 is the active substance (A) is a diagram showing the coordination of the SS bond, and FIG. 6 is a diagram showing the coordination of the SS bond of SPAI of the present invention.

Claims (1)

(57) [Claims] 1. A primary structural formula (Where X is Arg or Gly, and when X is Arg, Y is Se
When r and X are Gly, Y is Gly. N) as indicated by
a, Novel peptide having K-ATPase inhibitory activity.