JPS6320840B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel linear peptide having a reducing action and a method for producing the same. (Prior Art) It is well known that heavy metal elements such as cadmium, which are taken into the human body through oral ingestion, cause various disorders. Furthermore, when radiation therapy is applied to the human body, it is known that the irradiation of radiation induces radical reactions within the human body, producing harmful oxygen radicals. In the human body, L-cysteine and glutathione, which have active thiol groups (-SH), combine with the above-mentioned heavy metal elements or reduce the above-mentioned active radicals through redox reactions and become inactivated. By doing so, it is thought that the above-mentioned obstacles are alleviated to some extent. (Problems to be Solved by the Invention) However, the thiol group of L-cysteine is easily oxidized in the human body, and most of the thiol groups are formed by disulfide bonds (-S-S-) between the thiol groups.
becomes inactive L-cystine due to the formation of
The above-mentioned ability to bond with heavy metal elements and ability to reduce radicals were practically not very promising. In addition, glutathione actually has a
Although it plays a leading role in the reduction and disappearance of oxygen radicals, it cannot be said to have sufficient binding strength with heavy metals or reducing power. Therefore, the present invention is a novel chelating agent that binds to heavy metals and inactivates them, and a radical reducing agent that can maintain high activity in the human body and that does not cause any particular side effects or toxicity when administered to the human body. The technical problem to be solved is to provide substances and methods for their production. During the search for biological substances that neutralize heavy metal toxicity, the present inventor discovered that a low molecular weight peptide that binds cadmium was induced to be synthesized in yeast.
The invention has been completed. (Means for solving the problem) The technical means for solving the above problem is based on the chemical structural formula (wherein n represents an integer of 1 or 2);
and fission yeast (Schizosaccharomyces pombe)
By culturing the peptide in a medium containing a salt of a transition group heavy metal element, the linear peptide is induced to be synthesized as a complex compound with the transition group heavy metal element. The linear peptide of the present invention includes γ-L-glutamyl-L-cysteinyl-γ-L-glutamyl-L-cysteinyl-glycine, which is a condensation of five amino acids, and γ-L-glutamyl-L-cysteinyl-glycine, which is a condensation of five amino acids. becomes γ
-L-glutamyl-L-cysteinyl-γ-L-
It contains two types of peptides, glutamyl-L-cysteinyl-γ-L-glutamyl-L-cysteinyl-glycine, and γ-L-glutamyl-L-cysteinyl-, which is a condensation of nine amino acids. γ-L-glutamyl-L-cysteinyl-γ-L-glutamyl-L-cysteinyl-γ
-L-glutamyl-L-cysteinyl-glycine is considered. These linear peptides are described below.
They are collectively called cadeistein, and those consisting of the above seven amino acids are called cadeistein A, and those consisting of five amino acids are called cadeistein B. In addition, by the production method of the present invention, there is a possibility that a peptide in which n is an integer of 4 or more in the chemical structural formula described above can also be synthesized by induction. Next, the microorganism used in the production method of the present invention is Schizosaccharomyces pombe, and it is believed that the induced synthesis of heavy metal-binding proteins or peptides is a defense reaction universally recognized in living organisms. Therefore, it is considered possible to induce the synthesis of the linear peptide of the present invention using yeast other than fission yeast or other types of microorganisms. The medium may be one commonly used for culturing yeast, such as a nutrient medium using peptone or the like, or a synthetic medium with a known composition. It should be noted that using a liquid medium is more advantageous than an agar medium with no fluidity, since it is easier to propagate fission yeast and induce the synthesis of cadeistein. A salt of a transition group heavy metal element is added to the medium.
As the salt of this transition group heavy metal element, a salt with a high degree of dissociation such as cadmium, copper, zinc, lead, mercury, etc., for example, a salt with an inorganic acid is used. Less dissociated salts such as cadmium phosphate are not effective. As an example of the most effective salts, mention may be made of cadmium chloride _CdCl2 . The amount of transition group heavy metal element added varies depending on the type of heavy metal element, but is approximately 0.02 to 2 mmol concentration relative to the medium. If the amount added is less than the above range, induced synthesis of caddistain will not take place, and if it is more than the above range, yeast growth will be poor, which is not preferable. Yeast cultivation is carried out under aerobic conditions, such as shaking culture, at about 25 to 35°C, as in the usual case. The culture time is about 10 to 24 hours. Examples of possible structural models of the complex compound of caddistain and a transition group heavy metal element obtained by the production method of the present invention include those shown in FIG. 1 or 2. In FIGS. 1 and 2, the bold line indicates caddistain A, and cadmium is shown as a transition group heavy metal element. Incidentally, it is thought that Cadeistein B or the above-mentioned linear peptide composed of nine amino acids also forms a complex compound in a similar bonding manner. Regardless of their structure, these complex compounds, when adjusted to pH 2 with hydrochloric acid under strong acidity, dissociate to produce caddistain in a free state. It can be isolated by means of fractionation, such as a molecular sieve column that fractionates by molecular weight. (Function) Cadeistein contains at least two L-cysteines, and the amino groups of these L-cysteines each form a peptide bond with the carboxyl group at the γ-position of L-glutamic acid. Although not shown in the structural formula, the oxygen atom of the peptide bond forms a hydrogen bond with the thiol group of L-cysteine due to its spatial position, so the thiol group It is significantly more stabilized than the thiol group in cysteine or free cysteine, which constitutes the commonly known peptide. An example of the detoxifying effect of caddistain against transition group heavy metal elements based on such characteristics is shown in Table 1 and FIG. 3 below. Leucine transfer
LRS, an enzyme that attaches leucine to RNA
As shown in Figure 3, the activity of leucine transfer RNA synthetase (leucine transfer RNA synthetase) is inhibited by cadmium, but if caddestein is added to the reaction system before adding LRS, In, LRS
The inhibitory effect of cadmium on still,
In Table 1, leucine-tRNA indicates the amount of leucine-bound leucine transfer RNA produced;
This shows the activity of LRS. Also, in the same table,
"beforeE" and "afterE" indicate the case where caddistain was added before and after the addition of LRS, respectively, and caddistain in Table 1 refers to a mixture of caddistain A and caddistain B (3:
2) is shown.

[Table] From Table 1, it is clear that caddestein blocks the inhibitory effect of cadmium on enzyme activity by binding to cadmium. Next, FIG. 4 shows the stability of the complex compound of caddistain and heavy metal element thus formed. The figure shows the degree of dissociation of a complex compound of caddistain and cadmium observed by the decrease in specific absorbance at a wavelength of 250 nanometers (nm) depending on the pH.
The graphs of GSH and MT-1 are the substances related to the structural model shown in Figure 1 (hereinafter referred to as BP1), respectively.
Materials related to the structural model shown in Figure 2 (hereinafter referred to as BP)
Say 2. ), shows the absorbance changes of a complex compound of glutathione and cadmium, and a complex compound of mouse metallothionein-1 and cadmium, respectively.
The latter two substances are natural peptides known to have heavy metal binding activity similar to that of caddistain. According to these results, BP1 and BP2 are significantly more stable than glutathione complexes, and do not dissociate at all near neutral pH, which is the pH of the human body, and the stability of BP1 is particularly high because its molecular weight is approximately 10 times greater. It has been shown to be equivalent to mouse metallothionein-1, and it is believed that caddestein is extremely stable when administered to humans and forms complexes with cadmium. Furthermore, the affinity of caddeistein for cadmium suggests its strong reducing action, so to confirm this, we measured the redox potential of caddeistein and obtained the results shown in Table 2. .

[Table] The above measurements were carried out in a 50 mmolar sodium-phosphate buffer with a pH of 7.0 at 20°C using a Michaelis bath by titration with potassium ferricyanide as an oxidizing agent. It is something. Incidentally, the results of titration using sodium hydrosulfite as a reducing agent under the same conditions also showed similar values. Cadeistein A shown in Table 2,
The high negative potential of B indicates that caddistain has a significantly stronger reducing effect than glutathione. The above-mentioned affinity of cadeistein for transition group metal elements and its strong reducing action are brought about by the presence of the aforementioned thiol groups stabilized by hydrogen bonds, and for this reason cadeistein is harmful to the human body. cadmium, copper, zinc,
It has a detoxifying effect on heavy metal elements such as lead and mercury, and its reducing effect prevents the generation of oxygen radicals that are harmful to the human body due to radical reactions induced by radiation exposure to the human body. Next, both Cadeistein A and B are derivatives of glutathione in their chemical structure, and are composed of glycine, L-glutamic acid, and L-cysteine, which are amino acids found in the human body.
Since it is a substance that is induced and synthesized within living organisms, it is considered that it does not exhibit toxicity or harmful side effects to the human body. Next, the linear peptide of the present invention can be synthesized easily by administering a simple inorganic compound to microorganisms without using organic chemical synthesis means.
Can be manufactured quickly and cheaply. (Example) Next, an example of the present invention will be described. In this example, among fission yeast (Schizosaccharomyces pombe), L972h
- strains were used. This strain has been deposited with
American Type Culture Collection (abbreviation)
ATCC) catalog (16th edition, 1984), and the inventor is Dr. U. Leupold (Institute fu¨r
Allgemeine Microbiologie, University of
It was obtained from the laboratory of Dr. Bern, Bern, Switzerland, which was distributed to the Yanagishima laboratory of the Faculty of Science, Naiya University. 1 of fission yeast of the above strain in a flask plugged with cotton.
The cells were cultured with shaking at 29° C. in a liquid medium containing millimolar cadmium chloride Cdcl ₂ . Medium is 1%
Yeast extract (dry powder of yeast extract), 2%
Polypeptone (animal meat degraded by pepsin),
It was 2% glucose dissolved in water. Fission yeast continues to proliferate until around 20 hours have passed.
The amount of cadmium taken up reached approximately 5 μg per cell in 1 ml of medium. Bacteria were collected over time. The bacterial collection method was to precipitate the yeast by centrifugation and separate it from the liquid medium. Next, the collected yeast was ground in a mixture of 50 mmolar Tris-HCl buffer (PH7.6) and 0.1 molar potassium chloride, and the insoluble residue was removed by centrifugation to prepare a yeast extract. . When this extract was fractionated using a molecular sieve column (Sephadex G-50), most of the cadmium was found at the molecular weight position of approximately 4100 and the molecular weight position of approximately 1700.These substances are described below. The method was analyzed. Specifically, the former and latter substances were measured for cadmium by atomic absorption spectrometry, peptide quantification and amino acid analysis by the known so-called Kjeldahl-Nessler method, and it was found that the former substance contained four unit peptides and six unit peptides. The latter substance contained two unit peptides and two units of cadmium in one molecule. Also, there was knowledge that these substances seem to contain sulfur, so
Hydrochloric acid was added to an aqueous solution of these substances, and the dissociated sulfur was absorbed into zinc hydroxide to form zinc sulfide. This was reacted with P-aminodimethyl-aniline to produce methylene blue. Sulfur was measured by spectroscopically measuring the maximum absorption of the methylene blue produced, and it was found that the former substance contained one unstable sulfur element in one molecule. As a result, it was found that the former substance was BP1 shown in the structural model of FIG. 1, and the latter substance was BP2 shown in the structural model of FIG. 2. Next, in order to understand the structure of the unit peptides of BP1 and BP2, a dansyl group was added to the amino terminal amino acid of the peptide, followed by acid hydrolysis and reversed-phase high performance liquid chromatography (hereinafter abbreviated as HPLC). ) is a method for detecting dansylated amino acids, in which a unit peptide is digested by carboxypeptidase, which liberates amino acids at the carboxyl terminus of the peptide, and then the liberated amino acids are
Detection method using HPLC, aminoethylation of free carboxyl groups in glutamic acid, acid hydrolysis of the peptide, and determination by HPLC of whether the carboxyl group at the α or γ position of glutamic acid was aminoethylated. Amino acid sequence and structural analysis were carried out by a method in which the types and composition ratios of amino acids were determined by HPLC after peptides treated with the known Rainy-Nitzkel method were decomposed with carboxypeptidase. As a result, the unit peptides of BP1 and BP2 are the same substance, that is, the above-mentioned caddistain A, and
It was found that BP1 and BP2 also contained a small amount of the above-mentioned caddistain B. Therefore, in order to confirm the structures of Cadeistein A and B, peptides with the estimated structures were synthesized by the method shown in FIG. This method is known as a so-called stepwise organic synthesis method for peptides, and is a conventional organic synthesis method in which amino acids constituting a linear peptide are sequentially condensed, starting from the carboxyl terminal, while protecting a predetermined reactive group. It is a chemical synthesis method. In the figure, OB ZL is a benzyl group with a protected carboxyl group at the α position, B OC is a butyloxycarbonyl group with a protected amino group, MOB is a paramethoxybenzyl group with a protected thiol group, and Tr is an amino group of L-glutamine. represents a protected trityl group (triphenylmethyl group), and 1 in the figure is obtained by dissolving each amino acid or peptide in a 3:1 mixture of trifluoroacetic acid and anisole at 0°C, and adding hexane after 10 minutes. The operation of precipitating the product, 2 in the figure, is the operation of dissolving the precipitate in a mixture of dichlorohexyl carbodiimide and dichloromethane, and then drying it. 3 in the figure is shown as Protected 1 and Protected 2, respectively. Ta,
A procedure is shown in which Kadeistein A and Kadeistein B to which protective groups have been added are hydrolyzed in a solution of hydrofluoric acid to remove the protecting groups, and then Kadeistein A and Kadeistein B are isolated by ether extraction. Then, we investigated circular dichroism spectra, reversed-phase high performance chromatography, and protons in the molecules of Kadeistein A and Kadeistein B, which were induced and synthesized in fission yeast, and the corresponding organically synthesized Kadeistein A and Kadeistein B. Comparison of nuclear magnetic resonance spectra, Figures 6 to 1
Based on the results shown in Figure 0, it was confirmed that they were made from the same substance. In addition, in each figure, "synthetic CdA",
The graphs shown as "Synthetic CdB", "Natural CdA", and "Natural CdB" are respectively organically synthesized Cadeistein A and Cadeistein B, and induced synthesis of Cadeistein A and Cadeistein B in fission yeast.
This is a graph of Figures 6 and 7 show circular dichroism spectra (CD spectra),
The horizontal axis represents the wavelength in nanometers, and the vertical axis [θ] represents the optical activity of converting linearly polarized light into elliptical polarized light.
Moreover, FIG. 8 shows reverse phase high performance chromatography, and in the figure, the peaks of CdA and CdB coincided with those of synthetic CdA, natural CdA, synthetic CdB, and natural CdB, respectively. Next, Fig. 9 a, b and Fig. 10 a, b show synthetic CdA, natural CdA, synthetic CdB, and natural CdA, respectively.
This is a comparison of nuclear magnetic resonance spectra for protons of CdB, and the horizontal axis in the figure shows the so-called chemical shift in parts per million (ppm). Also, in Figures 9b and 10b, "imp"
The peak indicated by is BP1 or BP from the above-mentioned fission yeast.
This peak is due to the Tris-HCl buffer used in extracting 2 remaining as an impurity. Next, regarding the physical properties of Kadeistein A and Kadeistein B, when these substances are obtained as pure substances, their thiol groups form disulfide bonds between molecules and polymerize, making it difficult to measure their physical properties accurately. , measured with the thiol group protected by para-methoxybenzyl group, caddistain A has a melting point of 131°C and 1.5 g of trichloromethane/
The optical rotation measured by the D-line of sodium in a ml aqueous solution is -12.4°, and the melting point of Cadistein B is 107.
℃, and the optical rotation of the same as above was −1.05°. Next, the fission yeast Schizosaccharomyces pombe
In order to see whether the induced synthesis of cadeistein was generally observed in each strain, we tested seven known wild-type strains from different sources, namely L972h ^- mentioned above, IFO-0345, and L975h. ⁺ , L975h ⁹⁰ ,
Examples will be described for each of seven types of bacterial strains: M210h ⁺ , M216h ^- , and M216h ⁺ . Of the above strains, IFO-0345 strain is included in the catalog (7th edition, 1984) of the Fermentation Research Institute (abbreviated as IFO, 2-17-85 Jusanhonmachi, Yodogawa-ku, Osaka), which is the depositary institution. The L975h ⁺ strain is listed in the ATCC catalog mentioned above. In addition, the L975h ⁺ strain was obtained along with the L972h ^- strain by the route described above, and ⁹⁰ L975h strains, M210h ⁺ strain, and M216h ^-
strain, M216h ⁺ strain was obtained from Dr. H. Gutz (Institute for
Molecular Biology, The University of
The IFO-0345 strain was obtained from the above-mentioned IFO. In these Examples, taking into consideration that some bacterial strains may grow poorly in a medium containing cadmium, the concentration of cadmium chloride contained in the liquid medium was set to 0.1 mmol, and each bacterial strain in the logarithmic growth phase was The cells were cultured for 20 hours under the same conditions as in Example 1, and the induced and synthesized cadeistein was quantified. The results are shown in Table 3.

[Table] As shown in Table 3 above, we were able to observe the induced synthesis of caddistain in all seven strains.
pombe), it is thought that caddistain can be induced to be synthesized. (Effects of the Invention) The linear peptide of the present invention can act as a novel heavy metal antidote and a prognostic agent for radiation irradiation. or,
It can also be used to collect heavy metals in wastewater treatment. Moreover, the above-mentioned linear peptide can be produced easily, rapidly, and inexpensively by the production method of the present invention.

[Brief explanation of the drawing]

Figures 1 and 2 are explanatory diagrams showing the bonding state of a possible structural model of a complex compound of caddistein and cadmium, Figure 3 is a graph showing inhibition of enzyme LRS activity by administration of cadmium chloride, and Figure 4 is A graph showing the degree of dissociation at each pH of caddeistein and a complex compound of a reference substance and cadmium as specific absorbance. Figure 5 is a schematic diagram showing the organic chemical synthesis method of cadeistein. Figures 6 and 7 are the respective synthesis methods. and a graph showing a comparison of the circular dichroism spectra of natural CdA and CdB. Figure 8 shows a reversed phase high performance chromatograph of caddistain. Figure 9 a, b, and Figure 10 a, b. 1 is a graph showing comparative nuclear magnetic resonance spectra of synthetic and natural CdA and CdB, respectively.

Claims (1)

[Claims] 1. Chemical structural formula (wherein n represents an integer of 1 or 2). 2 γ-L-glutamyl-L-cysteinyl-γ
-L-glutamyl-L-cysteinyl-glycine.The linear peptide according to claim 1, which is -L-glutamyl-L-cysteinyl-glycine. 3 γ-L-glutamyl-L-cysteinyl-γ
-L-glutamyl-L-cysteinyl-γ-L-
The linear peptide according to claim 1, which is glutamyl-L-cysteinyl-glycine. 4 Schizosaccharomyces pombe
By culturing in a medium containing salts of transition group heavy metal elements, the chemical structural formula (In the formula, n represents an integer of 1 or 2.) A method for producing a linear peptide, which comprises induction synthesis of a linear peptide as a complex compound with the transition group heavy metal element. 5. The method for producing a linear peptide according to claim 4, wherein the transition group heavy metal element is cadmium.