JPH0159279B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to new peptide opioids belonging to the "big" enkephalin peptides. With the discovery of Met-enkephalin and Leu-enkephalin by Hughes et al. in 1975, the search for opioids (morphine-like substances) in the mammalian brain has progressed actively, and α-endorphin, β-endorphin,
Many "major" enkephalin opioids such as γ-endorphin, δ-endorphin, and the Met-enkephalin family have been discovered. These peptide opioids all have their N-
The terminal side has a peptide chain of Tyr-Gly-Gly-Phe-Met, that is, the structural formula, Number of amino acids constituting the peptide chain shown by 16
It is a long-chain polypeptide consisting of ~31 molecules, and is characterized by having a structure that, without exception, matches the partial sequence of the pituitary protein β-lipotropin (β-lipotropin), whose structure is known. Contrary to the above, Tyr-Gly-Gly-
The discovery of other Leu-enkephalin opioids having a Phe-Leu peptide chain was considerably delayed compared to the former, but α-neo-endorphin (α-enkephalin), which was first discovered by the present inventors in 1979
-neo-endorphin;Biochem.Biophys Res.
Commun., 86 , 153 ( 1979 )), and later Goldstein et al.
6666 (1979)) (the former structure was developed by the present inventors as Tyr-Gly-Gly-Phe-Leu-Arg
-Lys-Tyr-Pro-Lys, but the overall structure of the latter is still unknown. ). However, as a result of continuing the search for polypeptide opioids in the brain, the present inventors have now isolated and determined the structures of two types of Leu-enkephalin opioids from the pig hypothalamus. Endorphin (β-neo-
endorphin) and PH-8P (or dynorphin [1-8]).
According to the research conducted by the present inventors, these new peptide opioids have the following chemical structures. β-neo-endorphin: Tyr (N-terminus)
―Gly―Gly―Phe―Leu―Arg―Lys―Tyr―Pro
(C-terminus) PH-8P:Tyr (N-terminus)-Gly-Gly-Phe
-Leu-Arg-Arg-Ile (C-terminus) In other words, these peptides are the above-mentioned Leu-enkephalin peptide chain Tyr-Gly-Gly-Phe-
At the C-terminal carbon of Leu--Arg-Lys-Tyr-Pro
(C-terminus) and -Arg-Arg-Ile (C-terminus) short chain peptide residues are bonded together, so the general formula is Tyr-Gly-Gly-Phe-Leu-R (where R is Different peptide chains, -Arg-
Lys-Tyr-Pro (C-terminus) or -Arg-Arg-
Ile (meaning C-terminal)). (The following symbols for amino acids in the text shall be used in accordance with international regulations. Tyrosine: Tyr(Y) Glycine: Gly(G) Phenylalanine: Phe(F) Methionine: Met(M) Leucine: Leu (L) Arginine: Arg(R) Lysine: Lys(K) Proline: Pro(P) Isoleucine: Ile(I) The two types of enkephalin peptides according to the present invention, β-neo-endorphin and PH-8P, are shown below. The physical and chemical constants of β-neo-endorphin: - [α] ^20.5 _D = -33.5° (c, 0.49, H ₂ O), Rf =
0.66 (butanol:acetic acid:water=4:1:5
(v/v) (upper layer)), Rf=0.80 (butanol:
Acetic acid: water: pyridine = 15:3:12:10 (v/
v)) Elemental analysis: C ₅₄ H ₇₇ O ₁₂ N ₁₃・CH ₃ COOH・
As 5H ₂ O, Calculation: C, 53.16; H, 7.31; N, 13.89 Actual measurement: C, 53.45; H, 7.03; N, 13.75 Amino acid ratio (acid decomposition): P1.00, G2.00, L1.02, Y1.96, F0.98, K1.02, R1.01 Amino acid sequence: Note (→): Dansil-Edman degradation T: Trypsin degradation PH-8P: - [α] ^20.5 _D = -20.1° (c, 0.47, H ₂ O), Rf = 0.74
(Butanol: Acetic acid: Water = 4:1:5 (v/v)
(upper layer)), Rf = 0.82 (butanol: acetic acid: water: pyridine = 15:3:12:10 (v/v)) Elemental analysis: C ₄₆ H ₇₂ O ₁₀ N ₁₄・2CH ₃ COOH・
As 3H ₂ O, Calculated: C, 51.98; H, 7.50; N, 16.97 Measured: C, 51.68; H, 7.36; N, 17.36 Amino acid sequence The novel peptide according to the present invention is produced using pig hypothalamus as a raw material, for example, in the following manner. As shown in Figure 1, the hypothalamus of 30,000 pigs was defatted and extracted with 2N acetic acid.
25 (trade name of cross-linked dextran type chromatogel manufactured by Pharmacia) to reduce the void volume to 1.4~
Low molecular weight fraction (molecular weight 500-3000) that elutes 2.3 times
After adsorbing this on SP Sephadex C-25 (trade name of cross-linked sulfopropyl dextran type ion exchange chromatogel (cationic type) manufactured by Pharmacia), the fraction eluted with 1N-ammonia was extracted. , collect the G fraction (No. 115 to 129 in Figure 2) that elutes before α-neo-endorphin,
This is further gel-filtered through Sephadex G-25. Using high titer anti-[Arg ⁶ ]-Leu-enkephalin antiserum obtained by immunizing a rabbit with [Arg ⁶ ]-Leu-enkephalin/BSA conjugate, the eluate was prepared by the carbodiimide method. CM-52 (trade name of ion-exchange chromatogel manufactured by Watmann) and CM Sephadex C-25 (trade name of cross-linked carboxymethyldextran-type ion-exchange chromatogel manufactured by Pharmacia) (cationic type)) (Fig. 3) and purified by high-performance liquid chromatography (HLPC) using μ-Bondapak C-18 (trade name of reversed-phase HPLC packing material manufactured by Waters) (Fig. 4). When peaks #4 and #7 in the figure are subjected to HLPC again, β-neo-endorphin (30 n mol) is obtained from the former, and PH-8P (20 n mol) is obtained from the latter. This elution pattern is as shown in Figures 5 and 6, and shows that the synthetic β-neo-endorphin and
It was confirmed that the peak coincided completely with that of PH-8P. β-neo-endorphin according to the present invention and
PH-8P was determined by the guinea pig longitudinal ileal muscle test.
As shown in the table below, they have a relative activity of 760 and 390, respectively, taking Leu-enkephalin activity as 100, and are expected to be effective as a morphine-like analgesic. 【table】

[Brief explanation of drawings]

Figure 1 is a diagram schematically illustrating the extraction and purification method for the novel peptide of the present invention. Figure 2 is a chromatogram of a strongly basic fraction of a porcine hypothalamic low molecule using SP-Sephadex C-25 (column size: 42x 26cm, in advance
Equilibrate with 0.001N HCOOH. Sample loading: 13g of lyophilized material dissolved in 130ml of 0.1N HCOOH.
Fraction size: 20ml/tube. Elution system: A:
0.001N HCOOH, B-C: linear gradient from water until reaching solution (C), C: 0.5M ammonium formate (PH6.5), D: 0.5M ammonium formate (PH9.5),
E: _NNH4OH . 〓: Opiate active range. ), Figure 3 is a chromatogram of the immunoactive part of the G fraction in Figure 2 using CM Sephadex C-25 (sample load: CM:52 The immunoactive fraction purified by chromatography).
53.8mg. Column size: 0.95×100cm, 0.5M in advance
Equilibrate with pyridine-acetate (PH5.0). Fraction size: 2.0ml/min. Elution system: Pyridine-acetate (PH5.0) linear concentration gradient from 0.5M to 1.2M. ),
Figure 4 shows β-neo-endorphin and PH-8P
Elution pattern of reversed phase HLPC of containing fractions (sample loading: 3.21 mg of immunoactive fraction by CM-Sephadex C-25 ion exchange chromatography. Elution: 50 mM
Linear concentration gradient method (40 minutes) with KH ₂ PO ₄ -H ₃ PO ₄ buffer (PH2.0), 10-50% for CH ₃ CN system. (3rd
(See figure) Flow rate: 2.0ml/min. Column: μ-
Bondapack C-18 (manufactured by Waters) Column size: 3.9 x 300 mm. ), Figures 5 and 6 are reverse phase.
This is an elution pattern of β-neo-endorphin and PH-8P repurified by HLPC. In Figure 5, A: natural product, B: synthetic product.

Claims (1)

[Claims] 1 General formula Tyr-Gly-Gly-Phe-Leu-R (Formula Tyr, Gly, Phe and Leu represent tyrosine, glycine, phenylalanine and leucine residues, respectively, and R is the formula , -Arg-Lys-Tyr-Pro or the formula, -Arg-Arg-Ile, where Tyr has the above meaning and Arg, Lys, Pro and Ile are the residues of arginine, lysine, proline and isoleucine, respectively. C of Leu in the above formula represented by
a peptide chain attached to the terminal end). 2 Formula, Tyr-Gly-Gly-Phe-Leu-Arg-
Lys-Tyr-Pro (in the formula, Tyr, Gly, Phe, Leu,
Arg, Lys, Tyr and Pro are respectively tyrosine, glycine, phenylalanine, leucine, arginine,
2. The peptide opioid according to claim 1, which is represented by lysine and proline residues. 3 Formula, Tyr-Gly-Gly-Phe-Leu-Arg-
The peptide opioid according to claim 1, which is represented by Arg-Ile (wherein Tyr, Gly, Phe, Leu, Arg and Ile represent tyrosine, glycine, phenylalanine, leucine, arginine and isoleucine residues, respectively). .