Isolation and Structure of Pro-Methionine-Enkephalin: Potential Enkephalin Precursor from Porcine Hypothalamus (Endorphins/Pro-Hormones) WEI-YONG HUANG, ROBERT C
Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6177-6180, December 1979 Biochemistry Isolation and structure of pro-methionine-enkephalin: Potential enkephalin precursor from porcine hypothalamus (endorphins/pro-hormones) WEI-YONG HUANG, ROBERT C. C. CHANG, ABBA J. KASTIN, DAVID H. COY, AND ANDREW V. SCHALLY* Endocrine and Polypeptide Laboratory, Veterans Administration Medical Center, and Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70146 Contributed by Andrew V. Schally, September 12, 1979
ABSTRACT A hexapeptide that we have named pro-me- were used: (i) 1-butanol/acetic acid/water/ethyl acetate (1: thionine-enkephalin has been isolated from acid extracts of 1:1:1, vol/vol) and (ii) 1-butanol/pyridine/acetic acid/water porcine hypothalami and found to have the amino acid se- (15:10:3:12, vol/vol). The developed chromatograms were quence H-Tyr-Gly-Gly-Phe-Met(O)Arg-OH. This peptide is not a fragment of either porcine -1lipotropin or -endorphin, which visualized by spraying with ninhydrin/cadmium reagent. suggests that it could be a precursor of [Metienkephalin in the Amino Acid Analysis. Peptides (1-3yg) were hydrolyzed brain by a pathway differing from the one usually postulated. for 24 hr at 1100C with 4 M methanesulfonic acid/0.2% The existence of still larger precursors of [Metlenkephalin, tryptamine. The analyses were performed on a' Beckman 119 perhaps related to the recently reported a-neo-endorphin, is CL amino acid analyzer. strongly implied by these studies. Synthetic H-Tyr-Gly-Gly- Edman-Dansyl Procedure. The manual Edman degrada- Phe-Met-Arg-OH exhibited low, but significant, opiate activity tion was carried out according to the procedure described by in vitro. Hartley (10), and aliquots were removed for the dansylation The isolation from mammalian brain of the two pentapeptides, procedure. The dansyl amino acids were identified by TLC on [Met]- and [Leu]enkephalin, with morphipomimetic (opioid) polyamide sheets (10). (1) activity has stimulated a large number of biochemical, Carboxypeptidase Y Digestion. Peptide (5 jig) was dissolved neurophysiological and neuropharmacological studies. The in 50 Al of 0.1 M pyridine acetate buffer at pH 5.5, and 5 jig of endorphins (a, A3, and hy) structurally related to [Metlenkephalin carboxypeptidase Y was added. The resulting mixture was in- and f-lipotropin (fl-LPH),y have also been isolated and char- cubated at 370C for 5 hr and the amino acids released were acterized as endogenous ligands of opiate receptors (2-6). Re- identified by amino acid analysis. We have reported (11) the cently, a-neo-endorphin, a form of "big" [Leujenkephalin, was solid phase methodology used for peptide synthesis and puri- isolated from porcine hypothalami and proposed to be a pre- fication. cursor of the pentapeptide (7). Assay for Biological Activity. The opiate activity was In the course of fractionation of porcine hypothalamic ex- measured by the use of a mouse vas deferens assay (12). tracts in search for corticotropin-releasing factor (CRF), we have isolated and structurally characterized an opioid hexa- RESULTS peptide whose first five residues are the same as in [Metlen- Isolation. As we have mentioned in the introduction, pro- kephalin. However, the sixth residue differs from that present [Metlenkephalin was a by-prqduct from the purification of in the known endorphins, and the entire sequence is not com- CRF. After gel filtration of 2 M acetic acid extracts from mon to that of f3-LPH, which is considered to be a precursor of 470,000 pig hypothalami (lyophilized weight - 3.6 kg) on a the endorphins and [Metjenkephalin (2). preparative column of Seph.dex G-25 (8, 9),'CRF activity was eluted in several fractions. In this paper we shall be concerned MATERIALS AND METHODS only with fraction 8, located in tubes 1157-1256 with RF = Purification. The extract obtained from 470,000 pig hypo- 0.39-0.36 (e!ution volume/total volume = 0.88). (see figure 1 thalami was the same as that used for the isolation of porcine of ref. 8 or 9). This fraction (166 g) was desalted by phenol ex- somatostatin (8). All the extraction and purification procedures traction (9), which reduced the weight to 14.4 g. The phenol and most methods used for homogeneity, 'composition, and extract was purified by iop-exchange chromatography on a sequence studies have been described in detail (8, 9). CM-cellulose column, as in figure 3 of ref. 9 or figure 4 of ref. High-Pressure Liquid Chromatography (HPLC). The ap- 8. Again, the CRF activity appeared in several fractions, in- paratus used for HPLC consisted of a Waters Associates model cluding tubes 136-153 with a conductivity of 2.8-5.7 mS. 204 liquid chromatograph equipped with a UK6 injector, two Fractions from three such runs were combined (total yield = 6000A pumps, a 660 programmer, and Schoeffel vuriable- 2.6 g) and subjected to 50Q transfers by countercurrent distri- wavelength detector. Reverse-phase HPLC was performed on bution in the system 0.1% acetic acid/1-butanol/pyridine Au-Bondapak C18 columns (0.4 X 30 cm) by gradient elution. (11:5:3, vol/vol), as in figure 4 of ref. 9. Fractions 230-400 The buffer consisted of 0.01 M ammonium acetate adjusted to (mean partition coefficient K = 0.9), which contained CRF pH 4.0 with acetic acid and redistilled 2-propanol as the organic activity (98.3 mg) were repurified by chromatography on SE- eluent. Sephadex. The'separation pattern can be seen in Fig. 1. Frac- Homogeneity Tests. Thin-layer chromatography (TLC) was tions 131-142, with a conductivity of 5.1-5.6 mS, contained carried out on cellulose or silica gel plates. Two solvent systems CRF activity and were lyophilized to yield 19 mg of material. The publication costs of this article were defrayed in part by page Abbreviations: CRF, corticotropin-releasing factor; f3-LPH, f3-lipo- charge payment. This article must therefore be hereby marked "ad- tropin; HPLC, high-pressure liquid chromatography; TLC, thin-layer vertisement" in accordance with 18 U. S. C. §1734 solely to indicate chromatography. this fact. * To whom reprint requests should be addressed. 6177 Downloaded by guest on September 29, 2021 6178 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 76 (1979)
1.0 -
0.8
bI 0.6 .12
0 0 .10~1 °L 0.4 -8 - E .6
0.2 - .4 v 0 *0
50 100 150 200 250 Fraction FIG. 1. Chromatography of material from countercurrent distribution on SE-Sephadex column (1.5 X 140 cm) equilibrated with 0.1 M pH 5.3 pyridine acetate buffer. Fraction size, 10 ml. A linear gradient to 0.5 M pH 5.3 buffer was started at fraction 34. A stepwise gradient to 0.75 M pH 5.3 buffer was applied at fraction 238. Hatched bars at bottom indicate CRF. This was subjected to partition chromatography on a column this peptide were performed for five steps by the combined of Sephadex G-25, using as the solvent the upper phase of a Edman-dansyl procedure. This peptide was also subjected to system containing 1-butanol/acetic acid/water (4:1:5, vol/vol) carboxypeptidase Y digestion. On the basis of these data, the (Fig. 2). Fractions 165-201, RF = 0.07, weighed 2.65 mg and sequence was established as shown in Fig. 4. showed only one major spot by TLC. When this fraction was From the amino acid analysis of the sample hydrolyzed in submitted to amino acid analysis, it was found to contain 30% 4 M methanesulfonic acid, it was not possible to assess the peptide material; however, the amino acid ratios were not in- presence of methionine sulfoxide in this peptide, because it is tegral and the fraction appeared to be heterogeneous. This hydrolyzed back to free methionine (13). Therefore, the pres- material was therefore subjected to a final purification by ence of methionine sulfoxide was established by the amino acid HPLC. Fig. 3 illustrates the HPLC elution profile. The fraction analysis of the peptide sample after digestion with carboxy- containing pro-[Met]enkephalin was present in peak IV, which peptidase Y. To confirm the structure of this hexapeptide, it was had a retention time of 10.3 min and was well separated from synthesized by solid-phase methods (11), and the purified the other three peaks. peptide was then oxidized with H202 to convert methionine Composition and Sequential Analyses. Aliquots of all four to its sulfoxide (13). The RFS of oxidized natural and synthetic peaks were subjected to amino acid analyses. The results showed peptides were then compared by TLC on silica gel in two sol- -that the first three peaks did not contain peptides with amino vent systems as shown in Fig. 5 and found to be identical. The acid compositions related to enkephalins. However, the amino retention times on HPLC of both products were also the acid composition of peak IV was found to be Gly 1.8, Met 1.0, same. Tyr 1.0, Phe 1.1, Arg 1.0. Sequential analyses using 5-10 ,gg of Opiate Activity. The ability of pro-[Metlenkephalin to in-
0.20-
0.15-
0 j O0'0.10 uL 0.05
Fraction FIG. 2. Partition chromatography of material from SE-Sephadex on a Sephadex G-25 column (1.4 X 150 cm) in 1-butanol/acetic acid/water (4:1:5, vol/vol). Fraction size, 3 ml; hold-up volume, 42 ml. Downloaded by guest on September 29, 2021 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6179
0.007
III
0.005
C.)
E) G
0.003
Iv
;.; N S N
A B 0.001 FIG. 5. TLC of the synthetic (S) and natural (N) pro-[Met(O)]- enkephalin. (A) Solvent system: 1-butanol/acetic acid/water/ethyl acetate (1:1:1:1, vol/ vol); RF = 0.40. (B) Solvent system: 1-butanol/ pyridine/acetic acid/water (15:10:3:12, vol/vol); RF = 0.45.
0 6 12 18 fact that we did not follow the opiate activity in the course of Time, min the isolation so that some of the active material was probably FIG. 3. Reverse-phase HPLC of fraction 165-201 from partition distributed in other fractions. In addition, this hexapeptide may chromatography. The peptide (100 jig) was applied to agu-Bondapak be only a transient species present during the formation of C18 column (0.4 X 30 cm) and eluted at 1.0 ml/min with 2-propanol/ [Met]enkephalin. 0.01 M ammonium acetate (pH 4.0), using a linear gradient of 2- Although small amounts of [Metlenkephalin can be formed propanol from 5% to 25% for 25 min at 25°C. by digestion of the f3-LPH COGH-terminal fragment (2, 14) with brain homogenates, the Thr-Ser sequence by which hibit electrically induced contractions of the mouse vas deferens [Met]enkephalin is attached to the remainder of f3-endorphin is shown in Table 1. This activity was inhibited by pretreatment has always appeared to be a poor cleavage site for enzymatic with naloxone. production of the pentapeptide. In addition, [Met]enkephalin and f3-endorphin can be present in separate regions in the brain DISCUSSION (15-19), suggesting that fl-endorphin is not necessarily the main In the course of our work on the purification of CRF from pig precursor for the pentapeptide. With the isolation of a hypothalamic extracts, one of the side fractions, after amino a-neo-endorphin, a possible [Leulenkephalin precursor (7) that acid analysis, revealed a composition related to enkephalins. contains the NH2-terminal [Leu]enkephalin sequence joined From the structural analyses, it was readily determined that the by a readily cleavable Arg-Lys sequence to the remainder of peptide contained an extra arginine residue attached to the a chain totally unrelated to /3-endorphin, speculation arose that COOH terminal of the [Met]enkephalin sequence Tyr-Gly- the true precursor of [Metlenkephalin might also be unrelated Gly-Phe-Met. The relatively low biological activity (4% of that to fl-endorphin. Table 2 gives the sequences of peptides that of [Met]enkephalin) of this peptide, which we named pro- enter into such considerations. [Met]enkephalin, is in agreement with the results on the syn- The hexapeptide that we have isolated from hypothalamic thetic analog Tyr-Gly-Gly-Phe-Leu-Arg reported by Kangawa tissue supports the separate precursor theory. By analogy to et al. (7). The small yield of this peptide, 150,ug from 470,000 a-neo-endorphin, Tyr-Gly-Gly-Phe-Met-Arg could be cleaved pig hypothalami (dry weight 9.9 kg), is likely to be due to the quite readily by a trypsin-like enzyme and then converted to [Met]enkephalin by carboxypeptidase B-like enzymes. It re- Table 1. Opiate activities of pro-[Metlenkephalin in the Tyr-Gly-Gly-Phe-Met (O)-Arg mouse vas deferens assay Mean effective Activity, FIG. 4. Summary of the sequence studies on the hexapeptide. Peptide dose, nM % Residues were identified by: -, two-dimensional TLC of dansyl [Met]Enkephalin 7.1 100 amino -, Y acids; carboxypeptidase digestion and amino acid anal- 260 3.7 ysis. Pro-[Metlenkephalin Downloaded by guest on September 29, 2021 6180 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 2. Amino acid sequences of f3-endorphin, [Met]enkephalin, [Leujenkephalin, and related peptides Peptide Sequence fl-Endorphin (COOH-terminal 61 65 91 sequence of f3-LPH Tyr-Gly-Gly-Phe-Met-Thr-Ser ------Gln [Met]Enkephalin Tyr-Gly-Gly-Phe-Met Pro-[Met]enkephalin Tyr-Gly-Gly-Phe-Met-Arg a-Neo-endorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Arg--- [Leu]Enkephalin Tyr-Gly-Gly-Phe-Leu
mains to be established whether pro-[Met]enkephalin is cleaved 5. Bradbury, A. F., Smith, D. G., Snell, C. R., Birdsall, N. J. M. & physiologically or only during extraction. The possibility that Hulme, E. C. (1976) Nature (London) 260,793-795. this hexapeptide is a cleavage fragment of a still larger molecule 6. Ling, N., Burgus, R. & Guillemin, R. (1976) Proc. Nati. Acad. Sct. should be considered in the light of our previous experimental USA 73,3942-3946. 7. Kangawa, K., Matsuo, H. & Igarashi, M. (1979) Biochem. Bio- peptides; Thus, we found fragments cor- findings with other phys. Res. Commun. 86,153-160. responding to residues 33-46 of porcine hemoglobin a chain 8. Schally, A. V., Dupont, A., Arimura, A., Redding, T. W., Nishi, (20) as well as fragments derived from the NH2-terminal se- N., Linthicum, G. L. & Schlesinger, D. H. (1976) Biochemistry quence 1-10 of porcine hemoglobin ,B chain (21). These frag- 15,509-514. ments were most likely formed during the dissection of hypo- 9. Schally, A. V., Nair, R. M. G., Redding, T. W. & Arimura, A. thalami from pig brains and extraction in spite of the fact that (1971) J. Biol. Chem. 246,7230-7236. various protective measures were enforced, such as keeping the 10. Hartley, B. S. (1970) Biochem. J. 119,805-822. excised tissue on dry ice, extraction at 8°C with 2 M acetic acid, 11. Coy, D. H., Kastin, A. J., Schally, A. V., Morin, O., Labrie, F., and boiling to inactivate the proteolytic enzymes (9). We be- Walker, J. M., Fertil, R., Bernston, C. G. & Sandman, C. A. (1976) lieve that the fact that the peptide was isolated with methionine Biochem. Biophys. Res. Commun. 73,632-638. is significance, be- 12. Kastin, A. J., Coy, D. H., Schally, A. V. & Meyers, C. A. (1978) in the sulfoxide form of little physiological Pharmacol. Biochem. Behav. 9,673-676. were many steps cause tiny amounts of peptide subjected to of 13. Newmann, N. P. (1967) Methods Enzymol. 11, 485-490. purification during which oxidation of methionine to methio- 14. Austen, B. M. & Smyth, D. G. (1977) Nature (London) 267, nine sulfoxide in peptides and proteins frequently occurs (22, 619-621. 23). 15. Simantov, R., Kuhar, M. J., Uhl, G. R. & Snyder, S. H. (1977) Proc. Isolation of further possible larger forms of [Metlenkephalin Natl. Acad. Sci. USA 74,2167-2171. is desirable, in order to elucidate their relationships to ,B-LPH 16. Terenius, L. (1978) Annu. Rev. Pharmacol. Toxicol. 18, 189- and a-neo-endorphin. 204. 17. Snyder, S. H. (1978) Am. J. Psychiatry 135,645-652. We are grateful to Ms. Claudia Robin for biological tests. This in- 18. Bloom, F., Battenberg, E., Rossier, J., Ling, N. & Guillemin, R. vestigation was supported by the Veterans Administration and U.S. (1978) Proc. Natl. Acad. Sci. USA 75, 1591-1595. Public Health Service Grants AM 07467, NIDA 01806, and NSO 19. Rossier, J., Vargo, T. M., Minick, S., Ling, N., Bloom, F. & Guil- 7664. lemin, R. (1977) Proc. Nati. Acad. Sct. USA 74,5162-5165. 20. Schally, A. V., Huang, W. Y., Redding, T. W., Arimura, A., Coy, 1. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., D. H., Chihara, K., Chang, R. C. C., Raymond, V. & Labrie, F. Morgan, B. A. & Morris, H. R. (1975) Nature (London) 258, (1978) Biochem. Biophys. Res. Commun. 82,582-588. 577-579. 21. Schally, A. V., Baba, Y., Nair, R. M. G. & Bennett, E. (1971) J. 2. Cox, B. M., Goldstein, A. & Li, C. H. (1976) Proc. Nati. Acad. Sd. Biol. Chem. 246,6647-6650. USA 73, 1821-1823. 22. Lo, T., Dixon, J. S. & Li, C. H. (1961) Biochim. Biophys. Acta 53, 3. Li, C. H. & Chung, D. (1976) Proc. Natl. Acad. Sci. USA 73, 584-586. 1145-1148. 23. Schally, A. V., Arimura, A., Kastin, A. J., Uehara, T., Coy, D. H., 4. Li, C. H., Lemaire, S., Yamashiro, D. & Doneen, B. A. (1976) Coy, E. J. & Takahara, J. (1973) Biochem. Biophys. Res. Com- Biochem. Biophys. Res. Commun. 71,19-25. mun. 52, 1314-1319. Downloaded by guest on September 29, 2021