- Email: [email protected]
Novel opioid peptides derived from mitochondrial cytochrome b: Cytochrophins
European Journal of Pharmacology,
111 (1985) 293-294
293
Elsevier
Rapid communication N O V E L O P I O I D P E P T I D E S DERIVED F R O M M I T O C H O N D R I A L C Y T O C H R O M E b: CYTOCHROPHINS VICTOR BRANTL ¢, CHRISTIAN GRAMSCH *, FRIEDRICH LOTTSPEICH **, AGNES HENSCHEN ** KARL-HEINZ JAEGER *** and ALBERT HERZ * Boehringer Ingelheim KG, D - 6507 lngelheim am Rhein, F.R.G., * Max - Planck - Institut fftr Psychiatrie, D - 8033 Planegg- Martinsried, F.R.G., ** Max-Planck-lnstitut fftr Bioehemie, D-8033 Planegg-Martinsried, F.R.G., *** Organopharm AG, Alpenquai 40, CH-6005 Luzern, Switzerland
Received 3 April 1985, accepted 3 April 1985
Enzymatically treated protein preparations are known to contain opioid peptides such as flcasomorphins, which were originally isolated from casein peptone (Brantl, 1981). We have therefore evaluated whether a large variety of commercially available digests of protein-containing mammalian tissue and body fluids might contain novel opioid peptides. In a pharmaceutical preparation manufactured by Organopharm AG, CH-6005 Luzern, H~imUvocal ® (for injection), batch 012, which was obtained by treatment of bovine blood with gastrointestinal enzymes, we could recently demonstrate the presence of opioid activity by use of the electrically stimulated myenteric plexus/longitudinal muscle preparation of the guinea-pig ileum, G P I (detailed method, see Brantl, 1981). For the isolation and structure analysis of the opioid, we adopted methods previously employed for the isolation of fl-casomorphins (Brantl, 1981). A single peak displaying opioid activity could be obtained at the final purification step by use of high pressure liquid chromatography (HPLC). The material isolated proved to be pure and the following peptide structure could be determined by amino acid analysis/Edman degradation: Tyr-Pro-PheThr. A computer search for this sequence revealed that this tetrapeptide represents fragment 345-348 of mitochondrial cytochrome b (Anderson et al., * To whom all correspondence should be addressed: Boehringer Ingelheim KG, Abteilung Medizin, D-6507 Ingelheim am Rhein, F.R.G. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
1981). This tetrapeptide and the C-terminally extended pentapeptide (Tyr-Pro-Phe-Thr-Ile, fragment 345-349 of mitochondrial cytochrome b), were sythesized by Novabiochem AG, CH-4448 L~iufelfingen, Switzerland and tested for their opioid activity in the GPI. Table 1 presents the opioid activities of these novel opioid peptides Tyr-Pro-Phe-Thr and TyrPro-Phe-Thr-Ile in comparison to structurally related peptides (Brantl, 1984). In view of their origin, activity, and chain length the tetrapeptide and pentapeptide were named cytochrophin-4 and -5, respectively. The cytochrophins are less active
TABLE 1 Opioid activities of cytochrophins in comparison to human/bovine fl-casomorphinsand normorphine; values indicate concentrations (/LM) causing 50% inhibition (ICs0) of electrically induced contractions of the guinea-pig ileum myenteric plexus/longitudinal muscle preparation (GPI); mean valises from 4-5 determinations, standard deviations were less than 13% of mean values. Inhibition of GPI was both reversed and prevented by the specific opioid antagonist naloxone (0.5 tLM). Substance Cytochrophin-4 hfl-Casomorphin-4 fl-Casomorphin-4 Cytochrophin-5 hfl-Casomorphin-5 fl-Casomorphin-5 Normorphine
(Tyr-Pro-Phe-Thr) (Tyr-Pro-Phe-Val) (Tyr-Pro-Phe-Pro) (Tyr-Pro-Phe-Thr-Ile) (Tyr-Pro-Phe-Val-Glu) (Tyr-Pro-Phe-Pro-Gly)
* Data from Brantl (1984).
GPI 120.1 56.2 * 14.3 * 290.4 33.1 * 2.0 * 0.1
294
in the GPI (i.e. have higher IC50 values) than do bovine and human fl-casomorphins. Several types of opioids have been reported to occur in mammalian blood but have not been structurally characterized (Pert et al., 1976; Schulz et al., 1977). However, it is unlikely that these opioids were cytochrophin-4 and -5 since Pert et al. (1976) reported that hypophysectomised animals showed a reduction of 96-98% in opioid activity in the blood, indicating that the material originated from the hypophysis. The material characterized by Schulz et al. (1977) is furthermore pronase-resistant, whereas the cytochrophins are susceptible to rapid degradation by this enzyme (data not shown, pronase stability test performed as described, Brantl, 1981). Nevertheless, the cytochrophins, which represent degradation products of blood incubated with gastrointestinal enzymes such as pepsin and pancreatin, are evidently stable towards these latter types of enzyme. This is in good agreement with previous findings indicating that a proline residue in position 2 stabilizes the N-terminal tripeptide structure (Tyr-Pro-Phe, which is essential for opioid activity) against proteolytic enzymes. The question as to whether cytochrophins may
be released in the mammalian organism upon degradation of cytochrome b-containing material under physiological or pathological conditions (for example gastrointestinal bleeding) and whether they may exert their opioid actions remains to be clarified.
References Anderson, S., A.T. Bankier, B.G. Barrell, M.H.L. De Bruijn, A.R. Coulson, J. Drouin, I.C. Eperon, D.P. Nierlich, B.A. Roe, F. Sanger, P.H. Sehreier, A.J.H. Smith, R. Staden and I.G. Young, 1981, Sequence and organisation of the human mitochondrial genome, Nature 290, 457. Brantl, V., 1981, Isolation of pharmacologically active peptides by high pressure liquid chromatography (HPLC), in: HPLC in Protein and Peptide Chemistry, eds. F. Lottspeich, A. Henschen and K.-P. Hupe (Walter de Gruyter, Berlin) p. 365. Brantl, V., 1984, Novel opioid peptides derived from human fl-casein: Human fl-casomorphins, European J. Pharmacol. 106, 213. Pert, C.B., A. Pert and J.F. Tallmann, 1976, Isolation of a novel endogenous opiate analgesic from human blood, Proc. Natl. Acad. Sci. U.S.A. 73, 2226. Schulz, R., M. Wi~ster and A. Herz, 1977, Detection of a long acting endogenous opioid in blood and small intestine, Life Sci. 21, 105.
111 (1985) 293-294
293
Elsevier
Rapid communication N O V E L O P I O I D P E P T I D E S DERIVED F R O M M I T O C H O N D R I A L C Y T O C H R O M E b: CYTOCHROPHINS VICTOR BRANTL ¢, CHRISTIAN GRAMSCH *, FRIEDRICH LOTTSPEICH **, AGNES HENSCHEN ** KARL-HEINZ JAEGER *** and ALBERT HERZ * Boehringer Ingelheim KG, D - 6507 lngelheim am Rhein, F.R.G., * Max - Planck - Institut fftr Psychiatrie, D - 8033 Planegg- Martinsried, F.R.G., ** Max-Planck-lnstitut fftr Bioehemie, D-8033 Planegg-Martinsried, F.R.G., *** Organopharm AG, Alpenquai 40, CH-6005 Luzern, Switzerland
Received 3 April 1985, accepted 3 April 1985
Enzymatically treated protein preparations are known to contain opioid peptides such as flcasomorphins, which were originally isolated from casein peptone (Brantl, 1981). We have therefore evaluated whether a large variety of commercially available digests of protein-containing mammalian tissue and body fluids might contain novel opioid peptides. In a pharmaceutical preparation manufactured by Organopharm AG, CH-6005 Luzern, H~imUvocal ® (for injection), batch 012, which was obtained by treatment of bovine blood with gastrointestinal enzymes, we could recently demonstrate the presence of opioid activity by use of the electrically stimulated myenteric plexus/longitudinal muscle preparation of the guinea-pig ileum, G P I (detailed method, see Brantl, 1981). For the isolation and structure analysis of the opioid, we adopted methods previously employed for the isolation of fl-casomorphins (Brantl, 1981). A single peak displaying opioid activity could be obtained at the final purification step by use of high pressure liquid chromatography (HPLC). The material isolated proved to be pure and the following peptide structure could be determined by amino acid analysis/Edman degradation: Tyr-Pro-PheThr. A computer search for this sequence revealed that this tetrapeptide represents fragment 345-348 of mitochondrial cytochrome b (Anderson et al., * To whom all correspondence should be addressed: Boehringer Ingelheim KG, Abteilung Medizin, D-6507 Ingelheim am Rhein, F.R.G. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
1981). This tetrapeptide and the C-terminally extended pentapeptide (Tyr-Pro-Phe-Thr-Ile, fragment 345-349 of mitochondrial cytochrome b), were sythesized by Novabiochem AG, CH-4448 L~iufelfingen, Switzerland and tested for their opioid activity in the GPI. Table 1 presents the opioid activities of these novel opioid peptides Tyr-Pro-Phe-Thr and TyrPro-Phe-Thr-Ile in comparison to structurally related peptides (Brantl, 1984). In view of their origin, activity, and chain length the tetrapeptide and pentapeptide were named cytochrophin-4 and -5, respectively. The cytochrophins are less active
TABLE 1 Opioid activities of cytochrophins in comparison to human/bovine fl-casomorphinsand normorphine; values indicate concentrations (/LM) causing 50% inhibition (ICs0) of electrically induced contractions of the guinea-pig ileum myenteric plexus/longitudinal muscle preparation (GPI); mean valises from 4-5 determinations, standard deviations were less than 13% of mean values. Inhibition of GPI was both reversed and prevented by the specific opioid antagonist naloxone (0.5 tLM). Substance Cytochrophin-4 hfl-Casomorphin-4 fl-Casomorphin-4 Cytochrophin-5 hfl-Casomorphin-5 fl-Casomorphin-5 Normorphine
(Tyr-Pro-Phe-Thr) (Tyr-Pro-Phe-Val) (Tyr-Pro-Phe-Pro) (Tyr-Pro-Phe-Thr-Ile) (Tyr-Pro-Phe-Val-Glu) (Tyr-Pro-Phe-Pro-Gly)
* Data from Brantl (1984).
GPI 120.1 56.2 * 14.3 * 290.4 33.1 * 2.0 * 0.1
294
in the GPI (i.e. have higher IC50 values) than do bovine and human fl-casomorphins. Several types of opioids have been reported to occur in mammalian blood but have not been structurally characterized (Pert et al., 1976; Schulz et al., 1977). However, it is unlikely that these opioids were cytochrophin-4 and -5 since Pert et al. (1976) reported that hypophysectomised animals showed a reduction of 96-98% in opioid activity in the blood, indicating that the material originated from the hypophysis. The material characterized by Schulz et al. (1977) is furthermore pronase-resistant, whereas the cytochrophins are susceptible to rapid degradation by this enzyme (data not shown, pronase stability test performed as described, Brantl, 1981). Nevertheless, the cytochrophins, which represent degradation products of blood incubated with gastrointestinal enzymes such as pepsin and pancreatin, are evidently stable towards these latter types of enzyme. This is in good agreement with previous findings indicating that a proline residue in position 2 stabilizes the N-terminal tripeptide structure (Tyr-Pro-Phe, which is essential for opioid activity) against proteolytic enzymes. The question as to whether cytochrophins may
be released in the mammalian organism upon degradation of cytochrome b-containing material under physiological or pathological conditions (for example gastrointestinal bleeding) and whether they may exert their opioid actions remains to be clarified.
References Anderson, S., A.T. Bankier, B.G. Barrell, M.H.L. De Bruijn, A.R. Coulson, J. Drouin, I.C. Eperon, D.P. Nierlich, B.A. Roe, F. Sanger, P.H. Sehreier, A.J.H. Smith, R. Staden and I.G. Young, 1981, Sequence and organisation of the human mitochondrial genome, Nature 290, 457. Brantl, V., 1981, Isolation of pharmacologically active peptides by high pressure liquid chromatography (HPLC), in: HPLC in Protein and Peptide Chemistry, eds. F. Lottspeich, A. Henschen and K.-P. Hupe (Walter de Gruyter, Berlin) p. 365. Brantl, V., 1984, Novel opioid peptides derived from human fl-casein: Human fl-casomorphins, European J. Pharmacol. 106, 213. Pert, C.B., A. Pert and J.F. Tallmann, 1976, Isolation of a novel endogenous opiate analgesic from human blood, Proc. Natl. Acad. Sci. U.S.A. 73, 2226. Schulz, R., M. Wi~ster and A. Herz, 1977, Detection of a long acting endogenous opioid in blood and small intestine, Life Sci. 21, 105.