We describe the opioid properties of a tridecapeptide, the sequence of which corresponds to the NH2-terminal sequence of dynorphin, a novel porcine pituitary endorphin. It The first pituitary opioid peptide to be discovered (1, 2) had properties quite different from those of /3-endorphin. It had a lower apparent molecular weight, was more basic, and had a more persistent effect in the guinea pig ileum bioassay. Also, its biologic activity (unlike that of f3-endorphin) was resistant to destruction by cyanogen bromide. The final stages of purification presented special problems, which led to erroneous conclusions about composition and the presence of a blocked NH2 terminus (3). Losses due to adsorption were particularly troublesome, and the material proved to be remarkably potent; consequently, the yield of purified product was much less than anticipated. However, by means of microsequencing technology (4, 5), we have now been able to determine the sequence of the first 13 residues.To denote its extraordinary potency, the natural peptide has been named "dynorphin" (dyn-from Greek dynamis = power). Synthetic dynorphin-(1-13) proved to have approximately the same high potency as natural dynorphin. We describe here some properties of this synthetic tridecapeptide. MATERIALS AND METHODSPurification and Partial Sequence Determination of Porcine Dynorphin. Starting material was 100 g of melanotropin concentrate, the second oxycellulose adsorbate in commercial corticotropin production from porcine pituitary glands (6). The initial steps, with 25-g batches, have been described (2): extraction and back-extraction with butanol; separation from ,B-endorphin on Bio-Gel P-6; preparative reversed-phase high-performance liquid chromatography on C18 columns, first with a methanol gradient in trifluoroacetic acid and then with an acetonitrile gradient in Tris buffer at neutrality, followed by elution of the active material with trifluoroacetic acid. Every step was monitored by assay on the guinea pig ileum myenteric plexus longitudinal muscle preparation (7,8).Pooled material from four batches, obtained as above, was loaded on a Bio-Gel column (P-6/P-4, 4:1; 1.5 X 90 cm) in 0.1 M (NH4)2CO0 at pH 8.7. Fractions (1.7 ml, 15 min) were tested for activity in the bioassay. The peak of slow-reversing activity (2) (fractions 65-75) was lyophilized and then further purified on CM-Sephadex (0.9 X 30 cm) equilibrated with 12.5 mM sodium borate at pH 11.0. The material was eluted with a 160-ml linear gradient from the starting buffer to 0.1 M phosphate buffer at pH 12.0. Fractions (2.0 ml; 15 min) were collected and assayed. Slow-reversing activity emerged between 94 and 106 ml. The two peak tubes, containing 50% of the activity, were pooled and desalted on Bio-Gel P-2 (1.5 X 90 cm) in n-butanol/acetic acid/water, 2:1:4 (vol/vol).Finally, the desalted material was subjected to reverse-phase chromatography (high-performance liquid chromatography, C18 column) using a 10-50% acetonitrile gradient in 5 mM trifluoroacetic acid. The pea...
The full primary structure of the very potent opioid peptide dynorphin, from porcine pituitary, has been determined. It is (H)Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-LysLeu-Lys-Trp-Asp-Asn-Gln(OH). The synthetic peptide with this sequence behaves identically to natural dynorphin in a number ofways, and it has the same potency in the guiinea pig ileum myenteric plexus-longitudinal muscle bioassay. The potency is accounted for by the first 13 residues.
A method is described for analyzing the association of the opiate narcotic levorphanol with brain tissue into three components: nonsaturable, saturable nonspecific, and saturable stereospecific. The method may be of general applicability for the study of the interaction of drugs with body tissues. In mouse brain the stereospecific binding of levorphanol represents only 2% of the total association of drug with tissue, and it was found only in certain membrane fractions. The material responsible for the stereospecific binding might be the opiate receptor.
We describe nonpeptide opioids found in extracts of beef hypothalamus and adrenal, which are recognized by antisera raised against morphine. Four have been purified to homogeneity. One is morphine. The structures of the other three have not been determined yet. None of them are derived from morphine or normorphine after extraction from the tissues. It is not known whether the opiates described here are of endogenous or exogenous origin.In 1973 one of us (1) postulated the existence of an endogenous opioid in brain and attempted to detect it by immunoassay, using an antiserum raised against morphine. In mouse brain no morphine immunoreactivity (ir-morphine) could be found at a detection limit of 16 pmol per brain. In 1976, however, Gintzler et al. (2) reported the presence of irmorphine in rabbit and cat brain, and similar findings have been published by this (3-7) and other (8, 9) groups from time to time. To date, no proof of structure of any of these substances from mammalian tissues has been published. However, Hazum et al. (10) isolated a morphine-immunoreactive compound from cow's milk, which behaved identically to morphine in three HPLC systems and in mass spectrometry. They suggested the likelihood of a dietary source and commented that they had found ir-morphine in various animal fodders.The investigations reported here were stimulated by the conspicuous absence of an endogenous opioid with high selectivity for the p. opioid receptor (11,12). The positive findings cited above, as well as earlier ideas of Davis and Walsh (13) about a possible endogenous biosynthesis of morphine in mammalian brain, led us to resume the search interrupted a decade earlier. We have now identified several immunoreactive morphine-like substances in beef brain and adrenal. We have purified four of them to homogeneity and determined the structure of one, which is morphine.MATERIALS AND METHODS Materials. Peptides were obtained from Peninsula Laboratories (Belmont, CA), Bachem Fine Chemicals (Torrance, CA), Biosearch (San Rafael, CA), or Pierce. Purity of all compounds was verified by HPLC, either by the supplier or by us; when necessary, we purified them by reversed-phase HPLC. The following were gifts: metorphamide, from E. Weber; oripavine, from A. Jacobson and E. BrochmannHansen; thebaine, codeinone, and reticuline, from E. Brochmann-Hansen. Various opioids and related compounds were obtained from the indicated suppliers:
Recently, we described the presence of six immunoreactive (ir) morphinans in bovine adrenal and hypo-
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