A general procedure for analysis of proenkephalin B derived opioid peptides

Regulatory Peptides, 11 (1985) 65-76

65

Elsevier RPT 00365

A general procedure for analysis of proenkephalin B derived opioid peptides Ingrid Christensson-Nylander', Fred Nyberg ", Ulf Ragnarsson b and Lars Terenius b aDepartment of Pharmacology, University of Uppsala, Box 591, S-751 24 Uppsala, bDepartment of Biochemistry, University of Uppsala, Box 576, S-751 23 Uppsala, Sweden (Received 26 September 1984; revised manuscript received 18 December 1984; accepted for publication 4 March 1985)

Summary Tryptic digestion followed by radioimmunoassay for (Leu)enkephalin-Arg6 has been used in this study as a general method to detect the presence of all possible products containing the enkephalin sequence from the opioid peptide prohormone, proenkephalin B. Tissue extracts of human hypothalamus and pituitary were examined. Gel filtration was used to separate the different precursor products according to molecular weight. The elution profile was also monitored with highly sensitive radioimmunoassays for dynorphin A and dynorphin B, respectively. Immunoreactive dynorphin A appeared in three peaks with the approximate molecular weight of 1000, 2000 and 5000. Immunoreactive dynorphin B partly occurred in other peaks, 1500, 5000 and 10000 dalton. Profiles obtained by measuring immunoreactive (Leu)enkephalin-Arg6 in all fractions from gel filtration after trypsin digestion showed a more complex pattern compared to the profiles of immunoreactive dynorphin A and dynorphin B. The major peaks coincided with dynorphin A and dynorphin B but high levels of immunoreactive (Leu)enkephalin-Arg6 were also generated from higher molecular weight regions (MW > 5000). proenkephalin B; dynorphin; (Leu)enkephalin-Arg6; trypsin; hypothalamus; pituitary

Addressfor correspondence: Ingdd Nylander, Dept. of Pharmacology, Box 591, S-751 24 Uppsala, Sweden. Telephone: 018-174224. 0167-0115/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

66

Introduction

Proenkephalin B has been shown to be the precursor of three potent opioid peptides, ~-neoendorphin, dynorphin A (dyn A) and dynorphin B (dyn B = rimorphin) [1]. These peptides have been shown to be present in several species including man [2-9]. By using different chromatographic procedures and radioimmunoassays, multiple forms of proenkephalin B derived peptides have been found. Peptides of low molecular weight like dynorphin A(1-8) [9,10] as well as larger dynorphin-immunoreactive peptides [6,11-13] have been demonstrated. The processing of proenkephalin B into different peptides is still unclear but is commonly assumed to involve trypsin-like and carboxypeptidase B-like cleavage of the polypeptide into different fragments. Recently, a dynorphin converting enzyme was described [14] capable of cleavage at a single arginine residue, thus able to generate dyn A(1-8) and dyn B from dyn A and dyn-29, respectively. The opioid peptide sequences in proenkephalin B are all (Leu)enkephalin with C-terminal extension with an arginine residue. This sequence is also unique to this prohormone (Fig. 1). Digestion with trypsin followed by carboxypeptidase B, has previously been used to examine the presence of (Leu)enkephalin and (Met)enkephalin containing peptides in tissue extracts [15,16] as an indication of possible processing products of proenkephalin A (Fig. 1) in a particular tissue. A similar procedure has been used in order to examine proenkephalin B processing [17,18]. In the present study we have used trypsin digestion to generate (Leu)enkephalinArg ~ which is measured by radioimmunoassay as a general method to detect opioid peptides originating from proenkephalin B in human hypothalamus and pituitary. Gel infiltration was used to separate the different molecular sizes of precursor products in the tissue extract. Immunoreactive dyn A (ir-dyn A) and ir-dyn B were also measured with sensitive radioimmunoassays. PROENKEPHALIN

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Fig. I. Schematic structure of proenkephafin A and proenkephalin B, showing the location of enkephalin (Enk) sequences and their immediate C-terminal extensions.

67 Materials and Methods

Materials Dynorphin A, dynorphin B and (Leu)enkephalin-Arg ~ were purchased from Bachem Feinchemicalien AG, Bubendorf, Switzerland. SP-Sephadex C-25 and Sephadex G-50 (fine) were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden, and trypsin (type III) from Sigma, St. Louis, U.S.A.

Tissue extraction For the preparation of the two batches of tissue extract six human whole pituitaries and three human hypothalami without known pathology were used. The tissues were dissected out at autopsy and kept on ice until homogenizing. Tissue extraction was performed with 1 M acetic acid as follows: the acid was heated to 95"C and added to the tissue (approximately 1 ml per 150 mg), and the mixture was kept at 95"C for 5 min. After cooling on ice the tissue was homogenized, heated for another 5 min, cooled on ice and centrifuged for 20 min in a Beckman J 21 rotor at 9000 × g. The supernatant fraction was collected for further processing.

Gel filtration chromatography Gel filtration chromatography was performed on a Sephadex G-50 column (5 × 100 cm) eluted with 1 M acetic acid at a flow rate of 120 ml/h. The column was calibrated with protein and peptide standards as indicated in Fig. 3. Proteins were monitored by measuring UV absorbance at 230 nm, whereas labelled peptides were used. Fractions of 20 ml were collected. From each fraction a 10-ml aliquot was freeze-dried and later used for trypsin digestion, and 2 × 5 ml were taken to dryness in a vacuum centrifuge and assayed for ir-dyn A and ir-dyn B, respectively.

Digestion with trypsin Dried aliquots corresponding to 10 ml of each fraction from the gel filtration, were dissolved in 100 #1 0.4 M N-ethylmorpholine acetate, pH 8.2. Trypsin (100 #g) was added to each sample and incubation was performed at 37"C for 4 h. To terminate the reaction trypsin was inactivated in boiling water for 2 min, before the addition of 1-2 ml of ice-cold buffer I (0.018 M pyridine, 0.1 M formic acid), the buffer used to equilibrate the ion-exchange column. Control experiments were performed using dyn A and dyn B as substrate for trypsin. These experiments gave optimal results with 100 tag trypsin. The yield of ir(Leu)enkephalin-Arg ~ from these peptides was approximately 50%. Cerebellum, a region not containing any measurable amounts of ir-dyn material, gave no detectable ir-(Leu)enkephalin-Arg 6 in this study.

Ion-exchange chromatography To separate the mixture of peptides and fragments obtained by trypsin digestion, each sample was immediately chromatographed on a cation exchanger (SP-Sephadex C-25). The volume of the column was 1 ml and elution was performed in a stepwise manner with a mixture of pyridine and formic acid as previously described [19] (see Table I).

68 TABLE I Stepwiseelution of a cation exchangecolumn(SP-SephadexC-25) to separate peptides Fraction No. I II III IV V

Concentration (M)

Peptide eluted

Pyridine

Formic acid

0.018 0. I 0.35 0.8 1.6

0.1 0.1 0.35 0.8 1.6

(Met)- and (Leu)enkephalin (Leu)enkephalin-Arg~ Substance P, dyn B, dyn A(1--8) Dyn A, dyn B

Note howeverthat dynorphinB elutesin both fractionIV and V. Buffervolumewas 4 ml throughout.

After separation the fractions were evaporated in a vacuum centrifuge. Fraction III was redissolved in methanol/0.1 M HCI (1:1) and assayed for ir-(Leu)enkephalin-Arg 6.

Generation of antisera Conjugatkm of the peptides. Peptide (1 rag) and thyroglobulin (5 rag) were dissolved in 300 ~1 100 mM phosphate buffer, pH 7.5, and cooled to 0*C. Glutaraldehyde in 25% aqueous solution was diluted with ice-cold distilled water (1:100, v/v) and 180 /zl was added dropwise. The reaction mixture was stirred 30 rain at 0*C, then 24 h at room temperature. After extensive dialysis against 0.9% NaC1 the peptide conjugates were ready for injection into rabbits. Immunization. The conjugated peptides were emulsified with Freund's complete adjuvant and used for immunization in rabbits by intracutaneous injection (50-100 /zg) on the back of the animals at multiple sites. For booster doses, peptide conjugate emulsified in Freund's incomplete adjuvant was used as previously described [19].

Characteristics of the antisera Dynorphin A antiserum (84+). Crossreaction with dyn A(I-8), dyn A(1-13), ~neoendorphin, (Leu)enkephalin, (Leu)enkephafin-Arg~, (Met)enkephalin, neurotensin and dyn B was less than 0.1%. Crossreaction with dyn A(9-17) was 100%. Antiserum 84+ was used at a final dilution of 1:500000 in the radioimmunoassay. Detection limit was 5 fmol. Dynorphin B antiserum (113 B). Crossreaction with dyn A(1-13), dyn A(1-8), dyn A, (Leu)enkephalin, (Leu)enkephalin-Arg~, ~-neoendorphin, neurotensin and dyn A(9-17) was less than 0.01%. Antiserum 113 B was used at a final dilution of 1:500000. Detection limit was 5 fmol. (Leu)Enkephalin-Arg 6 antiserum (40 E). Crossreaction with (Met)enkephalin, (Leu)enkephalin, fl-endorphin, dyn A(1-8), dyn A, (Met)enkephalin-ArgtPhe7 was less than 0.1%; (Met)enkephalin-Lys~ 10%, (Met)enkephalin-Arg~ 50%. Antiserum

69

40 E was used in a final dilution of 1:600 in the radioimmunoassay. Detection limit was 50 fmol.

Labelled peptides Dynorphin A and B were both iodinated with the chloramine-T method. 5 #g peptide, 15/A sodium phosphate buffer (0.2 M), 0.5 mCi Na t25 I and 10 #1 chloramine-T solution in buffer (0.5 mg/ml) were allowed to react for 40 s before 10/zl sodium pyrosulfite (2 mg/ml in buffer) was added to terminate the reaction. The iodinated peptide was purified by analytical HPLC using a reversed phase column (#-Bondapak C-18, 4.5 x 250 mm, Waters) and a gradient from 15 to 40% acetonitrile in 0.04% trifluoroacetic acid. (Leu)Enkephalin-Arg° (3,5-I2) was synthetized by the solid-phase procedure [20,21] essentially, as described earlier for a set of other enkephalins [22]. Thus, Boc-Arg(NO2)-polymer, after removal of the protecting group (33% trifluoroacetic acid in CH2C12), was stepwise elongated to give Boc-Tyr(3,5-I2) (Bzl)-Gly-Gly-Phe-LeuArg(NO2)-polymer, from which the crude, free peptide could be obtained by treatment with liquid hydrogen fluoride. The peptide was purified by semi-preparative HPLC to give the pure product (Fig. 2). Amino acid analysis, after acid hydrolysis, which was accompanied by complete loss of iodine from Tyr(3,5-I2), gave: Tyr 0.97, Gly 2.03, Phe 1.00, Leu 0.99, Arg 1.00. The product was tritiated at The Radiochemical Centre, Amersham, England.

T T 0.01A 0.2 A J. .L

6

1'o

20

30rain ~

Fig. 2. HPLC ofiodinated hexapeptide: Upper trace semi-preparative purification (approx. 12 mg); lower trace rechromatography of purified product. Column:/~-Bondapak C-18 (10/an), 7.8 × 250 nun. Buffer: 0.010 M ammonium acetate/90% ethanol, gradient from 30 to 65% ethanol during 35 rain. Flow-rate 4 ml/min. Detection at UV25+.

70

Radioimmunoassays Radioimmunoassays for dyn A and dyn B followed the so-called non-immune globulin pre-precipitating method [23]. The samples from the gel filtration (a 5-ml aliquot from each fraction) were lyophilized and redissolved in 300/A methanol/0.1 M HCI (1:1). A 25-/A aliquot of the sample or standard, 100/zl antiserum, 100/~1 iodinated peptide (4500 cpm) were incubated overnight at 4"C. The antisera and labelled peptides were diluted in assay buffer containing 0.1% gelatin, 0.1% bovine serum albumin and 0.1% Triton X-100 in buffer A (see below). The samples were measured in three dilutions in order to determine parallelism with standard peptide in the radioimmunoassays. The sample dilution being closest to 50% inhibition was used to calculate the actual concentration in each sample. To separate antibodybound and free dynorphin, 50/~1 of an immunoprecipitate (see below) was added. The samples were incubated for 2 h at 4°C. After centrifugation in a Beckman Microfuge for 5 min the supernatant was discarded and the pellet was counted in a gamma counter. The immunoprecipitate used in the assay was prepared batchwise by addition of 1 ml normal rabbit serum and 10 ml sheep antirabbit serum (SBL, Solna, Sweden) to 100 ml buffer A (0.05 M sodium phosphate, 0.15 M NaC1, 0.02% sodium azide, pH 7.4). The mixture was incubated overnight at 4"C and centrifuged for 20 min at 1000 x g. After washing the pellet twice with buffer A, the pellet was resuspended in assay buffer (dependent of titre, the current batch 120 ml). Radioimmunoassay for (Leu)enkephalin-Arg 6 utilized fraction III (see Table I) of each sample from the ion-exchange procedure. Fraction III was lyophilized and redissolved in 250 #1 methanol/0.1 M HCI (1:1). A 25-/d aliquot of sample or standard, 100/zl antiserum, 100 pl labelled peptide (5500 cpm) were incubated overnight at 4°C. Antisera and labelled peptide were diluted in 0.01 M phosphate buffer containing 0.1% gelatin, 0.82% NaCI and 0.93% EDTA, pH 7.4. The samples were incubated 10 min with 200/~1 dextran-coated charcoal to separate antibody-bound and free peptide. After centrifugation for 1 min in a Beckman Microfuge, 300/A of the supernatant was counted in a liquid scintillation spectrometer.

HPLC-identification The immunoreactivity found in some fractions from the gel filtration was further identified by analytical HPLC. A reversed-phase column (/t-Bondapak C-18, 4.5 x 250 mm, Waters Assoc.) was used and eluted with a linear gradient from 20 to 45% methanol in 0.04% trifluoroacetic acid (Fig. 4). HPLC separation of enkephalyl hexapeptides was performed on a reversed-phase column (LC-18-DB, 4.6 x 250 mm, Supelco, Inc.), eluted with a linear gradient from 15 to 60% methanol in 0.04% trifluoroacetic acid. HPLC experiments were preceded with elution by buffer only, and fractions were analyzed for possible contamination by standard peptides in the column.

71

Experimental Results Elution profiles from Sephadex G-50 for hypothalamus and pituitary are shown in Fig. 3A and B, respectively. The figure illustrates the pattern obtained by measuring the ir-dyn A (upper panel, Fig. 3 A and B) and ir-dyn B (lower panel, Fig. 3 A and B) in each tissue extract. Gel filtration chromatography of the extract from hypothalamus revealed three peaks of ir-dyn A (Fig. 3 A, upper panel). Peak A appeared in the region of molecular weight 5000, peak B coeluted with synthetic dyn A and peak C, the predominant one, emerged in the low molecular weight region, i.e. approximately 1000. These results are consistent with previous reports from other authors [2,9]. HPLC was performed in order to identify these peaks more thoroughly (Fig. 4). Peak B coeluted with synthetic dyn A and peak C coeluted with dyn A(917) in this experiment. Ir-dyn B in hypothalamus showed another elution pattern (Fig. 3A, lower panel) with two minor peaks of higher molecular weight and a major peak coeluting with synthetic dyn B. The first minor peak was in the molecular weight region of 10000 and the second minor peak coeluted with peak A of ir-dyn A (MW 5000). Dilution curves of ir-dyn A and ir-dyn B of these peaks, were parallel to those of standard peptide in the radioimmunoassay.

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Fig. 3. Gel filtrationprofiles froma SephadexG-50 columnof the acetic acid extract fromhumanhypothalamus (A) and pituitary (B). The upperpanel in (A) and (B) shows the elution pattern obtainedby measuringthe ir-dyn A and the lower panel illustratesir-dyn B. The horizontalline in (13)representsthe detection limit in the radioimmunoassay.Note differencesin ordinates. Arrowsindicate: 1, void volume; 2, ribonuclease;3, #-endorphin;4, dyn A; 5, dyn B; 6, enkephalylhexapeptides;7, total volume.

72 A cO

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Fig. 4. HPLC identification of peaks A, B and C in hypothalamic extract (shown in Fig. 3A) run individually and detected with the dyn A antiserum. Column: p-Bondapak C-18, 4.5 x 250 ram. Gradient 20 to 45% methanol in 0.04% trifluoroacetic acid during 50 rain. Flow rate I ml/min. Detection at UV22o. Arrows indicate: I, dyn A(9-17); 2, dyn A(I-9); 3, dyn A(1-8); 4, dyn A; 5, ~-endorphin.

In the extract from pituitary, ir-dyn A appeared in one major peak (Fig. 3B, upper panel) coeluting with synthetic dyn A. Low levels of immunoreactivity were also detected in the same region as peak A in hypothalamus. Ir-dyn B could only be detected in pituitary at very low levels and the immunoreactivity emerged in two peaks (Fig. 3 B, lower panel): one coeluting with the synthetic peptide, and the other in the region of molecular weight 5000. In contrast to the extract from hypothalamus, no ir-dyn B was detected in the region of molecular weight 10000.

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Fig. 5. Gel filtration profile of extract from human hypothalamus (A) and pituitary (B). The figure ilh~. trates the concentration of ir-(Len)enkephalin-Arge liberated by trypsin digestion. Arrows indicate the same references as in Fig. 3,

73 Trypsin digestion of the fractions from the gel filtration, ion-exchange separation of the fragments formed by this procedure and finally radioimmunoassay of the (Leu)enkephalin-Arg6 in fraction III from the ion-exchange procedure, showed a more complex elution pattern as illustrated in Fig. 5. The profile generated from hypothalamus (Fig. 5A), shows one major peak, which appeared as expected in the region where dyn A and dyn B elute and corresponds well with the elution profiles of these peptides. HPLC analysis confirmed that 90% of the total immunoreactivity eluted as synthetic (Leu)enkephalin-Arg6. The gel filtration experiment revealed, in addition, relatively high levels of immunoreactivity in the high molecular weight region, especially in the region of 5000-10000, also in agreement with the profiles of ir-dyn A and B. A peak appearing near the total volume was also detected. Fig. 5B illustrates the ir-(Leu)enkephalin-Arg6 generated from the pituitary extract. Fractions 35-55 contained material interfering in the radioimmunoassay and are not represented in the figure. As compared with the profiles of ir-dyn A and ir-dyn B from this tissue, surprisingly high levels of the hexapeptide were detected, especially in the high molecular weight region. HPLC experiments of the immunoreactivity in this region showed one peak (approximately 75% of the total immunoreactivity) coeluting with synthetic (Leu)enkephalin-Arg~, but also other peaks were detected. Like the profile from hypothalamus, immunoreactivity also appeared in the same region as the dynorphin peptides and near the total volume.

Discussion

The present study illustrates the use of a general method to investigate proenkephalin B derived peptides containing the enkephahn sequence, in a particular tissue. Tryptic digestion of a tissue extract containing such peptides generates (Leu)enkephalin-Arg6 as a final product. Processing of proenkephalin B and also the further processing of the putative opioid peptide products still is unclear. The most general assay procedure to study the expression of a-neovndorphin/dynorphin peptides, is to digest them with trypsin and measure the generated (Leu)enkephalin-Arg~. As an application of this method, human hypothalamus and pituitary tissue extracts were chromatographically separated according to molecular weight, the fractions subjected to tryptic digestion and assayed for (Leu)enkephalin-Arg6. In hypothalamus the ir-(Leu)enkephalin-Arg6 profile corresponded to the profiles of ir-dyn A and ir-dyn B respectively, although rather high levels of immunoreactivity were also detected in the high molecular weight region. In contrast to the low levels of irdyn A and ir-dyn B in pituitary, high levels of ir-(Leu)enkephalin-Arg~ were detected in tryptic digests, especially in the high molecular weight region but also in the regions of dyn A and dyn B. These results could suggest the presence of high molecular weight dynorphin peptides in pituitary not crossreacting in the radioimmunoassays for dyn A and dyn B. Another explanation for the high levels of ir-(Leu)enkephalinArg 6, is the presence of peptide products other than (Leu)enkephalin-Arg~, for example (Met)-enkephalin-Arg~, crossreacting in the radioimmunoassay. Tryptic digestion also generates products from the other opioid peptide precursors, proen-

74

kephalin A and proopiomelanocortin. However, the use of an ion exchange separation step before the radioimmunoassay, reduces the possible crossreacting peptides. The HPLC experiments performed to identify the immunoreactivity found in hypothalamus and pituitary, showed that most of the ir-(Leu)enkephalin-Arg 6 (90%) found in hypothalamus, in fact coeluted with the synthetic peptide. In pituitary 75% of the ir-(Leu)enkephalin-Arg ~ coeluted with synthetic peptide, indicating that after all proenkephalin B derived peptides are in majority in the pituitary extracts. High levels of ir-dyn A and ir-dyn B were detected in human hypothalamus as compared to pituitary. Ir-dyn A separated into three peaks after gel filtration, the one coeluting with synthetic dyn A only representing approximately 30% of the total immunoreactivity. The predominant peak eluted in the same molecular weight region as dyn A(9-17), (a fragment crossreacting to 100% in the radioimmunoassay), and the peak also coeluted with dyn A(9-17) on the HPLC column. Dyn A(9-17) would form in the processing of dyn A into dyn A(1-8), a peptide shown to be present in several brain regions of rat and man [9,10]. In contrast to ir-dyn A, ir-dyn B eluted in one major peak (approximately 90% of the total immunoreactivity) coeluting with synthetic dyn B, suggesting dyn B to be more stable while dyn A is further processed. The possible processing of dyn A with a specific enzyme yielding dyn A(1-8) has been described [14], and this would be in agreement with the non-equivalent levels of ir-dyn A and ir-dyn B found in this study, with lower levels of dyn A compared to dyn B. Pituitary extracts contained very low levels of dynorphin immunoreactivity. Ir-dyn A appeared in one major peak coeluting with synthetic dyn A and ir-dyn B emerged in two barely detectable peaks, one coeluting with the synthetic peptide while the other eluted in the high molecular weight region. Studies in the rat have shown that whereas the hypothalamus contains both cell bodies and terminals of ir-dyn fibers [24,25], ir-dyn in the pituitary is primarily in terminals of a hypothalamic-hypophyseal pathway [26]. Ir-dyn of the human pituitary is also essentially in the neurointermediate lobe [2]. A corresponding hypothalamic-hypophyseal pathway with the neuropeptide vasopressin has been studied extensively [27]. By comparison there seems to be relatively more high molecular weight precursors for dynorphin peptides than for vasopressin in the pituitary. The comparatively very low levels of dyn A and B in pituitary suggest a different processing of proenkephalin B in the hypothalamichypophyseal pathway, than in CNS pathways contributing to the observed immunoreactivity in the hypothalamus.

Acknowledgements The authors are grateful to Ms. Inga Hansson for skillful technical assistance. This work was supported by the Swedish Medical Research Council (Grant No. 04X3766), the National Institute on Drug Addiction (Grant No. DA 1503), the Bank of Sweden Tercentenary Foundation (Grant No. 83/208) and the Swedish Natural Science Research Council (Grant No. K-KU 3020-112).

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References 1 Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, Y., Hirose, T., Asai, M., Inayama, S., Nakanishi, S. and Numa, S., Cloning and sequence analysis of cDNA for porcine ~-neo-endorphin/dynorphin precursor, Nature, 298 (1982) 245-249. 2 Gramsch, C., H611t, V.,/,asi, A., Mehraein,/,. and Herz, A., Immunoreactive dynorphin in human brain and pituitary, Brain Res., 233 (1982) 65-74. 3 Suda, T., Tozawa, F., Tachibana, S., Demura, H., Shizume, K., Sasaki, A., Mouri, T. and Miura, Y., Multiple forms of immunoreactive dynorphin in human pituitary and pheochromocytoma, Life Sci., 32 (1983) 865-870. 4 Goldstein, A. and Ghazarossian, V. E., Immunoreactive dynorphin in pituitary and brain,/'roe. Nail. Acad. Sci. USA, 77 (1980) 6207-6210. 5 Cone, R.I., and Goldstein, A., A specific radioimmunoassay for the opioid peptide dynorphin B in neural tissues, Nenropeptides, 3 (1982) 97-106. 6 Cone, R.I., Weber, E., Barchas, J.D. and Goldstein, A., Regional distribution of dynorphin and neo-endorphin peptides in rat brain, spinal cord, and pituitary, J. Neurosci., 3 (1983) 2146-2152. 7 Kilpatrick, D.L., Wahlstr6m, A., Lahm, H.W., Blacher, R. and Udenffiend, S., Rimorphin, a unique, naturally occurring (Leu)enkephalin containing peptide found in association with dynorphin and ~neo-endorphin, Proc. Natl. Acad. Sci. USA, 79 (1982) 6480-6483. 8 Maysinger, D., H611t, V., Seizinger, B.R., Mehracin, P., Pasi, A. and Herz, A., Parallel distribution of immunoreactive ~-neo-endorphin and dynorphin in rat and human tissue, Nenropeptides, 2 (1982) 211-225. 9 Pittius, C.W., Seizinger, B.R., Pasi, A., Mehraein, P. and Herz, A., Distribution and characterization of opioid peptides derived from proenkephaiin A in human and rat central nervous system, Brain Res., 304 (1984) 127-136. 10 Weber, E., Evans, C.J. and Barchas, J.D., Predominance of the amino-terminal octapeptide fragment of dynorphin in rat brain regions, Nature, 299 (1982) 77-79. 11 Fischli, W., Goldstein, A., Hunkapiller, M.W. and Hood, L.E., Isolation and amino acid sequence analysis of a 4,000-dalton dynorphin from porcine pituitary,/,roe. Natl. Acad. SCI. USA, 79 (1982) 5435-5437. 12 Seizinger, B.R., Grimm, C., Htllt, V. and Herz, A., Evidence for a selective processing of proenkephalin B into different opioid peptide forms in particular regions of rat brain and pituitary, J. Neurochem., 42 (1984) 447-457. 13 Zhu, Y.X., H611t, V. and Loh, H., Immunoreactive peptides related to dynorphin B (= rimorphin) in the rat brain, Peptides, 4 (1983) 871-874. 14 Devi, L. and Goldstein, A., Dynorphin converting enzyme with unusual specificity from rat brain, Proc. Nail. Acad. Sci. USA, 81 (1984) 1892-1896. 15 Kojima, K., Kilpatrick, D.L., Stern, A.S., Jones, B.N. and Udenfriend, S., Proenkephalin: A general pathway for enkephalin biosynthesis in animal tissues, Arch. Biochem. Biophys., 215 (1982) 638--643. 16 Lewis, R.V., Stern, A.S., Kimura, S., Rossier, J., Stein, S. and Udenfriend, S., An about 50,000-dalton protein in adrenal medulla: a common precursor of (Me0- and (Leu)-enkephalin, Science, 208 (1980) 1459-1461. 17 Nakao, K., Yoshimasa, T., Suda, M., Sakamoto, M., Ikeda, Y., Hayashi, K. and Imura, H., Rimorphin (dynorphin B) exists together with a-neo-endorphin and dynorphin (dynorphin A) in human hypothalamus, Biochem. Biophys. Res. Commun., 113 (1983) 30-34. 18 Kangawa, K., Mizuno, K., Minamino, N. and Matsuo, H., Radioimmunoassay for detecting prc~ Lcu-enkephalins in tissue extracts: purification and identification of (Argt)-Leu-enkephalin in porcine pituitary, Biocbem. Biophy. Res. Commun., 95 (1980) 1467-1474. 19 Bergstrtm, L., Christensson, I., Folkesson, R., Stenstr6m, B. and Tereuius, L., An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P., Life Sci., 33 (1983) 1613-1619. 20 Merrifield, R.B., Solid phase peptide synthesis. I. The synthesis of a tetrapeptide, J. Am. Chem. Soc., 85 (1963) 2149-2154.

76 21 Barany, G. and Merrifleld, R.B., Solid phase peptide synthesis. In E. Gross and J. Meinhofer (Eds.), The Peptides, Vol. 2, Academic Press, New York, 1980, pp. 1-284. 22 Terenius, L., Wahlstr6m, A., Lindeberg, G., Karlsson, S. and Ragnarsson, U., Opiate receptor atfmity of peptides related to Leu-enkephalin, Biochem. Biophys. Res. Commun., 71 (1976) 175-179. 23 Midgley, A.R. and Hepburn, M.R., Use of the double-antibody method to separate antibody bound from free ligand in radioimmunoassay. In H.V. Vunakis and J.J. Langine (Eds.), Methods in Enzymology, Vol. 70, Immunochemical techniques part A, Academic Press, New York, 1980, pp. 273-274. 24 Vincent, S.R., H6kfelt, T., Christensson, I. and Terenius, L., Dynorphin-immunoreactive neurons in the central nervous system of the rat, Neurosci. Lett., 33 (1982) 185-190. 25 Watson, S.J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van WimersmaGreidanus, Tj.B., Dynorphin and vasopressin: common localization in magnoceUular neurons, Science, 216 (1982) 85-87. 26 Palkovits, M., Brownstein, M.J. and Zamir, N., Immunoreactive dynorphin and a-neo-endorphin in rat hypothalamo-neurohypophyseal system, Brain Res., 278 (1983) 258--261. 27 Brownstein, M.J., Russell, J.T. and Gainer, H., Synthesis, transport and release of posterior pituitary hormones, Science, 207 (1980) 373-378.