Proc. NatL Acad. Sci. USA Vol. 79, pp. 6480-6483, November 1982 Biochemistry

Rimorphin, a unique, naturally occurring tLeulenkephalin- containing found in association with -and a-neo-endorphin* (posterior pituitary gland/microanalysis of sequence) D. L. KILPATRICKt, A. WAHLSTROMt, H. W. LAHMt, R. BLACHERt, AND S. UDENFRIENDt tRoche Institute of Molecular Biology, Nutley, New Jersey 07110; and tDepartment of Molecular Genetics, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110 'Contributed by Sidney Udenfriend, August 2, 1982

ABSTRACT The tridecapeptide NH2-Tyr-Gly-Gly-Phe-Leu- 20,000 8,000 2)000 500 Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-COOH has been purified from 1400 4 4 4 extracts ofbovine posterior pituitary glands. This unique peptide, I which has been given the name "rimorphin," is a major [Leu]- 1200 H -containing peptide in all tissues examined that contain 0 dynorphin and a-neo-endorphin. However, except for the initial hexapeptide sequence, it is structurally unrelated to the other two 0.> 1000 K 0.2 0 ...... . 0...... 800 F I The [Leu]enkephalin-containing peptides ([Leu]ECPs) dynor- a phin and a-neo-endorphin have been shown to possess potent (3 ^ cc activity in certain in vitro bioassay systems (1, 2). These 600 H r_ peptides have been detected in several tissues, including hy- *1 Q) 0.1 pothalamus, spinal cord, and posterior pituitary gland (1, 3-6). P. 400 H 0 However, there have been no studies attempting to character- cWa) Ioq ize the other ECPs present in tissues that contain a-neo-en- r- dorphin and dynorphin. During such studies in the bovine pos- 200 - terior pituitary gland we observed a unique [Leu]ECP that did not correspond to any previously identified peptide. Details of o the isolation and structural characterization ofthis new [Leu]ECP, which we have given the trivial name "rimorphin," are pre- 17 29 41 53 65 77 sented. The distribution ofrimorphin in tissues from three dif- Fraction ferent species indicates a close relationship to dynorphin and a-neo-endorphin. FIG. 1. Sephadex G-75 chromatography of bovine posterior pitui- tary extracts. Tissues were extracted in 1 M~acetic acid/20 mM HC1 containing 0.1% 2-mercaptoethanol and 1 ,ug each of phenylmethane- METHODS sulfonyl fluoride and pepstatin A per ml and chromatographed. Col- Fresh bovine pituitary glands were obtained from the local umn fractions were then assayed for . Results are shown slaughterhouse and dissected into posterior and anterior lobes. as pmol/g of tissue. The posterior lobes were carefully scraped to remove the pars intermedia and were then frozen in liquid nitrogen. A total of Edman degradations were performed in a modified Beckman 60 posterior pituitary glands were used in three separate iso- 890C sequencer (13-15) on 2 nmol of pure rimorphin. Opiate lation experiments. Tissue extraction and fractionation on Seph- activity of rimorphin was assayed in the guinea pig ileum lon- adex G-75 were carried out as described (7). Reverse-phase gitudinal muscle preparation (16) and by a radioreceptor assay HPLC was performed on an Ultrasphere octadecylsilane (ODS) (17). column (5 ,Am particle size; 4.6 x 250 mm) and a Macherey-Nagel Nucleosil phenyl column (7 ,um particle size; 4.6 X 250 mm) RESULTS (Rainen Instruments, Ridgefield, NJ). Peptides were eluted Chromatography ofbovine posterior pituitary extracts on Seph- from the columns with gradients ofl-propanol in 0.9 M pyridine adex G-75 revealed the presence of many ECPs, ranging from acetate at pH 4.0. A fluorescamine detection system was used 500 to 8,000 in Mr (Fig. 1). Rechromatography of the Mr to monitor peptide content of the column eluants (8) and ali- 500-2,000 region on a calibrated Ultrasphere ODS column re- quots of each fraction were digested with trypsin and carboxy- vealed the presence ofmany ECPs, including [Leu]enkephalin, peptidase B prior to assay for [Met]- and [Leu]enkephalin im- [Met]enkephalin, [Met]enkephalin-Arg6-Phe7, [Met]enkephalin- munoreactivity (9, 10). Crossreactivities between the two RIAs Arg6-Gly7-Leu8, a-neo-endorphin, dynorphin-(1-8), and dy- were approximately 10% for each pentapeptide. Amino acid norphin-(1-17). In addition, there consistently was observed a analysis was carried out in a fluorescamine analyzer (11) on 50- [Leu]ECP that elutedjust after [Met]enkephalin-Arg6-Phe7 and to 200-pmol samples. Carboxypeptidase Y time course hydro- did not correspond to any previously characterized ECP. This lysis was carried out according to Jones et al. (12). Automated Abbreviations: ODS, octadecylsilane; [Leu]ECP, [Leu]enkephalin- The publication costs ofthis article were defrayed in part by page charge containing peptide. payment. This article must therefore be hereby marked "advertise- * Part of this work was presented at the International Re- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. search Conference, June 14-18, 1982, North Falmouth, MA.

6480 Downloaded by guest on September 30, 2021 Biochemistry: Kilpatrick et aL Proc. Nati. Acad. Sci. USA 79 (1982) 6481

; 15 O 10 100 0 5 ;O e-1

C.)a) o _ a)

4-4

E a) R e

._ Xd 5: 4) 0._C)_4"1

50 60 70 80 90 100 Fraction FIG. 2. Purification of rimorphin on an Ultrasphere ODS column. Pooled fractions from Sephadex G-75, corresponding to Mr 500-2,000, were applied to the column. Peptides were eluted with a linear gradient of 1-propanol (----) in 0.9 M pyridine acetate (pH 4.0) at a flow rate of 24 ml/hr. Aliquots of each fraction (1 ml) were assayed for enkephalins. A [Leu]ECP (denoted here as L-5) was eluted shortly after [Metlenkephalin- Arg6-Phe7. is designated as L-5 in Fig. 2. This unique [Leu]ECP was pu- a small peptide ofapproximately 13 residues containing amino rified further by HPLC on a reverse-phase phenyl column. A acids that are not present in dynorphin or a-neo-endorphin-i. e., single symmetrical peak offluorescence was obtained that co- valine and threonine. This conclusion was confirmed by se- incided with the peak of [Leu]enkephalin immunoreactivity quence analysis utilizing both automated Edman degradation (Fig. 3), indicative ofhomogeneity. In addition, automated Ed- and carboxypeptidase Y time course hydrolysis (Fig. 4). The man degradation ofthe purified peptide yielded a single amino tridecapeptide rimorphin resembles dynorphin and a-neo-en- terminal residue, tyrosine. Approximately 7 nmol ofpure pep- dorphin in having an amino-terminal [Leu]enkephalin se- tide was recovered from 16 g of tissue representing 60 glands. quence followed by paired basic residues, in this case Arg-Arg. Amino acid analysis ofthe purified material (Table 1) revealed However, the remainderofthe rimorphin sequence differs con-

15

av 1010 _ -___-______-- 5 -/ _ 60 O ' _ 50

40

4 - - 30 ag 3-3 20 "w Ca

Fraction FIG. 3. Purification of rimorphin by HPLC on a reverse-phase phenyl column. The fractions containing peak L-5 from Ultrasphere ODS chro- matography were dried, redissolved in 4 M urea/0.9 M pyridine acetate, pH 4.0, and injected onto the column. Peptides were eluted and assayed for enkephalins as described in Fig. 2. A single peak of [Leulenkephalin immunoreactivity was obtained. Downloaded by guest on September 30, 2021 6482 Biochemistry: Kilpatrick et aL Proc. Natl. Acad. Sci. USA 79 (1982)

Table 1. Amino acid composition of rimorphin Table 2. Opiate activity of rimorphin Amino acid Residues, no./mol IC50,* nM Asx 0.07 (0) Guinea pig Receptor binding Thr 0.95 (1) Peptide ileum assay assay Ser 0.05 (0) [Leu]Enkephalin 99.2 6.6 Glx 1.05 (1) Rimorphin 3.0 12 Pro 0.03 (0) Gly 1.93 (2) Values reported are from individual experiments. Each experiment Ala 0.04 (0) was done three times. Cys 0.00 (0) * Concentration that inhibits maximal response by 50%. Val 1.75 (2) Met 0.03 (0) dynorphin, suggested that it too would exhibit potent activity Ile 0.02 (0) in the guinea pig ileum preparation. Rimorphin was indeed Leu 1.04 (1) in this Tyr 1.03 (1) found to be far more active than free [Leu]enkephalin Phe 2.02 (2) system, with an IC50 of3 nM (Table 2). This is more active than His 0.03 (0) a-neo-endorphin (1) and approximately 1/10th as active as dy- Lys 0.96 (1) norphin (21). Rimorphin was also found to bind with high af- Arg 2.03 (2) finity to the opiate receptor of NG108-15 cells (Table 2). Trp 0.01 (0) The co-occurrence ofa-neo-endorphin and dynorphin in tis- Total 13 sues has been noted (22, 23). It was ofinterest to examine tissues other than the bovine posterior gland to determine whether Rimorphin samples (200 pmol) were hydrolyzed at 110HC for 22 hr rimorphin is generally associated with the two other [Leu]ECPs. in 200 ,ul of constant boiling HCl. For tryptophan analysis, hydrolysis was carried out in HOl containing 4% (vol/vol) thioglycolic acid. Each Tissues from several species were extracted and fractionated on value represents the mean of five separate analyses. The numbers in Sephadex G-75 followed by HPLC. Eluted fractions were di- parentheses have been rounded off to the nearest integral value. gested with trypsin and carboxypeptidase B and assayed for enkephalins. A [Leu]ECP eluting in the position of rimorphin siderably from the corresponding sequences in dynorphin and was observed in all the tissues (Fig. 5). The amount ofrimorphin a-neo-endorphin and from any sequences in (18, in each tissue was generally greater than that of dynorphin- 19). The presence ofthe Val-Val sequence in rimorphin explains (1-17) and a-neo-endorphin (not shown). As with dynorphin the relatively low yields of valine that were obtained on acid (3), the neurointermediate lobe ofthe pituitary gland contained hydrolysis (Table 1) because this bond is known to be hydro- the highest concentrations ofrimorphin, approximately 500 and lyzed slowly under the conditions used (20). 400 pmol/g for cow and pig, respectively. Although a rimorphin The presence ofthe Tyr-Gly-Gly-Phe-Leu-Arg- sequence at peak was observed in rat neurointermediate lobe extracts, the amino terminus of rimorphin, as in a-neo-endorphin and quantitation was complicated by the presence oflarge amounts 1 5 10

TYR-GLY-GLY-PHE-LEU-ARG-ARG-GLN-PHE-LYS-VAL-VAL-THR RIMORPHIN: -% - - % _ -- -I -- - =

1 5 10 15

DYNORPHIN: TYR-GLY-GLY-PHE-LEU-ARG-ARG-ILE-ARG-PRO-LYS-LEU-LYS-TRP-ASP-ASN-GLN

5 10

O(-NEO-ENDORPHIN: TYR-GLY-GLY-PHE-LEU-ARG-LYS-TYR-PRO-LYS

5 10 15

PEPTIDE E: TYR-GLY-GLY-PHE-MET-ARG-ARG-VAL-GLY-ARG-PRO-GLU-TRP-TRP-MET-ASP-TYR-GLN-

20 25

LYS-ARG-TYR-GLY-GLY-PHE-LEU

FIG. 4. Amino acid sequence of rimorphin. Residue assignments in rimorphin that were established by automated Edman analyses are indicated by and those determined by carboxypeptidase Y hydrolysis are indicated by -. Sequences for dynorphin (2), a-neo-endorphin (1), and peptide E (16) are also shown for comparison. Downloaded by guest on September 30, 2021 Biochemistry: Kilpatrick et aL Proc. Nati. Acad. Sci. USA 79 (1982) 6483 phin") or a large fragment thereoffrom which the three smaller [Leu]ECPs are derived. Dynorphin, a-neo-endorphin, and ri- morphin thus may represent three independent humoral sub- stances that arise from a common precursor, just as ACTH, (B- endorphin, and MSH arise from pro-opiomelanocortin (24). After this manuscript was completed, a report appeared of the cloning of a cDNA from porcine hypothalamus that con- E Swine SpinalCodC tained in it the three enkephalin sequences a-neo-endorphin, dynorphin, and rimorphin (25).

X 4 Bot ypo l Note Added in Proof. Fischli et aL (26) have just reported the isolation 0'U of a 32-residue ECP from pig pituitary gland that contains both the dynorphin and rimorphin sequences coupled through the sequence 4a 2 s i -Lys-Arg-. 0 -1 We thank Ms. L. D. Gerber and Mr. L. Brink for their excellent Rat Spinal Cord technical assistance and Ms. S. Andriola for preparation of this 4 manuscript.

2 1. Kangawa, K., Minamino, N., Chino, N., Sakakibara, S. & Mat- suo, H. (1981) Biochem. Biophys. Res. Commun. 99, 871-878. lbcine e urointermediateLobe 2. Goldstein, A., Fischli, W., Lowney, L. I., Hunkapillar, M. & 600 Hood, L. (1981) Proc. NatL Acad. Sci. USA 78, 7219-7223. 3. Goldstein, A. & Ghazarossian, V. E. (1980) Proc. NatL Acad. Sci. 300 USA 77, 6207-6210. 4. Botticelli, L. J., Cox, B. M. & Goldstein, A. (1981) Proc. Natl. 116 120 124 128 132 134 138 Acad. Sci. USA 78, 7783-7786. Fraction 5. H6llt, V., Haarmann, I., Bovermann, K., Jerlicz, M. & Herz, A. (1980) Neuro-Sci. Lett. 18, 149-153. FIG. 5. Determination of rimorphin in tissues. Bovine posterior 6. Ito, S., Iwanaga, T., Ryogo, Y., Yamaguchi, K., Hama, H., pituitary, hypothalamus, and spinal cord, rat spinal cord and neuroin- Kyuji, K. & Shibata, A. (1981) Life Sci. 29, 1457-1461. termediate lobe, and porcine neurointermediate lobe were each ex- 7. Lewis, R. V., Stem, A. S., Rossier, J., Stein, S. & Udenfriend, tracted. Each extract was chromatographed on Sephadex G-75 and the S. (1979) Biochem. Biophys. Res. Commun. 89, 822-829. region ofMr 500-2,000 was subjected to HPLC on an Ultrasphere ODS 8. Bohlen, P., Stein, S., Stone, J. & Udenfriend, S. (1975) AnaL column with a shallow propanol gradient. Fractions were assayed for Biochem. 67, 438-445. [Met]- and [Leu]enkephalin immunoreactivity and are reported with- 9. Lewis, R. V., Stem, A. S., Kimura, S., Rossier, J., Stein, S. & out correction for recovery. Only that portion of the chromatogram in Udenfriend, S. (1980) Science 208, 1459-1461. which rimorphin anddynorphin-(1-17) are eluted is shown. Small vari- 10. Kojima, K., Kilpatrick, D. L., Stem, A. S., Jones, B. N. & Uden- ations in elution position have been corrected for by use of the internal friend, S. (1982) Arch. Biochem. Biophys. 215, 638-642. standard "2I-labeled [Leulenkephalin whose position is designated by 11. Stein, S., Bohlen, P., Stone, J., Dairman, W. & Udenfriend, S. the arrow. Identification of the dynorphin peak was corroborated in (1973) Arch. Biochem. Biophys. 155, 203-212. some experiments by a dynorphin radioimmunoassay. 12. Jones, B. N., Paabo, S. & Stein, S. (1981)J. Liq. Chromatogr. 4, 565-586. of fendorphin-related peptides in the same fractions. For this 13. Shively, J. E., Hawke, D. & Jones, B. N. (1982) AnaL Biochem. 120, 312-322. reason the data for this tissue are not shown. 14. Hawke, D., Yuan, P.-M. & Shively, J. E. (1982) AnaL Biochem. 120, 302-311. DISCUSSION 15. Hunkapiller, M. W. & Hood, L. E. (1978) Biochemistry 17, 2124-2133. It is clear that the rimorphin sequence is distinct from that of 16. Kilpatrick, D. L., Taniguchi, T., Jones, B. N., Stem, A. S., dynorphin or a-neo-endorphin and thus represents a third Shively, J. E., Hullihan, J., Kimura, S., Stein, S. & Udenfriend, unique [Leu]ECP in the posterior pituitary gland that possesses S. (1981) Proc. NatL Acad. Sci. USA 78, 3265-3268. high opiate activity. However, rimorphin does resemble dy- 17. Gerber, L. D., Stein, S., Rubinstein, M., Wideman, J. & Uden- norphin, a-neo-endorphin, and adrenal peptide E in certain friend, S. (1978) Brain Res. 151, 117-126. structural aspects and in its potency in the guinea pig ileum 18. Gubler, U., Kilpatrick, D. L., Seeburg, P. S., Gage, L. P. & Udenfriend, S. (1981) Proc. NatL Acad. Sci. USA 78, 5484-5487. assay. Each of these peptides starts with an enkephalin se- 19. Noda, M., Furutani, Y., Takahashi, H., Toyosato, M., Hirose, quence (either [Met]- or [Leu]-) at its amino terminus, followed T., Inayama, S., Nakanishi, S. & Numa, S. (1982) Nature (Lon- by paired basic residues at positions 6 and 7 (Fig. 4). In addition, don) 295, 202-206. a basic residue (either arginine or lysine) is present at position 20. Duggan, E. L. (1957) Methods EnzymoL 79, 31-38. 10 or 11. Both structural features may be important in deter- 21. Chavkin, C. & Goldstein, A. (1981) Proc. NatL Acad. Sci. USA 78, mining the high potencies ofall ofthese peptides in the guinea 6543-6547. 22. Weber, E., Roth, K. A. & Barchas, J. D. (1981) Biochem. Bio- pig ileum assay, as was demonstrated for dynorphin (21). phys. Res. Commun. 103, 951-958. Dynorphin and a-neo-endorphin appear to exist within the 23. Weber, E., Roth, K. A. & Barchas, J. D. (1982) Proc. NatL Acad. same cells and it has been proposed that they arise from a com- Sci. USA 79, 3062-3066. mon precursor (22, 23). The parallel distribution of rimorphin, 24. Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. dynorphin, and a-neo-endorphin reported here suggests that Y., Cohen, S. N. & Numa, S. (1979) Nature (London) 278, all three peptides may be derived from a single precursor mol- 423-427. 25. Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, ecule, analogous to adrenal proenkephalin (18, 19). In support Y., Hirose, T., Asai, M., Inayama, S., Nakanishi, S. & Numa, S. of this concept, we have observed a [Leu]ECP having a Mr of (1982) Nature (London) 298, 245-249. about 20,000-30,000 in extracts of bovine spinal cord (unpub- 26. Fischli, W., Goldstein, A., Hunkapiller, M. W. & Hood, L. E. lished data). This larger ECP may be the gene product ("pronor- (1982) Proc. NatL Acad. Sci. USA 79, 5435-5437. Downloaded by guest on September 30, 2021