A 4,000-dalton dynorphin was isolated from porcine pituitary. It has 32 amino acids (Mr = 3,986), with the previously described heptadecapeptide (now called dynorphin A) at its amino terminus and a related tridecapeptide, dynorphin B, at its carboxyl terminus. The two peptides are separated by the "processing signal" Lys-Arg

Fischli, Walter

Goldstein, Avram

Hood, Leroy E.

Hunkapiller, Michael W.

English

Caltech Authors - Main

Proc. NatL Acad. Sci. USA
Vol. 79, pp. 5435-5437, September 1982
Neurobiology
Isolation and amino acid sequence analysis of a 4,000-dalton
dynorphin from porcine pituitary
(opioid/peptide/endorphin/neuropeptide/prohormone)
WALTER FISCHLI*, AVRAM GOLDSTEIN*, MICHAEL W. HUNKAPILLERt, AND LEROY E. HOODt
*Addiction Research Foundation and Stanford University, Palo Alto, California 94304; and tCalifornia Institute of Technology, Pasadena, California 91125
Contributed by Avram Goldstein, June 17, 1982
ABSTRACT A 4,000-dalton dynorphin was isolated from por-
cine pituitary. It has 32 amino acids (Mr = 3,986), with the pre-
viously described heptadecapeptide (now called dynorphin A) at
its amino terminus and a related tridecapeptide, dynorphin B, at
its carboxyl terminus. The two peptides are separated by the
"processing signal" Lys-Arg.
Dynorphin was isolated from porcine pituitary (1, 2) and gut
(3) and identified as a heptadecapeptide with Mr 2,148. It
showed the highest potency of all the known opioid peptides
in the guinea pig ileum preparation and a high selectivity for
the K opioid receptor (4, 5). We report here the isolation and
characterization of a longer dynorphin-containing peptide from
porcine pituitary.
MATERIALS AND METHODS
The starting material for this purification was the same porcine
melanotropin concentrate from which the dynorphin hepta-
decapeptide was isolated previously (1, 2). This powder is a
dried oxycellulose adsorbate of an extract of whole porcine pi-
tuitaries (generously furnished by J. D. Fisher, Armour, Kan-
kakee, IL). The purification itself was followed by radioim-
munoassay using synthetic dynorphin-(1-13) (Peninsula
Laboratories, San Carlos, CA) as the standard for measurement
of immunoreactive dynorphin (ir-dynorphin); the "Lucia" anti-
serum, which displays an affinity in the low picomolar range,
was used (6).
In the course of isolating the dynorphin heptadecapeptide
it had been observed that neutralization ofthe acetic acid extract
from porcine pituitary precipitated 60-70% of ir-dynorphin,
which could then be redissolved only partly. Gel permeation
analysis of this material on Sephadex G-50 showed that it con-
sisted mainly of ir-dynorphin of higher apparent Mr. For pu-
rification of this larger ir-dynorphin, methods and conditions
were chosen that did not precipitate any immunoreactive
material.
Table 1 shows the scheme of the purification. Details are
given in the table legend. ir-Dynorphin from a crude acetic acid
extract was adsorbed to CM-Sephadex and eluted with a sodium
chloride gradient in acetic acid containing Triton X-100 (step
2a). The immunoreactive material was then extracted with an
immunoaffinity resin (step 3a). Antibodies ofthe same type used
in radioimmunoassay were first purified by affinity chromatog-
raphy on dynorphin-(1-13) coupled to cyanogen bromide-acti-
vated Sepharose 4B (Pharmacia) and then coupled to Sepharose
4B (2). Adsorption of ir-dynorphin to this resin and subsequent
washes with phosphate buffer (pH 6.0) containing 2.5% Triton
X-100 could be done without loss of the larger ir-dynorphin, as
proved by Sephadex G-50 chromatography. A purification factor
of close to 100 and a yield of >50% was achieved at this step.
Concentration and desalting was done on CM-Sephadex
(steps 2b and 3b). ir-Dynorphin was adsorbed onto a minimum
amount ofCM-Sephadex and eluted in a small volume ofmeth-
anol/0.3 M HCl, 1:1 (vol/vol). Lyophilization, which gave un-
acceptable losses (2), could thus be avoided during the purifi-
cation procedure. Purification by HPLC on a C18 column (step
4, see Fig. 1) finally yielded five peaks of absorbance and im-
munoreactivity. A228/A2,- ratios were 14.3 (peak 1), 20.0 (peak
2), 18.3 (peak 3), 14.8 (peak 4), and 15.9 (peak 5).
RESULTS AND DISCUSSION
The pattern shown in Fig. 1 represents a late (basic) fraction of
ir-dynorphin from CM-Sephadex chromatography (step 2a) con-
taining mainly peak 3, whereas earlier fractions contained
mostly peak 1 (data not shown). Thus, ir-dynorphin from peak
3 is more basic than that from peak 1 and also is eluted with
higher acetonitrile concentrations upon HPLC. The absor-
bance, at 228 nm, of the total ir-dynorphin from CM-Sephadex
was 3 times greater for peak 1 than for peak 3, indicating that
peak 1 is the major component in porcine pituitary.
Peak 1 coeluted from HPLC as a single symmetrical absor-
bance peak when mixed with synthetic heptadecapeptide. Peak
1, peak 2, and synthetic heptadecapeptide showed the same
elution pattern on Sephadex G-50 and approximately the same
potency (per immunoreactive dynorphin equivalent) in the
guinea pig ileum bioassay. Relative potencies, expressed as per-
cents relative to that of the synthetic heptadecapeptide tested
on the same strips, were (mean ± SEM): Peak 1, 103 + 4 (n
= 8); peak 2, 66 ± 8 (n = 4). Peak 1 was therefore assumed to
be the previously isolated heptadecapeptide and peak 2, a de-
rivatized product (possibly oxidized at tryptophan-14). They
were not analyzed further.
On gel permeation analysis, HPLC peak 3 proved to be the
larger dynorphin with apparent Mr of approximately 3,000. Au-
tomatic Edman degradation at Pasadena (8) gave the sequence
shown in Fig. 2, corresponding to actual Mr 3,986. The first 31
amino acids were established without ambiguity; there was
some uncertainty in regard to threonine-32 because ofthe small
signal. Cycles 33, 34, and 35 yielded no additional amino acid.
To estimate the carboxyl-terminal amino acids of the peptide
in peak 3, it was digested with carboxypeptidase A (see legend
to Fig. 1). After 10- and 60-min incubations, only the following
amino acids were released (as nmol/nmol of peptide): threo-
nine, 0.22 and 0.50; valine, 0.41 and 1.15. The simultaneous
release of threonine and valine was seen even after 2-min in-
cubation. Carboxypeptidase Y digestion (10 ng of enzyme and
200 pmol of peptide in 0.025 M pyridinium acetate at pH 5.5)
gave, after 60 and 120 min, the following released amino acids
Abbreviation: ir-dynorphin, immunoreactive dynorphin.
5435
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payment. This article must therefore be hereby marked "advertise-
ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5436 Neurobiology: Fischli et al.
Table 1. Purification scheme for dynorphin peptides in
porcine pituitary
SpecificDynorphin activity,
Amount, Recovery, nmol/mg
Step nmol % protein
1. Extract 198 - 0.038
2a. Eluate from CM-
Sephadex
chromatography 162 82 0.23
2b. Eluate from CM-
Sephadex adsorption 128 79 0.48
3a. Immunoextract 67 52 *
3b. Eluate from CM-
Sephadex adsorption 57 85 37
4. Eluate from HPLC 32 56 250
Melanotropin concentrate powder (10 g) was extracted in 1 M acetic
acid (180 ml, 9000,10 min). The ir-dynorphin was then adsorbedto CM-
Sephadex (Pharmacia, C-25,8.5 g, prewashed with 1M acetic acid) and
eluted by a linear gradient of NaCl (0.066-0.6 M; total volume, 1,540
ml) in 1 M acetic acid/1% Triton X-100. The fractions containing ir-
dynorphin were pooled (850 ml) and diluted to 0.025 M NaCl with 1
M acetic acid/1% Triton X-100, adsorbed again on CM-Sephadex (2.5
g), and eluted in 30 ml of methanol/0.3 M HCl, 1: 1 (vol/vol). After
dilution with 150 ml of 0.25 M sodium phosphate, pH 6.0/2.5% Triton
X-100, the ir-dynorphin was adsorbed in batches of 25-30 nmol to the
immunoaffinity resin (30 min, 400). The resin was then washed six
times with 50 ml of incubation buffer (30 min per wash, 400) and the
ir-dynorphin was eluted with four 10-ml portions of 0.5 M acetic acid
(30 min per elution, 370C). ir-Dynorphin was concentrated again on
CM-Sephadex (0.5 g), and the methanol/HCl eluate (5-6 ml) was in-jected, in batches of 1-2 ml, onto the HPLC column. Amounts of dy-
norphin were determined by radioimmunoassay (see text); protein was
measured by the method of Lowry et al. (7). Specific activity of pure
dynorphin-32 was calculated on the basis of Mr = 3,986.
* Protein was not determined at this step in order to save material.
(nmol/nmol of peptide): threonine, 0.68 and 0.74; valine, 1.14
and 1.18; lysine, 0.16 and 0.23; phenylalanine, 0.24 and 0.28.
The nmol ofamino acid released per nmol ofpeptide was always
below the theoretical value because the amount of peptide was
measured before lyophilization. The estimated amount thus did
not account for the usual losses during lyophilization and re-
constitution (due to incomplete dissolution in incubation buffer
and adsorption to the incubation vial). The results proved that
the peptide is not amidated because the carboxypeptidase A
used here has no amidase activity (2). Hydrazinolysis (1-100 hr,
100°C), which would give an estimate of the carboxyl-terminal
amino acid, showed no free amino acid. The same negative re-
sult was obtained with synthetic dynorphin-32-(20-32), in
which the carboxyl-terminal amino acid is threonine. Thus, all
the results are consistent with the carboxyl-terminal sequence
-Phe2'-Lys29-Val30-Val31-Thr32(OH). However, tryptophan and
proline would not be detectable in our procedure.
10
'--4
x
00
c4
04
OI
28 33 38
Acetonitrile, %
FIG. 1. HPLC of porcine pituitary dynorphins. The figure repre-
sents a typical elution pattern of concentrated ir-dynorphin from step
3b. Concentrate (1.5 ml) containing 500 pmol of ir-dynorphin was in-jected onto a pBondapak C18 column (3.9 x 300 mm). Elution was
achieved by a linear gradient of acetonitrile (20-50%, 30 min, 1.5 ml/
min, 0.6-ml fractions) in 5 mM trifluoroacetic acid. The five peaks
emerged at the following acetonitrile concentrations: 31.6% (peak 1),
32.4% (peak 2), 33.6% (peak 3), 34.8% (peak 4), and 35.1% (peak 5).
Fractions containing the peaks were acidified with 10 ,ul of 6 M HCl,
pooled,and lyophilized. Theresiduewastakenup in 1.0ml ofmethanol/
0.1 M HCl, 1: 1 (vol/vol) and stored at -70°C.
Immunoreactive peak 4 was eluted from HPLC as a minor
component. Sephadex G-50 chromatography showed ir-dynor-
phin with apparent Mr = 2,500, intermediate between that of
the heptadecapeptide and that ofdynorphin-32. Sequence anal-
ysis gave the first 24 amino acids of dynorphin-32 (actual Mr
= 2,970) without ambiguity. Carboxypeptidase A digestion for
60 and 120 min gave the following released amino acids (nmol/
nmol of peptide): leucine, 0.53 and 0.54; phenylalanine, 0.60
and 0.58; glycine, 0.02 and 1.44. Hydrazinolysis (9 hr, 100°C)
indicated. that leucine was the carboxyl-terminal amino acid.
We conclude, therefore, that peak 4 is dynorphin-32-(1-24),
1 5 10 17
(H)Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-Lys-Arg-
- -* -* -* -* -* -* --*-.- -4- -.- - - -
20 24 32
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr(OH)
FIG. 2. Sequence of porcine pituitary dynorphin-32. -*, Automated Edman degradrtion on 200-300 pmol of peptide in the gas/liquid/solid
phase peptide Sequenator (8) with a sensitivity limit of about 5 pmol of cleaved amino acid. +-, Carboxypeptidase A digestion of peak 3 or [4-] peak
4. Peptide (100-200 pmol) was dissolved-in 50-100 ,ul of 0.02 M sodium borate, pH 7.15/0.2 M NaCl/0.1% Triton X-100 and incubated with 100
ng of enzyme at 370C. Samples (10 ,ul) were withdrawn, diluted with commercially available sample diluent (30 ,l, "Na-S," Dionex), and injected
directly into the amino acid analyzer (Dionex, Sunnyvale, CA). Zero-time samples provided correction for free amino acids present initially. The
sequence 20-32 is dynorphin B. The original heptadecapeptide 1-17 is now referred to as dynorphin A.
Proc. Natl. Acad. Sci. USA 79 (1982)
Proc. Natl. Acad. Sci. USA 79 (1982) 5437
with carboxyl-terminal [Leu]enkephalin, as reported elsewhere
(9).
Peak5 was recovered only in trace amounts. Gel permeation
analysis showed that the apparent Mr of this material was 5,700
(calibration with the heptadecapeptide and dynorphin-32). It
was set apart for further characterization.
The finding of dynorphin-32 is interesting because it seems
to be a precursor of two related peptides. Enzymatic attack at
Lys18-Arg19, which proves to be a processing signal in proopi-
omelanocortin (10), preproglucagon (11), preprocalcitonin (12),
and preproenkephalin (13-15), would generate an amino-ter-
minal heptadecapeptide (dynorphin A) and a carboxyl-terminal
tridecapeptide (dynorphin B). The placement of Lys-Arg shows
that dynorphin A is a naturally processed product that termi-
nates at glutamine-17.
Dynorphin B represents the third "big [Leu]enkephalin"
besides dynorphin A and a-neo-endorphin-(1-10) (16). Inter-
estingly, it is related to both of them. It shares the sequence
1-7 with dynorphin A but has lysine [which seems to be im-
portant for potency and receptor selectivity (17)] shifted from
position 11 in dynorphin A to position 10. The homology with
a-neo-endorphin-(1-10) (Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-
Lys) is also clear; again the sequence 1-7 is the same, except
for a conservative change from lysine-7 in a-neo-endorphin to
arginine-7 in dynorphin B; and both these peptides have lysine
at position 10.
Dynorphin-32-(1-24) could be another naturally processed
product from dynorphin-32. It is similar to preproenkephalin
peptide E (18), which contains 25 amino acids and carries
[Met]enkephalin at the amino terminus and [Leu]enkephalin
at the carboxyl terminus (9).
Dynorphin B was synthesized (Peninsula Laboratories, San
Carlos, CA) and all the peptides were compared in the guinea
pig ileum preparation (19). The concentration of the dynorphin-
32 solution used for the bioassay dilutions was estimated by ra-
dioimmunoassay and by amino acid analysis of the acid hy-
drolysates. According to radioimmunoassay referred to dynor-
phin-(1-13) as standard, concentration was found to be 2.5 ,M.
The prominent peaks from amino acid analysis of valine, iso-
leucine, leucine, and phenylalanine yielded estimates of 2.5
+ 0.2 AM. Therefore, the "Lucia" antiserum is equally reactive
with dynorphin-(1-13) (against which it was raised) and dynor-
phin-32. It can be concluded that the intermediate-size peptide
dynorphin-32-(1-24) is also fully crossreactive.
In the bioassay, to minimize variability, potencies relative to
dynorphin A were estimated by IC50 ratios on the same muscle
strips. The IC50 value for dynorphin A (geometric mean) was
0.40 nM in 58 different preparations, with 95% confidence in-
terval 0.34-0.47 nM. Potencies expressed as percents relative
to dynorphin A were (mean ± SEM; n = number of observa-
tions): dynorphin-32-(1-24) (peak 4), 42 + 2 (n = 15); dynor-
phin-32 (peak 3), 27 ± 3 (n = 8); dynorphin-32-(20-32) (dy-
norphin B), 7.8 + 0.5 (n = 16). The naloxone Ke values for all
these peptides were found to be the same, 24-28 nM (data not
shown). Because naloxone Ke is a measure ofreceptor selectivity
(20) the results indicate that all the peptides act through the
same opioid receptor type in guinea pig ileum myenteric plexus,
which contains functional ,u and K but not 8, receptors (21).
Selective protection experiments (4, 5) and crosstolerance stud-
ies (22) have shown that dynorphin interacts with a single highly
specific receptor in guinea pig myenteric plexus and brain as
well as in mouse vas deferens. That receptor proved to be iden-
tical with the previously known K opioid receptor. That dynor-
phin B displays the same high K opioid selectivity as dynorphin
A is surprising. Lysine-li in dynorphin was shown to be es-
sential not only for high potency but also for receptor selectivity.
The outcome here suggests that lysine-10 in dynorphin B can
fulfill the same requirement for selectivity as does lysine-l1 in
dynorphin A. We propose that dynorphin B is a dynorphin-like
K opioid agonist.
Interestingly, a-neo-endorphin-(1-10), which is colocalized
with dynorphin A (23) and has important structural similarities
to dynorphin B, also shows selectivity for K opioid receptors
(unpublished data). It therefore is likely that a-neo-endorphin-
(1-10), dynorphin A, and dynorphin B are components of the
same precursor and, thus, that the dynorphin gene would en-
code a set of K opioid agonists.
Note Added in Proof. At the International Narcotic Research Confer-
ence, North Falmouth, MA, June 14-18, 1982, Udenfriend's group (24)
reported the isolation of a tridecapeptide from bovine hypothalamus
with sequence identical to that of porcine dynorphin B.
We acknowledge the skilled technical assistance provided by Keiko
Otsu and Patricia Lowery and the valuable participation of Louise I.
Lowney at many stages of this investigation. The work was supported
by grants from the National Institute on Drug Abuse (DA-1199 to A.G.),
the Swiss National Foundation (to W.F.), the National Institutes of
Health (GM-06965 to J. P. Revel), and the Weingart Foundation (to
L.E.H.).
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Neurobiology: Fischli et al.


Isolation and amino acid sequence analysis of a 4,000-dalton dynorphin from porcine pituitary

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Isolation and amino acid sequence analysis of a 4,000-dalton dynorphin from porcine pituitary

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