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A specific radioimmunoassay for the novel opioid peptide dynorphin
PergamonPress
Life Sciences,Vol. 27,~~.75-86 Printedin the U.S.A.
A SPECIFIC RADIOIMMUNOASSAY FOR THE NOVEL OPIOID PEPTIDE .DYNORPHIN Vartan E. Ghazarossian, Charles Chavkin and Avram Goldstein Addiction Research Foundation and Stanford Dniversity, Palo Alto, California 94304 (Received in final form May 1,
1980)
Summary Dynorphin was recently isolated from porcine pituitary extracts and shown to be the most potent known Antisera *were prepared to synthetic opioid peptide. dynorphin-(l-13), the biologically active NH2-terminal A high-titer, sensitive fragment of the peptide. antiserum was characterized with fragments from dynorphin-(l-13). Leucine-enkephalin, which is contained in dynorphin, is not recognized at all by the antiserum. To study distribution in tissue, a procedure using hot acidified methanol extraction of rat pituitary neuroinmediate lobe preparations was developed and validated. M I-labelled dynorphin-(l-13), when added to tissue, remained intact throughout this extraction procedure, and added dynorphin-(l-13) was almost completely recovered. There was no destruction of radiolabelled peptide during incubation in the radioimmunoassay. Serial dilutions of pituitary extracts yielded curves that were parallel to The immunorethe dynorphin-(l-13) standard curve. activity from tissue was completely destroyed by papain treatment. Dynorphin is a recently described leucine-enkephalin containing peptide isolated from porcine pituitary extracts (1). Its NH -terminal fragment, dynorphin-(l-13) (YGGFLRRIRPKLK)*, when tegted in the guinea pig ileum myenteric plexus-longitudinal muscle bioassay, is approximately 200 times more potent than normorphine and 50 times more potent than B -endorphint. We now report the developmsnt of a highly specifig and sensitive radioimmunoassay (RIA), using antiserum raised to dynorphin-(l-13) coupled to thyroglobulin. We also describe an extraction procedure for the measurement of immunoreactive (ir-) dynorphin in biological material. Materials and Methods Peptides Dynorphin-(l-13), D-Ala2-dynorphin-(l-13)amide, D-Ala'-dynorphin-(l-11), dynorphin-(l-9), dynorphin-(6-13) and N-acetyl * Y=Tyr, G=Gly, F=Phe, L=Leu, R=Arg, I=Ile, P=Pro, K=Lys. t Subscripts: c = camel, h = human. 0024-3205/80/270075-12$02.00/O Copyright(c) 1980Pergamon Press Ltd.
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Dynorphin Radioimmunoassay
Vol. 27, No. I, 1980
dynorphin-(6-13) % were synthesized by Peninsula Laboratories (San Carlos, CA). All peptides were demonstrated to be pure by thinlayer chromatography, by e lectrophoresis, and by amino-acid analysis. In addition, the sequences of dynorphin-(l-13) and of dynorphin-(6-13) were verified by Edman degradation on a sequenator, the former by Drs. L.E. Hood and M. Hunkapiller (Caltech), the latter by A. Smith (Univ. of Calif., Davis). Dynorphin-(l-12), -(i-ii) , -(i-i0) , -(1-7) , -(1-6) and -(2-13) were prepared from dynorphin-(l-13) enzymatically, as follows: Dynorphin-(l-12) was obtained by treatment with carboxypeptidase B, -(i-ii) by treatment with carboxypeptidase B followed by carboxypeptidase A, and -(1-7) and -(1-6) by trypsin digestion. Dynorphin-(2-13) was obtained by treatment with leucine aminopeptidase, dynorphin-(l-10) by treatment with prolyl endopeptidase (2) generously furnished by Dr. Marian Orlowski, dynorphin-(l-8) by carboxypeptidase B treatment of -(1-9). All of the above peptides were purified to homogeneity by reversed-phase high performance liquid chromatography (HPLC) (CIR column, various elution solvents), and their amino acid composl ~ tions were verified following acid hydrolysis. Detailed data on the preparation of these dynorphin fragments and proofs of structure will be published elsewhere (C. Chavkin and A. Goldstein, in preparation). A totally blocked derivative of dynorphin-(l-13) was synthesized by Peninsula Laboratories, with methyl ester blocking the terminal carboxyl group. This was catalytically dehydrogenated, and the resulting dynorphin-(l-13) methyl ester was purified to homoaeneitv bv HPLC. Its identitv was confirmed by amino acid analysis, conversion to dynorphin-(l'-13~ on saponification, and resistance to car boxypeptidase B. --H-le uc ine-en kepha lin (41 Ci/mmol) was purchased from Ame r sham, le ucine-en kephalin from Biosearch (San Rafael, CA), ~-endorphin from Pierce Chemical Co. (Rockford, IL) . All other opioid peptides were obtained from Peninsula Laboratories. Thin Layer Chromatography
(TLC)
Silica gel G plates (polygram Sil G, Brinkmann, Inc., Westbury, NY) were developed with butanol : acetic acid : ethyl acetate : water (1:1:2:1), System i; or with chloroform : methanol : acetic acid : water (15:10:2:3), System 2. Cellulose plates (13255 Cellulose, Eastman Kodak Company, Rochester, NY) were developed with butanol : acetic acid : water (4:1:5), System 3. Conjugation of dynorphin-(l-13)
to bovine
thyroglobulin
Dynorphin-(l-13) (5.2 mg) containing a trace quantity of 125Idynorphin-(l-13) (see below) and thyroglobulin (26 mg) were dissolved in 2 ml of i00 mM phosphate buffer (pH 7.5) and cooled to 0 °. Glutaraldehyde (Sigma, 25% aqueous solution) was diluted with icecold distilled water (i:i00) and 1 ml was added dropwise to the reaction mixture which was stirred 30 min at 0 ° then 2 h at room temperature. After extensive dialysis against 0.9% NaCI and then water, the conjugated peptide was lyophilized and stored at -20 ° . The molar ratio o~2~ynorphin-(l-13) to thyroglobulin was 92:1, as determined from [ I]dynorphin-(l-13) remaining in the dialysis bag.
%Terminal s-amino group acetylated.
Vol. 27, No. i, 1980
Immunization
Dynorphin Radioimmunoassay
77
Procedure
Male and female New Zealand White and New Zealand White x San Juan rabbits were used. The conjugated peptide (30-50 ~g) was suspended in saline and emulsified with an equal volume of Freund's complete adjuvant (incomplete adjuvant after the first injection). After a series of four immunizations at two-week intervals, the rabbits were injected at monthly intervals and bled 10-12 days after each booster injection following the fifth immunization. Several of the rabbits yielded useful antiserum; one with highest titer, good sensitivity, and high specificity, "Lucia 9/14", was chosen for the RIA described here. Iod ination
Procedure
125I-labelled dynorphin-(1-13) was prepared according to a procedure described for ~-endorphin (3). The radiolabelled peptide was separated from iodide by chromatography on Sephadex G-15 ~ i g . i). It was stable on storage at -20 ° for up to i0 weeks. ~I labelled leucine-enkephalin was prepared and purified as described by Miller et al. (4).
10
w~ O X u
1 J I
I
~L_I__£_TTT___ I 10
I
I 20
I
I 30
Fraction
I
No.
I 40
I
I 50
I
I 60
I
(1-ml.)
Fig. i. Gel filtration following iodination of dynorphin-(l-13). Sephadex G-15, 35 x 1.2 cm column, 0.25 M acetic aci~2~n 0.1% BSA. First peak of radioactivity contained + ~I-labelled dynorphin-(l-13), which chromatographed as a single component in TLC systems 1 and 3. Second peak contained unreacted iodide.
78
Dynorphin Radioimmunoassay
Vol. 27, No. I, 1980
Tissue Extraction and RIA Procedure Tissue was weighed and homogenized immediately in i0 volumes (but not less than 1 ml) of acidified methanol (MeOH-HCI, consisting of equal parts of methanol and 0.i M HCI, v/v) at 70 ° , incubated I0 rain at 70 ° , cooled on ice, centrifuged (15,000 x g), and the supernatant was transferred to a polypropylene tube and stored at -20 ° . All subsequent operations were carried out on ice (0°-4 °). Tissue extracts or dynorphin-(l-13) (10 mM stock solution in MeOHHCI stored at -20 ° ) were diluted serially in MeOH-HCI in polypropylene tubes for immunoassay. The 300-~i RIA incubation mixture consisted of: i00 ~i of a dilution of dynorphin-(l-13) or tissue extract (see above); 100 ~i of a 1:10,000 dilution of antiserum in 150 mM phosphate buffer (pH 7.5), containing 0 ~ ) bovine serum albumin (BSA) and 0.1% Triton X-100; i00 ~i of ['z=I]dynorphin-(l-13) (approximately 5000 cpm) in the same buffer as the antiserum. All components were mixed in polypropylene tubes in an ice bath§, and incubated 18-24 h at 0o-4 ". At the end of this period unbound radiolabelled dynorphin-(l-13) was removed by adding 1.2 ml of a suspension of ice-cold dextrancoated charcoal (5 g Norit A + 0.3 g dextran + 15 ml horse serum in i00 ml of 150 mM phosphate buffer, pH 7.4). The tubes were centrifuged (5000 x g, 4 ° , 20 rain), and radioactivity was determined in lml aliquots of each supernatant. Papa in Treatment Papain (Worthington Biochemical Corp., Freehold, NJ, 26.3 U/mg) was obtained as a suspension in 50 mM sodium acetate. It was activated by incubating the following mixture at 37 ° for 20 min: Papain (7.4 rag), 10 mM EDTA (2 ml), 12 mM mercaptoethanol (0.i ml), 50 mM cysteine-HCl (2 ml), glass distilled water (14 ml). A MeOHHCI extract of six pooled rat pituitary glands was lyophilized, dissolved in 10 mM EDTA (250 ~i), and divided into two ~ortions. One was treated eight times at 10-12 h intervals with freshly activated papain (i ml). The control was treated simultaneously with papain that had been inactivated at 100 ° for 10 min. At the end of the 90-h digestion, both reaction mixtures were brought to 100 ° for 15 min, lyophilized, dispersed in MeOH-HCI, centrifuged, and the supernatants were assayed as described above. Results and Discussion Procedures for minimizing loss of dynorphin peptides Significant loss of opioid peptides to surfaces has been described previously (3). Acidified methanol prevents the loss of dynorphin-(l-13) or of ir-dynorphin f ~ tissues to surfaces of containers. Table 1 shows that with [ I]dynorphin-(l-13) there is no loss to glass or polypropylene tubes in MeOH-HCI, whereas in the presence of salts, major losses occur. Iodinated peptide that has been lost from solution can be recovered, after drying, by addition of MeOH-HCI.
~A large volume of antiserum and mixed.
[1251]dynorphin-(l~13) can b e p r e -
Vol. 27, No. i, 1980
Dynorphin Radioimmunoassay
Table
i
125 Los3 of I-labelled dynorphin-(l-13) means of quadruplicate determinations. 750 ~I.
to surfaces. Data are Volume of solutions was
Polypr opylene * Solvent
79
Glass t
Percent of initial radioactivity remaining in solution after 60 min at 23 °
50 mM NaC1 in 50 mM phosphate buffer " + 0.1% BSA " + 0.1% Triton X-100 " + 0.1% BSA + 0.1% Triton X-100 MeOH-HCl
51 91 105
54 58 72
104 I01
75 96
Percent of radioactivity.recovered from tube walls ~ 50 mM NaCl in 50 mM phosphate buffer MeOH-HCI
15 93
33 96
"12 x 82 mm polypropylene tubes (Sarstedt). %13 x 100 mm borosilicate glass culture tubes (Scientific Products). %After loss of half the radioactivity from solution in phosphate buffer saline (first row), solution was aspirated, tubes were dried, and the indicated recovery solvent (750 ~l) was added.
Properties
of the antiserum
Antiserum "Lucia 9/14" binds 30% of radiolabelled peptide at a final dilution of about 1:50,000 (Fig. 2). Usefully, the antiserum tolerates 17% MeOH in the final incubation mixture; although the binding of radiolabelled dynorphin-(l-13) is reduced by 30%, there is no effect on the slopes of competition curves (see below). The antiserum does not contain antibo~i~@ that recognize leucinee~kephalin, since no binding of [~'~I] ledcine-enkephalin or [ H]leucine-enkephalin was observed (Fig. 2). Typical RIA protocols for leucine-enkephalin employ polyethylene glycol rather than charcoal to separate bound from free ligand after the incubation. Ey~ with polyethylene glycol, however, there was no binding of [~ I]leucine-enkephalin to antiserum "Lucia" (1:30 dilution). Under the same conditions, when an antiserum specific for leucineenkephalin was used ("DBD-3", kindly furnished by Drs. June Dahl and Iris Lindberg), there was 60% binding of radiolabelled leucineenkephal!n. The absence of binding sites for leucine-enkephalin in antiserum "Lucia" is especially important for the specific immunohistochemical localization of dynorphin.
80
Dynorphin Radioimmunoassay
Vol. 27, No. 1,1980
100
"o c
50
125 I-Dyn--(]--I3)
~'~O~o
\
Z
1251_Leu_Enk
~ ~ | ~ O ~ o I
101
I
102
3H_Leu_En~
I
l
103
104
I
105
Antiserum Dilution-1 Fig. 2. Binding of 125I-labelled dynorphin-(1-13) at different dilutions of antiserum "Lucia 9/14". Shown for contrast are results with radioactive leucine-enkephalin (Leu-enk). Dyn = dynorphin.
The antiserum is very sensitive. At a final dilution of 1:30,000 an IC~ ¶ of about 50 pM is routinely observed with dynorphin-(l-13) ~ i g . 3) ; this concentration is equivalent to about 17 fmol per assay tube. There is only modest ~ of sensitivity in a fast RIA, in which a 2-h incubation with [ I]dynorphin-(l-13) follows a l-h pre-incubation of the other two components (R.R.M. Dent, unpublished observations). Reproducibility of the RIA was assessed from IC~ 0 values for dynorphin-(l-13) determined over a three-month period. All dilution curves were symmetrical sigmoids, as in Fig. 3, with midpoint slope consistent with that predicted from the mass-law equation for i:i competition at a binding site (5). Mean IC~0 was 50 pM, with coefficient of variation 17% in 13 experiments, uslng a concentration of dynorphin-(l-13) near the IC.^, the triplicate values of i00 x B / B (see Fig. 3 legend) in e a ~ of these same experiments were anal~zed. The mean of all 39 values was 46%, with standard deviation 5%. The standard deviation within triplicates was 3%, and the variance ratio (between experiments/within triplicates) was 7.5. Thus, all variances were small, and the major (and significant) source of variance was between experiments.
¶ICon = concentration of competing [125I]dynorphin-(l-13) by 50%.
peptide
that reduces binding of
Vol. 27, No. I, 1980
Dynorphin Radioimmunoassay
81
Fig. 3 also shows that leucine-enkephalin [dynorphin-(l-5)] shows no crossreactivity at concentrations more than 10 million times thein IC
100
\ ~Dyn
o ca
~Dyn_( 6 -13)
1-13)
50
0
~
\ i I
I 10 pM
I 100
N_Acetyl - Dyn_(6_13
I 1
I 10 nM
I 100
I 1
I 10 pM
~ 100
I 1
I 10 mM
Concentration
Fig. 3. Immunoreactivity ~ y n o r p h i n - ( l - 1 3 ) and related peptides. Displacement of ~I-la~elled dynorphin-(l-13) was measured as described under Materials and Methods. B / B is binding in presence of competing peptide (x-axis sho~s concentration in incubation mixture) relative to binding of [ I]dynorphin-(1-13) alone, expressed as percent.
82
Dynorphin Radioimmunoassay
Table Crossreactivity
of dynorphin
Vol. 27, No. I, 1980
2
fragments and related peptides.
Pept ide
Crossreactivity
Dynorphin
(%)
fraqments and analoques
Dynorphin- (1-13) "
-(1-13)
"
-(1-12)
"
- (i-ii)
"
-(1-10)
"
-(1-9)
[YGGFLRRIRPKLK] methyl ester
i00 115 115 i0 4.8 3.8
"
-(1-8)
2.5 x i0 -I
"
- (1-7)
8.3 x 10 -3
"
-(1-6)
"
-(1-5) (le ucine enkephalin)
"
-(2-13)
"
-(6-13)
6.0 x 10 -5
"
-(6-13) , N-acetyl
2.4 x 10 -4
5.2 x 10 -3 < 1.0 x 10 -6 52
D-AIa 2-dynor ph in - (1-13 ) amide
9.6
D-Ala2-dynorphin
4.7
- (1-11)
Other opioid peptides
-3
s-endorph in
-3
8h-endorphin [Leu 5] 8h-endorphin
-3
<3.3xi0
-2
Me th ion ine -en ke pha i in
e - n e o - e n d o r p h i n - (1-8) [YGGFLRKR]
3.6xi0
-2
Data are based on molar ICon values obtained from dilution curves, all of which were p a r a l l e l to dynorphin-(l-13) standard curve. All p e p t i d e s shown here were dissolved and diluted in 50 m M phosphate buffer (pH 7.5) c o n t a i n i n g 0.1% BSA and 0.1% Triton X-100.
indicate that the antigenic d e t e r m i n a n t occupies a region of the peptide e x t e n d i n g toward the N H 2 - t e r m i n a l at least through residue 5, and p o s s i b l y as far as residue 2, and toward the C O O H - t e r m l n a l as far as (but not beyond) residue 12. This complete c h a r a c t e r i z a t i o n of s p e c i f i c i t y indicates that n a t u r a l l y - o c c u r r i n g porcine p i t u i t a r y dynorphin, which is a C O O H - t e r m i n a l l y e x t e n d e d dynorphin-(1-13)
Vol. 27, No. i, 1980
Dynorphln Radiolmmunoassay
83
(1), should very likely be recognized by this antiserum. Moreover, if precursors for dynorphin have extensions from the NH 9 terminus [in analogy with 8-endorphin (6)] they may also be recdgnized by the antiserum. Table 2 also shows that the antiserum does not react significantly with any of the other known opioid peptides or their fragments. Tissue extraction and assay of ir-dynorphin Serial dilutions of rat pituitary posterior lobe extracts in MeOH-HCI exhibited parallelism with standard dynorphin-(l-13) (Fig. 4A). When portions of the same rat pituitary extract were assayed on different occasions, the coefficient of variation was 14%, about the same as for replicate assays of dynorphin-(l-13) in the absence of tissue extract. The immunoreactivity was completely destroyed on treatment with papain, whereas similar extracts treated with heat-inactivated papain showed little loss (Fig. 4B). Thus, the immunoreactive material appears to be a peptide. No differences in ir-dynorphin content could be observed between posterior lobes allowed to stand for 30 rain at 23 ° before homogenizing, and those homogenized a few minutes after decapitation. In contrast to this stability in intact tissue, homogenizing in Tris buffer (50 mM, pH 7.5, 23 ° ) resulted in significant degradation in 10 min. This was e v i ~ _ ~ e d by appearance of a new component in TLC (system i) when I-labelled dynorphin-(l-13) had been added to the Tris buffer, and also by a loss of about 25% in ir-dynorphin as measured by RIA. Degradation did not occur when tissue was homogenized in MeOH-HCI at 70 °. MeOH-HCI extracts are completely stable on storage at -20 ° for at least 30 days. Recovery of dynorphin-(l-13) added to rat pituitary extracts was estimated as follows. A rat pituitary extract was divided into two parts, and dynorphin-(l-13) was added to one -- just enough to increase the total immunoreactivity by one-third to one-half. Two dilution curves resulted, both _parallel to the dvnorDhin-(l-13) standard curve, yielding two ICon values from which recovery of added peptide was computed to be 75-100% in three different experiments. In a separate set of experiments, complete recovery was also observed when dynorphin-(l-13) was added to every tube in a tissue extract dilution series. It is significant that satisfactory recoveries were obtained with addition of relatively small amounts of dynorphin-(l-13), but it does not necessarily follow that dynorphin in tissue extracts is recovered equally well. This uncertainty can only be resolved when the complete primary structure of dynorphin is known. Although it was unlikely that degradative enzymes would be present in hot MeOH-HCI extracts of rat pituitary neural lobe, it was necessary to demonstrate directly that radiolabelled dynorphin(1-13) was not degraded in the presence of tissue extracts in 24 hr at 4 ° in the RIA incubation mixture. TLC analysis of radioactivity from the incubation mixture at 0 and 24 hr revealed no change in R F, but certain fragments might not have been detected -- especially dynorphin-(l-ll), which is only weakly immunoreactive (cf. Table 2) -- since they are poorly separated from dynorphin-(l-13).
84
Dynorphin Radioimmunoase~y
100
Extract 1/10 2
1/10 3
Vol. 27, No. 1,1980
Concentration
I,/1
1/10 I
|
l
i,,L
v
'
A Dyn-(1J3~)e ~
50
0
I I
I
.....w/:
I00
|
I 103
8
e•O
50
0
I
101 102 Concentrat ion ( pM )
Contl~ro~
i i/i03
I I 1/ 102 1/101 Extract Concentration
! 1/ I
Fig. 4. A: Dilution curve of rat pituitary neurointermediate lobe extract and dynorphin-(l-13) standard curve. Scale of x-axis: Upper = extract concentration; lower = concentration of standard in incubation mixture. B: Papain treatment of rat pituitary extract. See Materials and M~thods for details. Control was extract treated with heat-inactivated papain.
If the apparent immunoreactivity of a tissue extract were an artifact due to degradation of radiolabelled dynorphin-(l-13), a decrease in B / B would be observed at longer incubation times. Accordingly, we ~arried out the experiment summarized in Table 3, the results of which show that there was no such degradation artifact. The evident degradation by rabbit plasma provides an instructive contrast. That the immunoreactive material extracted from rat pituitary posterior lobe is very likely natural dynorphin is indicated by the following : (l~25The possibility was ruled out that reduction in binding of [ I]labelled dynorphin-(l-13) was due to a degradative enzyme in the tissue extract.
Vnl. 27, No. i, 1980
Dynorphin Radioirmnunoassay
Table
3
Stability of r a d i o l a b e l l e d
dynorphin-(l-13)
the RIA incubation Incubation Addition
85
in
mixture.
time with r a d i o l a b e l l e d peptide at 0-4 °
0-24 hr
0-48 hr
24-48 hr
Dynorphin-(l-13)
35 + 1
40 + 1
40 + 4
Rat p i t u i t a r y extract
50 + 1
50 -+ 1
54 _+ 3
Normal rabbit plasma
38 _+ 1
26 -+ 2
--
Data are means of values of B/B o (%) -+ s.e.m, from triplicate incubations. D y n o r p h z n - (1-13) , final concentration 66 pM, rat p i t u i t a r y e x t r a c t diluted i:i0 in MeOH-HCl, n o r m a l rabbit plasma diluLted 1:3 in same buffer as for a n t i s e r u m (see M a t e r i a l s and Methods). First two columns represent incubations in which all c o m p o n e n t s were mixed at zero time. Third column represents e x p e r i ment in which dynorphin-(l-13) or p i t u i t a r y e x t r a c t was incubated first for 24 hr, followed by incubation with r a d i o l a b e l l e d dynorphin-(l-13) and a n t i s e r u m for another 24 hr. This d e l a y e d - a d d i t i o n e x p e r i m e n t was to rule out the p o s s i b i l i t y that a degradative enzyme in tissue e x t r a c t was inactivated during the first 24-hr incubation.
(2) I m m u n o r e a c t i v i t y of the tissue e x t r a c t was d e s t r o y e d by a protease, papain. (3) Dilution curves for the tissue e x t r a c t were p a r a l l e l to those o b t a i n e d with d y n o r p h i n - ( l - 1 3 ) . (4) A n t i s e r u m was highly specific for a sequence of at least e i g h t residues of d y n o r p h i n - ( l - 1 3 ) , did not react significantly with any other opioid peptide, and did not recognize statherin ~a peptide c o n t a i n i n g a sequence of five residues identical to d y n o r p h i n - ( 4 - 8 ) ] , making it improbable that tissue i m m u n o r e a c t i v i t y was due to any unrelated peptide. (5) There should be n o impediment tO the detection of longer p e p t i d e s c o n t a i n i n g d y n o r p h i n - ( l - 1 3 ) , since n e i t h e r the COOH- nor the N H 2 - t e r m i n a l residue is required for immunoreactivity. The tissue e x t r a c t i o n and RIA p r o c e d u r e s d e s c r i b e d here have been used to determine the distribution of immunoreactive dynorphin in p i t u i t a r y and brain of several species (A. Goldstein and V.E. Ghazarossian, in preparation) . I m m u n o h i s t o c h e m i c a l localization studies using "Lucia" a n t i s e r u m have shown intensive staining of fibers and terminals in pars n e r v o s a of rat p i t u i t a r y and have
86
Dynorphin Radioimmunoassay
Vol. 27, No. I, 1980
identified magnocellular immunoreactive dynorphin in rat supraoptic nucleus (S. Watson et al., unpublished observations). As long as supplies last, we are willing to furnish reasonable amounts of the antiserum described here to investigators who request it. Acknowledgments We thank Asha Naidu and Keiko Otsu for technical assistance of the highest quality, Dr. S. Tachibana for helpful discussion, Dr. F. Leslie for purification and confirmation of structure of dynorphin-(l-13) methyl ester, Dr. Marian Orlowski for prolyl endopeptidase, Drs. June Dahl and Iris Lindberg for leucine-enkephalin antiserum, and Debbie Knapp for preparation of this manuscript. These studies were supported by grant DA-II99 from the National Institute on Drug Abuse. Re fe fence s i.
A. GOLDSTEIN, S. TACHIBANA, L.I. LOWNEY, M. HUNKAPILLER, L. HOOD Proc. Natl. Acad. Sci. USA 76 6666-6670 (1979).
2.
M. ORLOWSKI, E. WILK, S. PEARCE and S. WILK 461-469 (1979).
3.
V.E. GHAZAROSSIAN, R.R. DENT, K. OTSU, M. ROSS, B. COX and A. GOLDSTEIN Analyt. Biochem. 102 80-89 (1980).
4.
R.J. MILLER, K.J. CHANG, Sci. 22 379-387 (1978).
5.
A. GOLDSTEIN, L. ARONOW and S.M. KA£~4AN, Principles of Druq Action, 2nd Edition, pp. 89-96, Wiley, New York (1974).
6.
A. GOLDSTEIN and B.M. COX, Progress in Molecular and Subcellular Bioloqy, Vol. 6, eds. F.E. Hahn, H. Kersten, W. Kersten and W. Szybalski, pp. 113-157, Springer-Verlag Berlin Heidelberg (1978).
J. Neurochem.
and 33
J. LEIGHTON and P. CUATRECASAS Life
Life Sciences,Vol. 27,~~.75-86 Printedin the U.S.A.
A SPECIFIC RADIOIMMUNOASSAY FOR THE NOVEL OPIOID PEPTIDE .DYNORPHIN Vartan E. Ghazarossian, Charles Chavkin and Avram Goldstein Addiction Research Foundation and Stanford Dniversity, Palo Alto, California 94304 (Received in final form May 1,
1980)
Summary Dynorphin was recently isolated from porcine pituitary extracts and shown to be the most potent known Antisera *were prepared to synthetic opioid peptide. dynorphin-(l-13), the biologically active NH2-terminal A high-titer, sensitive fragment of the peptide. antiserum was characterized with fragments from dynorphin-(l-13). Leucine-enkephalin, which is contained in dynorphin, is not recognized at all by the antiserum. To study distribution in tissue, a procedure using hot acidified methanol extraction of rat pituitary neuroinmediate lobe preparations was developed and validated. M I-labelled dynorphin-(l-13), when added to tissue, remained intact throughout this extraction procedure, and added dynorphin-(l-13) was almost completely recovered. There was no destruction of radiolabelled peptide during incubation in the radioimmunoassay. Serial dilutions of pituitary extracts yielded curves that were parallel to The immunorethe dynorphin-(l-13) standard curve. activity from tissue was completely destroyed by papain treatment. Dynorphin is a recently described leucine-enkephalin containing peptide isolated from porcine pituitary extracts (1). Its NH -terminal fragment, dynorphin-(l-13) (YGGFLRRIRPKLK)*, when tegted in the guinea pig ileum myenteric plexus-longitudinal muscle bioassay, is approximately 200 times more potent than normorphine and 50 times more potent than B -endorphint. We now report the developmsnt of a highly specifig and sensitive radioimmunoassay (RIA), using antiserum raised to dynorphin-(l-13) coupled to thyroglobulin. We also describe an extraction procedure for the measurement of immunoreactive (ir-) dynorphin in biological material. Materials and Methods Peptides Dynorphin-(l-13), D-Ala2-dynorphin-(l-13)amide, D-Ala'-dynorphin-(l-11), dynorphin-(l-9), dynorphin-(6-13) and N-acetyl * Y=Tyr, G=Gly, F=Phe, L=Leu, R=Arg, I=Ile, P=Pro, K=Lys. t Subscripts: c = camel, h = human. 0024-3205/80/270075-12$02.00/O Copyright(c) 1980Pergamon Press Ltd.
76
Dynorphin Radioimmunoassay
Vol. 27, No. I, 1980
dynorphin-(6-13) % were synthesized by Peninsula Laboratories (San Carlos, CA). All peptides were demonstrated to be pure by thinlayer chromatography, by e lectrophoresis, and by amino-acid analysis. In addition, the sequences of dynorphin-(l-13) and of dynorphin-(6-13) were verified by Edman degradation on a sequenator, the former by Drs. L.E. Hood and M. Hunkapiller (Caltech), the latter by A. Smith (Univ. of Calif., Davis). Dynorphin-(l-12), -(i-ii) , -(i-i0) , -(1-7) , -(1-6) and -(2-13) were prepared from dynorphin-(l-13) enzymatically, as follows: Dynorphin-(l-12) was obtained by treatment with carboxypeptidase B, -(i-ii) by treatment with carboxypeptidase B followed by carboxypeptidase A, and -(1-7) and -(1-6) by trypsin digestion. Dynorphin-(2-13) was obtained by treatment with leucine aminopeptidase, dynorphin-(l-10) by treatment with prolyl endopeptidase (2) generously furnished by Dr. Marian Orlowski, dynorphin-(l-8) by carboxypeptidase B treatment of -(1-9). All of the above peptides were purified to homogeneity by reversed-phase high performance liquid chromatography (HPLC) (CIR column, various elution solvents), and their amino acid composl ~ tions were verified following acid hydrolysis. Detailed data on the preparation of these dynorphin fragments and proofs of structure will be published elsewhere (C. Chavkin and A. Goldstein, in preparation). A totally blocked derivative of dynorphin-(l-13) was synthesized by Peninsula Laboratories, with methyl ester blocking the terminal carboxyl group. This was catalytically dehydrogenated, and the resulting dynorphin-(l-13) methyl ester was purified to homoaeneitv bv HPLC. Its identitv was confirmed by amino acid analysis, conversion to dynorphin-(l'-13~ on saponification, and resistance to car boxypeptidase B. --H-le uc ine-en kepha lin (41 Ci/mmol) was purchased from Ame r sham, le ucine-en kephalin from Biosearch (San Rafael, CA), ~-endorphin from Pierce Chemical Co. (Rockford, IL) . All other opioid peptides were obtained from Peninsula Laboratories. Thin Layer Chromatography
(TLC)
Silica gel G plates (polygram Sil G, Brinkmann, Inc., Westbury, NY) were developed with butanol : acetic acid : ethyl acetate : water (1:1:2:1), System i; or with chloroform : methanol : acetic acid : water (15:10:2:3), System 2. Cellulose plates (13255 Cellulose, Eastman Kodak Company, Rochester, NY) were developed with butanol : acetic acid : water (4:1:5), System 3. Conjugation of dynorphin-(l-13)
to bovine
thyroglobulin
Dynorphin-(l-13) (5.2 mg) containing a trace quantity of 125Idynorphin-(l-13) (see below) and thyroglobulin (26 mg) were dissolved in 2 ml of i00 mM phosphate buffer (pH 7.5) and cooled to 0 °. Glutaraldehyde (Sigma, 25% aqueous solution) was diluted with icecold distilled water (i:i00) and 1 ml was added dropwise to the reaction mixture which was stirred 30 min at 0 ° then 2 h at room temperature. After extensive dialysis against 0.9% NaCI and then water, the conjugated peptide was lyophilized and stored at -20 ° . The molar ratio o~2~ynorphin-(l-13) to thyroglobulin was 92:1, as determined from [ I]dynorphin-(l-13) remaining in the dialysis bag.
%Terminal s-amino group acetylated.
Vol. 27, No. i, 1980
Immunization
Dynorphin Radioimmunoassay
77
Procedure
Male and female New Zealand White and New Zealand White x San Juan rabbits were used. The conjugated peptide (30-50 ~g) was suspended in saline and emulsified with an equal volume of Freund's complete adjuvant (incomplete adjuvant after the first injection). After a series of four immunizations at two-week intervals, the rabbits were injected at monthly intervals and bled 10-12 days after each booster injection following the fifth immunization. Several of the rabbits yielded useful antiserum; one with highest titer, good sensitivity, and high specificity, "Lucia 9/14", was chosen for the RIA described here. Iod ination
Procedure
125I-labelled dynorphin-(1-13) was prepared according to a procedure described for ~-endorphin (3). The radiolabelled peptide was separated from iodide by chromatography on Sephadex G-15 ~ i g . i). It was stable on storage at -20 ° for up to i0 weeks. ~I labelled leucine-enkephalin was prepared and purified as described by Miller et al. (4).
10
w~ O X u
1 J I
I
~L_I__£_TTT___ I 10
I
I 20
I
I 30
Fraction
I
No.
I 40
I
I 50
I
I 60
I
(1-ml.)
Fig. i. Gel filtration following iodination of dynorphin-(l-13). Sephadex G-15, 35 x 1.2 cm column, 0.25 M acetic aci~2~n 0.1% BSA. First peak of radioactivity contained + ~I-labelled dynorphin-(l-13), which chromatographed as a single component in TLC systems 1 and 3. Second peak contained unreacted iodide.
78
Dynorphin Radioimmunoassay
Vol. 27, No. I, 1980
Tissue Extraction and RIA Procedure Tissue was weighed and homogenized immediately in i0 volumes (but not less than 1 ml) of acidified methanol (MeOH-HCI, consisting of equal parts of methanol and 0.i M HCI, v/v) at 70 ° , incubated I0 rain at 70 ° , cooled on ice, centrifuged (15,000 x g), and the supernatant was transferred to a polypropylene tube and stored at -20 ° . All subsequent operations were carried out on ice (0°-4 °). Tissue extracts or dynorphin-(l-13) (10 mM stock solution in MeOHHCI stored at -20 ° ) were diluted serially in MeOH-HCI in polypropylene tubes for immunoassay. The 300-~i RIA incubation mixture consisted of: i00 ~i of a dilution of dynorphin-(l-13) or tissue extract (see above); 100 ~i of a 1:10,000 dilution of antiserum in 150 mM phosphate buffer (pH 7.5), containing 0 ~ ) bovine serum albumin (BSA) and 0.1% Triton X-100; i00 ~i of ['z=I]dynorphin-(l-13) (approximately 5000 cpm) in the same buffer as the antiserum. All components were mixed in polypropylene tubes in an ice bath§, and incubated 18-24 h at 0o-4 ". At the end of this period unbound radiolabelled dynorphin-(l-13) was removed by adding 1.2 ml of a suspension of ice-cold dextrancoated charcoal (5 g Norit A + 0.3 g dextran + 15 ml horse serum in i00 ml of 150 mM phosphate buffer, pH 7.4). The tubes were centrifuged (5000 x g, 4 ° , 20 rain), and radioactivity was determined in lml aliquots of each supernatant. Papa in Treatment Papain (Worthington Biochemical Corp., Freehold, NJ, 26.3 U/mg) was obtained as a suspension in 50 mM sodium acetate. It was activated by incubating the following mixture at 37 ° for 20 min: Papain (7.4 rag), 10 mM EDTA (2 ml), 12 mM mercaptoethanol (0.i ml), 50 mM cysteine-HCl (2 ml), glass distilled water (14 ml). A MeOHHCI extract of six pooled rat pituitary glands was lyophilized, dissolved in 10 mM EDTA (250 ~i), and divided into two ~ortions. One was treated eight times at 10-12 h intervals with freshly activated papain (i ml). The control was treated simultaneously with papain that had been inactivated at 100 ° for 10 min. At the end of the 90-h digestion, both reaction mixtures were brought to 100 ° for 15 min, lyophilized, dispersed in MeOH-HCI, centrifuged, and the supernatants were assayed as described above. Results and Discussion Procedures for minimizing loss of dynorphin peptides Significant loss of opioid peptides to surfaces has been described previously (3). Acidified methanol prevents the loss of dynorphin-(l-13) or of ir-dynorphin f ~ tissues to surfaces of containers. Table 1 shows that with [ I]dynorphin-(l-13) there is no loss to glass or polypropylene tubes in MeOH-HCI, whereas in the presence of salts, major losses occur. Iodinated peptide that has been lost from solution can be recovered, after drying, by addition of MeOH-HCI.
~A large volume of antiserum and mixed.
[1251]dynorphin-(l~13) can b e p r e -
Vol. 27, No. i, 1980
Dynorphin Radioimmunoassay
Table
i
125 Los3 of I-labelled dynorphin-(l-13) means of quadruplicate determinations. 750 ~I.
to surfaces. Data are Volume of solutions was
Polypr opylene * Solvent
79
Glass t
Percent of initial radioactivity remaining in solution after 60 min at 23 °
50 mM NaC1 in 50 mM phosphate buffer " + 0.1% BSA " + 0.1% Triton X-100 " + 0.1% BSA + 0.1% Triton X-100 MeOH-HCl
51 91 105
54 58 72
104 I01
75 96
Percent of radioactivity.recovered from tube walls ~ 50 mM NaCl in 50 mM phosphate buffer MeOH-HCI
15 93
33 96
"12 x 82 mm polypropylene tubes (Sarstedt). %13 x 100 mm borosilicate glass culture tubes (Scientific Products). %After loss of half the radioactivity from solution in phosphate buffer saline (first row), solution was aspirated, tubes were dried, and the indicated recovery solvent (750 ~l) was added.
Properties
of the antiserum
Antiserum "Lucia 9/14" binds 30% of radiolabelled peptide at a final dilution of about 1:50,000 (Fig. 2). Usefully, the antiserum tolerates 17% MeOH in the final incubation mixture; although the binding of radiolabelled dynorphin-(l-13) is reduced by 30%, there is no effect on the slopes of competition curves (see below). The antiserum does not contain antibo~i~@ that recognize leucinee~kephalin, since no binding of [~'~I] ledcine-enkephalin or [ H]leucine-enkephalin was observed (Fig. 2). Typical RIA protocols for leucine-enkephalin employ polyethylene glycol rather than charcoal to separate bound from free ligand after the incubation. Ey~ with polyethylene glycol, however, there was no binding of [~ I]leucine-enkephalin to antiserum "Lucia" (1:30 dilution). Under the same conditions, when an antiserum specific for leucineenkephalin was used ("DBD-3", kindly furnished by Drs. June Dahl and Iris Lindberg), there was 60% binding of radiolabelled leucineenkephal!n. The absence of binding sites for leucine-enkephalin in antiserum "Lucia" is especially important for the specific immunohistochemical localization of dynorphin.
80
Dynorphin Radioimmunoassay
Vol. 27, No. 1,1980
100
"o c
50
125 I-Dyn--(]--I3)
~'~O~o
\
Z
1251_Leu_Enk
~ ~ | ~ O ~ o I
101
I
102
3H_Leu_En~
I
l
103
104
I
105
Antiserum Dilution-1 Fig. 2. Binding of 125I-labelled dynorphin-(1-13) at different dilutions of antiserum "Lucia 9/14". Shown for contrast are results with radioactive leucine-enkephalin (Leu-enk). Dyn = dynorphin.
The antiserum is very sensitive. At a final dilution of 1:30,000 an IC~ ¶ of about 50 pM is routinely observed with dynorphin-(l-13) ~ i g . 3) ; this concentration is equivalent to about 17 fmol per assay tube. There is only modest ~ of sensitivity in a fast RIA, in which a 2-h incubation with [ I]dynorphin-(l-13) follows a l-h pre-incubation of the other two components (R.R.M. Dent, unpublished observations). Reproducibility of the RIA was assessed from IC~ 0 values for dynorphin-(l-13) determined over a three-month period. All dilution curves were symmetrical sigmoids, as in Fig. 3, with midpoint slope consistent with that predicted from the mass-law equation for i:i competition at a binding site (5). Mean IC~0 was 50 pM, with coefficient of variation 17% in 13 experiments, uslng a concentration of dynorphin-(l-13) near the IC.^, the triplicate values of i00 x B / B (see Fig. 3 legend) in e a ~ of these same experiments were anal~zed. The mean of all 39 values was 46%, with standard deviation 5%. The standard deviation within triplicates was 3%, and the variance ratio (between experiments/within triplicates) was 7.5. Thus, all variances were small, and the major (and significant) source of variance was between experiments.
¶ICon = concentration of competing [125I]dynorphin-(l-13) by 50%.
peptide
that reduces binding of
Vol. 27, No. I, 1980
Dynorphin Radioimmunoassay
81
Fig. 3 also shows that leucine-enkephalin [dynorphin-(l-5)] shows no crossreactivity at concentrations more than 10 million times thein IC
100
\ ~Dyn
o ca
~Dyn_( 6 -13)
1-13)
50
0
~
\ i I
I 10 pM
I 100
N_Acetyl - Dyn_(6_13
I 1
I 10 nM
I 100
I 1
I 10 pM
~ 100
I 1
I 10 mM
Concentration
Fig. 3. Immunoreactivity ~ y n o r p h i n - ( l - 1 3 ) and related peptides. Displacement of ~I-la~elled dynorphin-(l-13) was measured as described under Materials and Methods. B / B is binding in presence of competing peptide (x-axis sho~s concentration in incubation mixture) relative to binding of [ I]dynorphin-(1-13) alone, expressed as percent.
82
Dynorphin Radioimmunoassay
Table Crossreactivity
of dynorphin
Vol. 27, No. I, 1980
2
fragments and related peptides.
Pept ide
Crossreactivity
Dynorphin
(%)
fraqments and analoques
Dynorphin- (1-13) "
-(1-13)
"
-(1-12)
"
- (i-ii)
"
-(1-10)
"
-(1-9)
[YGGFLRRIRPKLK] methyl ester
i00 115 115 i0 4.8 3.8
"
-(1-8)
2.5 x i0 -I
"
- (1-7)
8.3 x 10 -3
"
-(1-6)
"
-(1-5) (le ucine enkephalin)
"
-(2-13)
"
-(6-13)
6.0 x 10 -5
"
-(6-13) , N-acetyl
2.4 x 10 -4
5.2 x 10 -3 < 1.0 x 10 -6 52
D-AIa 2-dynor ph in - (1-13 ) amide
9.6
D-Ala2-dynorphin
4.7
- (1-11)
Other opioid peptides
-3
s-endorph in
-3
8h-endorphin [Leu 5] 8h-endorphin
-3
<3.3xi0
-2
Me th ion ine -en ke pha i in
e - n e o - e n d o r p h i n - (1-8) [YGGFLRKR]
3.6xi0
-2
Data are based on molar ICon values obtained from dilution curves, all of which were p a r a l l e l to dynorphin-(l-13) standard curve. All p e p t i d e s shown here were dissolved and diluted in 50 m M phosphate buffer (pH 7.5) c o n t a i n i n g 0.1% BSA and 0.1% Triton X-100.
indicate that the antigenic d e t e r m i n a n t occupies a region of the peptide e x t e n d i n g toward the N H 2 - t e r m i n a l at least through residue 5, and p o s s i b l y as far as residue 2, and toward the C O O H - t e r m l n a l as far as (but not beyond) residue 12. This complete c h a r a c t e r i z a t i o n of s p e c i f i c i t y indicates that n a t u r a l l y - o c c u r r i n g porcine p i t u i t a r y dynorphin, which is a C O O H - t e r m i n a l l y e x t e n d e d dynorphin-(1-13)
Vol. 27, No. i, 1980
Dynorphln Radiolmmunoassay
83
(1), should very likely be recognized by this antiserum. Moreover, if precursors for dynorphin have extensions from the NH 9 terminus [in analogy with 8-endorphin (6)] they may also be recdgnized by the antiserum. Table 2 also shows that the antiserum does not react significantly with any of the other known opioid peptides or their fragments. Tissue extraction and assay of ir-dynorphin Serial dilutions of rat pituitary posterior lobe extracts in MeOH-HCI exhibited parallelism with standard dynorphin-(l-13) (Fig. 4A). When portions of the same rat pituitary extract were assayed on different occasions, the coefficient of variation was 14%, about the same as for replicate assays of dynorphin-(l-13) in the absence of tissue extract. The immunoreactivity was completely destroyed on treatment with papain, whereas similar extracts treated with heat-inactivated papain showed little loss (Fig. 4B). Thus, the immunoreactive material appears to be a peptide. No differences in ir-dynorphin content could be observed between posterior lobes allowed to stand for 30 rain at 23 ° before homogenizing, and those homogenized a few minutes after decapitation. In contrast to this stability in intact tissue, homogenizing in Tris buffer (50 mM, pH 7.5, 23 ° ) resulted in significant degradation in 10 min. This was e v i ~ _ ~ e d by appearance of a new component in TLC (system i) when I-labelled dynorphin-(l-13) had been added to the Tris buffer, and also by a loss of about 25% in ir-dynorphin as measured by RIA. Degradation did not occur when tissue was homogenized in MeOH-HCI at 70 °. MeOH-HCI extracts are completely stable on storage at -20 ° for at least 30 days. Recovery of dynorphin-(l-13) added to rat pituitary extracts was estimated as follows. A rat pituitary extract was divided into two parts, and dynorphin-(l-13) was added to one -- just enough to increase the total immunoreactivity by one-third to one-half. Two dilution curves resulted, both _parallel to the dvnorDhin-(l-13) standard curve, yielding two ICon values from which recovery of added peptide was computed to be 75-100% in three different experiments. In a separate set of experiments, complete recovery was also observed when dynorphin-(l-13) was added to every tube in a tissue extract dilution series. It is significant that satisfactory recoveries were obtained with addition of relatively small amounts of dynorphin-(l-13), but it does not necessarily follow that dynorphin in tissue extracts is recovered equally well. This uncertainty can only be resolved when the complete primary structure of dynorphin is known. Although it was unlikely that degradative enzymes would be present in hot MeOH-HCI extracts of rat pituitary neural lobe, it was necessary to demonstrate directly that radiolabelled dynorphin(1-13) was not degraded in the presence of tissue extracts in 24 hr at 4 ° in the RIA incubation mixture. TLC analysis of radioactivity from the incubation mixture at 0 and 24 hr revealed no change in R F, but certain fragments might not have been detected -- especially dynorphin-(l-ll), which is only weakly immunoreactive (cf. Table 2) -- since they are poorly separated from dynorphin-(l-13).
84
Dynorphin Radioimmunoase~y
100
Extract 1/10 2
1/10 3
Vol. 27, No. 1,1980
Concentration
I,/1
1/10 I
|
l
i,,L
v
'
A Dyn-(1J3~)e ~
50
0
I I
I
.....w/:
I00
|
I 103
8
e•O
50
0
I
101 102 Concentrat ion ( pM )
Contl~ro~
i i/i03
I I 1/ 102 1/101 Extract Concentration
! 1/ I
Fig. 4. A: Dilution curve of rat pituitary neurointermediate lobe extract and dynorphin-(l-13) standard curve. Scale of x-axis: Upper = extract concentration; lower = concentration of standard in incubation mixture. B: Papain treatment of rat pituitary extract. See Materials and M~thods for details. Control was extract treated with heat-inactivated papain.
If the apparent immunoreactivity of a tissue extract were an artifact due to degradation of radiolabelled dynorphin-(l-13), a decrease in B / B would be observed at longer incubation times. Accordingly, we ~arried out the experiment summarized in Table 3, the results of which show that there was no such degradation artifact. The evident degradation by rabbit plasma provides an instructive contrast. That the immunoreactive material extracted from rat pituitary posterior lobe is very likely natural dynorphin is indicated by the following : (l~25The possibility was ruled out that reduction in binding of [ I]labelled dynorphin-(l-13) was due to a degradative enzyme in the tissue extract.
Vnl. 27, No. i, 1980
Dynorphin Radioirmnunoassay
Table
3
Stability of r a d i o l a b e l l e d
dynorphin-(l-13)
the RIA incubation Incubation Addition
85
in
mixture.
time with r a d i o l a b e l l e d peptide at 0-4 °
0-24 hr
0-48 hr
24-48 hr
Dynorphin-(l-13)
35 + 1
40 + 1
40 + 4
Rat p i t u i t a r y extract
50 + 1
50 -+ 1
54 _+ 3
Normal rabbit plasma
38 _+ 1
26 -+ 2
--
Data are means of values of B/B o (%) -+ s.e.m, from triplicate incubations. D y n o r p h z n - (1-13) , final concentration 66 pM, rat p i t u i t a r y e x t r a c t diluted i:i0 in MeOH-HCl, n o r m a l rabbit plasma diluLted 1:3 in same buffer as for a n t i s e r u m (see M a t e r i a l s and Methods). First two columns represent incubations in which all c o m p o n e n t s were mixed at zero time. Third column represents e x p e r i ment in which dynorphin-(l-13) or p i t u i t a r y e x t r a c t was incubated first for 24 hr, followed by incubation with r a d i o l a b e l l e d dynorphin-(l-13) and a n t i s e r u m for another 24 hr. This d e l a y e d - a d d i t i o n e x p e r i m e n t was to rule out the p o s s i b i l i t y that a degradative enzyme in tissue e x t r a c t was inactivated during the first 24-hr incubation.
(2) I m m u n o r e a c t i v i t y of the tissue e x t r a c t was d e s t r o y e d by a protease, papain. (3) Dilution curves for the tissue e x t r a c t were p a r a l l e l to those o b t a i n e d with d y n o r p h i n - ( l - 1 3 ) . (4) A n t i s e r u m was highly specific for a sequence of at least e i g h t residues of d y n o r p h i n - ( l - 1 3 ) , did not react significantly with any other opioid peptide, and did not recognize statherin ~a peptide c o n t a i n i n g a sequence of five residues identical to d y n o r p h i n - ( 4 - 8 ) ] , making it improbable that tissue i m m u n o r e a c t i v i t y was due to any unrelated peptide. (5) There should be n o impediment tO the detection of longer p e p t i d e s c o n t a i n i n g d y n o r p h i n - ( l - 1 3 ) , since n e i t h e r the COOH- nor the N H 2 - t e r m i n a l residue is required for immunoreactivity. The tissue e x t r a c t i o n and RIA p r o c e d u r e s d e s c r i b e d here have been used to determine the distribution of immunoreactive dynorphin in p i t u i t a r y and brain of several species (A. Goldstein and V.E. Ghazarossian, in preparation) . I m m u n o h i s t o c h e m i c a l localization studies using "Lucia" a n t i s e r u m have shown intensive staining of fibers and terminals in pars n e r v o s a of rat p i t u i t a r y and have
86
Dynorphin Radioimmunoassay
Vol. 27, No. I, 1980
identified magnocellular immunoreactive dynorphin in rat supraoptic nucleus (S. Watson et al., unpublished observations). As long as supplies last, we are willing to furnish reasonable amounts of the antiserum described here to investigators who request it. Acknowledgments We thank Asha Naidu and Keiko Otsu for technical assistance of the highest quality, Dr. S. Tachibana for helpful discussion, Dr. F. Leslie for purification and confirmation of structure of dynorphin-(l-13) methyl ester, Dr. Marian Orlowski for prolyl endopeptidase, Drs. June Dahl and Iris Lindberg for leucine-enkephalin antiserum, and Debbie Knapp for preparation of this manuscript. These studies were supported by grant DA-II99 from the National Institute on Drug Abuse. Re fe fence s i.
A. GOLDSTEIN, S. TACHIBANA, L.I. LOWNEY, M. HUNKAPILLER, L. HOOD Proc. Natl. Acad. Sci. USA 76 6666-6670 (1979).
2.
M. ORLOWSKI, E. WILK, S. PEARCE and S. WILK 461-469 (1979).
3.
V.E. GHAZAROSSIAN, R.R. DENT, K. OTSU, M. ROSS, B. COX and A. GOLDSTEIN Analyt. Biochem. 102 80-89 (1980).
4.
R.J. MILLER, K.J. CHANG, Sci. 22 379-387 (1978).
5.
A. GOLDSTEIN, L. ARONOW and S.M. KA£~4AN, Principles of Druq Action, 2nd Edition, pp. 89-96, Wiley, New York (1974).
6.
A. GOLDSTEIN and B.M. COX, Progress in Molecular and Subcellular Bioloqy, Vol. 6, eds. F.E. Hahn, H. Kersten, W. Kersten and W. Szybalski, pp. 113-157, Springer-Verlag Berlin Heidelberg (1978).
J. Neurochem.
and 33
J. LEIGHTON and P. CUATRECASAS Life