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Purification of turkey prolactin and the development of a homologous radioimmunoassay for its measurement
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
40, 297-307 (1980)
Purification of Turkey Prolactin and the Development of a Homologous Radioimmunoassay for Its Measurement W.H.
BURKE* AND HAROLDPAPKOFF~
*Department of Animal Science, University of Minnesota St. Paul, Minnesota Research Laboratory and Reproductive Endocrinology Center, University San Francisco, California 94143
55108 and tHormone of California,
Accepted September 24, 1979 Prolactin has been purified from pituitary glands of the turkey (Meleagris gallopavo) and a radioimmunoassay has been developed for its measurement. The prolactin is a molecule of about 26,000 MW. It is high in glutamic and aspartic acids and in leucine, while it contains few methionine and half-cystine residues. Turkey prolactin elutes from Sephadex G-100 with a Ve/Vo ratio of 2.0. It displays multiple bands in disc electrophoresis, when run at pH 8.3. The most prominent prolactin band is at a RI value of 0.83-0.85. Prolactin is active in stimulating pigeon crop-sac development, but does not give a parallel dose-response to ovine prolactin. An antiserum was developed against turkey prolactin and was used to develop a homologous RIA using 1251-labeled turkey prolactin as the radioligand. The antiserum binds about 20% of the radioligand at a dilution of 1:7500. The least detectable dose of prolactin was 0.42 * 0.13 ng when tracer and sample were simultaneously mixed with antiserum. The 50% bound point averaged 3.8 f 0.32 ng. Considerably greater sensitivity was achieved by incubating the sample with the antiserum for 1 or 2 days prior to addition of tracer. The within-assay coefficient of variation was 7.9% for samples in the 45-65% bound range. Between-assay coefficient of variation was 8.9%. Binding is inhibited by sera from turkeys in some, but not all, stages of growth and reproductive development. Serial dilutions of sera and pituitary extracts yield dose-response curves which do not differ in slope from the purified turkey prolactin standard. Potency estimates of pituitary preparations by cropsac assay and by the prolactin RIA are in excellent agreement. Rat pituitary extract, rat prolactin, ovine prolactin, turkey FSH, LH, and GH all show no inhibition of binding even at very high doses. The antiserum neutralizes crop sac-stimulating activity of turkey prolactin.
A number of indirect studies implicate prolactin in a wide variety of physiological functions in avian species i.e., an involvement in incubation and the associated gonadal regression in Galliformes, the regulation of nasal gland secretion in Anseriformes, fat deposition in Galliformes and Passerines, growth promotion in Columbiformes, and stimulation of crop-sac development in this same group (see Nicoll, 1974 for a review). The general unavailability of avian prolactins has made the development of homologous radioimmunoassays for their detection impossible. Prolactin from the chicken has recently been purified by Scanes et al. (1975) and with it they developed a radioimmunoassay (RIA) for its measurement (Scanes et al.,
1976). McNeilly ef al. (1978) have reported on a mammalian prolactin RIA which was useful for measurement of turkey prolactin. It was the purpose of these studies to obtain highly purified turkey prolactin suitable for the development of a homologous radioimmunoassay. The purification of turkey prolactin, its chemical characteristics and the development of a radioimmunoassay for its measurement are described. MATERIALS AND METHODS Pituitaries and Fractionation Pituitary glands used in these studies were described Burke et al. (1979) as were the initial stages of purification. In brief, glands were homogenized in a Waring Blendor with ice-cold water and the homogenate was adjusted to pH 9.5 with Ca(OH),. After removal of the residual tissue by centrifugation, the suby
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298
BURKE
AND
pernatant was adjusted to 0.15 M (NH&SO, and the pH was reduced to 4.0 by the addition of metaphosphoric acid. The precipitate which formed was removed by centrifugation. It was taken up in H,O, dialyzed against water, and lyophilized. This fraction, termed MP, was extracted for 1 hr with pH 9.50.1 N NaCl and then centrifuged. The supernatant, which contained the GH and prolactin, was brought to a concentration of 0.2 saturated ammonium sulfate (SAS) and adjusted to pH 5.1. The precipitate which formed was removed by centrifugation and the supernatant was then adjusted to 0.5 SAS. The precipitate which formed at 0.5 SAS contained the bulk of the GH and prolactin and was removed by centrifugation. The supernatant was adjusted to 0.8 saturated ammonium sulfate and the precipitate that formed was also collected. The precipitated materials were dialyzed against water and lyophilized. The fraction precipitating between 0.2 and 0.5 SAS was further fractionated using a DEAE-cellulose column. The DEAE-cellulose was equilibrated with 0.03 M Tris-HCI, pH 7.4, and the hormone-containing fraction was dissolved in the same buffer and applied to the column. After the first protein peak was eluted with this buffer, the column was eluted with the same buffer adjusted to 0.07 M NaCl. The column was then eluted with 1 M NH,HCO,. The prolactin-rich fraction that eluted from the DEAE column with 1 M NH,HCO, was dialyzed against water, lyophilized, and then reapplied to a DEAE column as described previously. The column was again eluted with 0.03 M Tri-HCl pH 7.4, then with 0.03 M Tri-HCl pH 7.4 adjusted to 0.07 M NaCl, and finally with the same Tris buffer adjusted to 0.17 M NaCl. The protein emerging with the latter buffer had most of the prolactin activity and it was dialyzed against water and lyophilized. It was then taken up in 0.05 M NH,HCO, and chromatographed on a 2.5 x 90-cm Sephadex G-100 column equilibrated with the same buffer. The prolactin emerged with a Ve/Vo ratio of 2.0. A yield of 185 mg/kg wet wt of pituitaries was obtained.
Assays Fractions obtained during purification were monitored for prolactin by the local pigeon crop-sac assay, using the procedure of Nicoll(1%7) and for GH by the anti-snapping turtle GH radioimmunoassay (RIA) described by Hayashida et al. (1975). They were also examined by polyacrylamide gel disc electrophoresis (DE) in 7.5% gels using Tris-glycine buffer at pH 8.3. The gels were stained with Coomassie brilliant blue G250 according to the procedure of Blakesly and BoeLi (1977). Distribution of GH was also confirmed by the homologous turkey GH RIA of Proudman and Wentworth (1978). The amino acid composition of the purified preparation was determined as described by Spackman et al.
PAPKOFF
(1958) on a Beckman Model 119C amino acid analyzer following oxidation in performic acid (Li, 1957) and hydrolysis in constant boiling HCl (20 hr at 105”). Tryptophan and tyrosine determinations were made by the spectroscopic method of Beaven and Holiday (1952). Amino terminal amino acid analyses were done according to the method of Gray (1967) and Woods and Wang (1%7). Late in the course of these studies an RIA was developed for the measurement of turkey prolactin (see below). It was then used to examine precursor fractions and purified preparations.
Prolactin Antisera Production During the course of the studies a fraction with substantial crop sac-stimulating activity and low GH contamination was obtained and used to immunize several rabbits. Before use as an antigen, 200 pg of the preparation was applied to each of five polyacrylamide gel columns prepared as described above. After electrophoresis, one gel was stained and a prominent band was seen at an Rf value of 0.83, similar to the R, of chicken prolactin (Nicoll and Nichols, 1971: Scanes et al., 1975) and other avian prolactins (Nicoll and Nichols, 1971). A 6-mm region centered on an R, of 0.83 was cut from each of the unstained gels. Each gel piece was forced through a l-ml syringe into 1 ml of saline. The gel-saline suspensions were stored frozen until used for immunization. At immunization a suspension was thawed and emulsified with Freund’s complete adjuvant. Immunizations were done using the procedure of Vaitukaitus et al. (1972). After the first two immunizations, further boosters were given using highly puritied turkey prolactin, which was then available, without prior electrophoresis.
Prolactin RIA: Iodination Turkey prolactin (B15lB) which resulted from these studies was iodinated using a slight modification of the method of Goldfine et al. (1974). Five micrograms of the hormone was dissolved in 5 ~1 of 0.03 M NH,HCO, and diluted to 30 ~1 with 0.5 M phosphate buffer, pH 7.4. lp51, 0.5 mCi, was added to the vessel, followed by 10 ~1 of chloramine-T (0.4-0.8 pg/lO ~1). The reaction was monitored as described by Goldfine et al. (1974) and terminated when approximately 60% of the lz51 was precipitatable by trichloroacetic acid. The free iodine was separated from bound by chromatography on a disposable Bio-Gel P-60 column (0.7 x 18 cm) previously equilibrated with 0.05 M phosphate buffer-0.25% bovine serum albumin, pH 7.4. The column was eluted with the same buffer and 0.4 ml fractions were collected. Protein-bound iodine peaked in fraction 8. The ability of contents of this tube and others on each side of it to bind to antisera was quite low. Rechromatography of tubes on the descending limb of the peak (tubes 9 and 10) on a Sephadex G-100 column (1.5 x 50 cm) resulted in the resolution of a large peak with a Ve/Vo ratio of 1.35 and a second
TURKEY
PROLACTIN
PURIFICATION
smaller peak with a Ve/Vo ratio of 1.79. A third peak with a VelVoratio of about 3.0 was also obtained. Only the second peak showed good binding to the antisera. It was diluted to about 15,000 cpml50 ~1 with a pH 7.4 0.1 M phosphate-O.15 M NaCl-0.05 M EDTA buffer containing 0.6% normal rabbit serum.
Prolactin
RIA Protocol
Assay tubes were set up containing a maximum of 100 ~1 of sample or sample diluted in pH 7.4, 0.1 M phosphate-O. 15 M NaCl buffer containing 0.1% bovine serum albumin. To this was added 100 ~1 of a 1:3000 dilution (final dilution 1:7500) of the antiserum in 0.1 M phosphate-O.15 M NaCl-0.05 M EDTA buffer adjusted to pH 7.4 with NaOH. Fifty microliters of the radioligand was added to the assay tubes immediately before the antiserum or following a 24- or 48-hr delay. Twenty-four hours after addition of the radioligand, 100 ~1 of a 1:30 dilution of sheep anti-rabbit y-globulin was added. The tubes were allowed to incubate overnight and were then diluted with 1 ml of the phosphate-buffered saline described above and centrifuged at 24OOgfor 30 min. Supematants were poured off; the tubes were allowed to drain and the radioactivity in the pellets was determined. Samples were kept at 5”, except for brief intervals when reagents were added. In each assay triplicate sets of tubes containing tracer, tracer plus buffer (NSB), and tracer plus buffer plus antiserum (Bo) were run. The mean NSB counts were subtracted from the Bo tubes and from all standard or unknown samples. All standards or unknowns were expressed as a percentage of the Bo tubes (B/B0 x 100) and dose-response curves were obtained by calculating the linear regression of the logit percentage B vs the log of the hormone dose. Potency of unknowns were obtained by comparison with the turkey prolactin standard (Bl5lB). The standard was run in triplicate in six to eight serial dilutions, while unknowns were run in duplicate at multiple doses.
Prolactin
RIA Validation
Hormone-neutralizing activity of the antiserum was tested by incubating partially purified prolactin (150 pg or 50 pg) in 0.4-ml aliquots of undiluted antiserum or in normal rabbit serum for 24 hr at 5”. Six pigeons received antiserum-hormone mixtures over one hemicrop and the normal serum-hormone mixture over the other hemicrop. Injections were done and responses were determined as described by Nicoll (1%7). Control pigeons received injections of normal rabbit serum. Responses obtained from the two hemicrops of the hormone-injected pigeons were compared and neutralization effects were then assessed on a withinpigeon basis. Pituitary tissue was obtained from five laying turkeys and from five which had been nesting for several weeks (i.e., broody turkeys). A blood sample was ob-
AND
RIA
299
tamed from the hens and they were then killed by cervical dislocation. The anterior pituitary glands were quickly removed, separated into cephalic and caudal lobes, weighed, and quick frozen on dry ice. The glands were later individually homogenized in pH 7.4 phosphate-buffered saline with a Brinkmann Polytron Model PCU-2. The homogenates were kept at 5” for 2 hr and then centrifuged at 11,OOOgfor 30 min. The supematant fluids were diluted in assay buffer and frozen until used for assay. To control for nonspecific effects of tissue extracts, several rat pituitary glands were comparably processed and assayed. Sera from five broody and five laying hens were stored frozen until assayed. During the course of purification many samples were assayed in the crop-sac bioassay, but few of these were retained. When the prolactin RIA was developed, fractions of widely different bioactivity which had been assayed in the same crop-sac assay were assayed in a single immunoassay to determine the relationship between immuno- and bioactivity. Many other fractions which had been bioassayed in different crop-sac assays, and were known to differ vastly in bioactivity, were assayed by RIA and the responses were compared. Many fractions which had been monitored by DE and found either to have prolactin bands or be free of them were run in the RIA and the results were compared to expectations based on DE. Specificity of the assay was tested by assaying available turkey pituitary hormones. Turkey LH, FSH, and GH were tested in the prolactin RIA at doses 40- to 50-fold higher than the maximum assayable dose of turkey prolactin. Likewise ovine prolactin (NIH P-S15) and rat prolactin (NIH RP-1) were also tested at doses up to 1000 ng. Sera from turkeys of a variety of ages, sexes, and reproductive states were assayed at multiple doses to determine if serum per se would inhibit binding of radioligand and to determine parallelism of serum samples with the purified hormone. Sera from two surgically hypophysectomized turkeys were kindly donated by Dr. Howard Opel and were tested at multiple doses in this assay.
RESULTS
As determined by crop-sac assay (Table 1) all of the prolactin was precipitated by metaphosphoric acid at pH 4. From this MP material the most enriched prolactin was obtained at SAS concentrations between 0.2 and 0.5 and a slight amount above 0.5 SAS. Prolactin was absorbed onto the DEAE-Tris column at 0.03 M Tris pH 7.4 and was eluted. by the Tris-0.17 M NaCl buffer or by 1 M NH,HCO,. The prolactin RIA (see validation below) strongly sup-
DEAE-cellulose 1.05 1.13 2.34 1.52 Sephadex G- 100 2.12 2.30
200 150 150 150 100 20
13.8 mg 17.0 mg 22.8 mg 7.6 mg
21.8 mg 24.0 mg
0.03 M Tris 0.03 M Tris-0.07 M NaCl 0.03 M T&-0.17 M NaCl 1 M NHIHCO,
Fraction A Fraction B
(NH&SO,
fractionation 1.12 2.60 1.72
0.17 1.0
0.04 0.17 0.02
0.05 0.003
Potency
13.4 86.6
%.4 3.5
-
26.9 69.2 3.8
97.6 2.3
Percentage
0.15 0.04 0.009 0.006
0.03 0.44 0.02
0.11 0.04
Potency
69.2 22.7 6.7 1.3
6.1 89.8 4.1
87.4 12.6
Percentage
Not assayed Not assayed
GH RIA
a Examples of the distribution of hormones in the various purification steps. b Mucosal dry weight of test substance divided by mucosal dry weight of control. Three to four hemicrops per treatment. c Percentage of the activity in each fraction. The potency of each fraction in a given purification procedure was multiplied by the mass of that fraction to obtain the units of activity in each fraction. The units of activity in a given fraction were divided by the total units of activity obtained in that procedure and the quotient was multiplied by 100.
50 50 100
3.6 g 2.1 g 0.8 g
acid precipitation 1.51 1 .Ol
O-O.2 SAS 0.2-0.5 SAS 0.5-0.8 SAS
Metaphosphoric 100 100
12.0 g 4.8 g
MP MS
Mucosal dry weight*
Dose (IL@
Yield
Prolactin RIA
TABLE 1 GROWTH HORMONE IN VARIOUS PURIFICATION STEPS
Crop-sac assay
AND
Fraction
DISTRIBUTION” OF PROLACTIN
TURKEY
PROLACTIN
PURIFICATION
ported the interpretation obtained by the bioassays which in turn was supported by disc gel electrophoresis. The RIA indicated that nearly 98% of the prolactin was precipitated by metaphosphoric acid and nearly 70% of that went into the 0.2-0.5 SAS fraction. Over 96% of the immunoreactive prolactin absorbed to the DEAE-Tris column and was eluted with the Tris buffer adjusted to 0.17 M NaCl. Disc electrophoresis clearly showed the presence of GH in the MP fraction with lit-. tle apparent in the MS. The homologous GH RIA indicated that the bulk of the GH did in fact go into the MP fraction (Table 1). About 90% of the GH precipitated between 0.2 and 0.5 SAS as assessed by GH RIA. Complete separation of the prolactin and GH was achieved by the chromatography on DEAE-cellulose. This was confirmed by both disc electrophoresis and the homologous turkey GH RIA (Table 1). The most potent turkey prolactins consistently showed a cross-reaction in the heterologous GH RIA (Hayashida et al., 1975), exhibiting potencies of 0.02-0.04 times the rat standard and also in the turkey GH RIA with a potency of about 0.02 times the turkey GH. These preparations, when run in DE at 100 /.&gel, showed no stain-
FIG.
90-cm
1. Gel filtration of the 0.17 M NaCl column; 3.6 ml collected per tube.
fraction
AND
301
RIA
able bands in the region in which GH migrates. Biochemical
Characterization
The gel filtration elution pattern of prolactin (Fig. 1) shows a major peak, designated B, with a Ve/Vo ratio of 2.0 and an additional larger molecular component designated A. Both were active in the pigeon crop assay. The disc electrophoretic pattern of turkey prolactin is seen in Fig. 2 and compared with turkey GH. Characteristically, both display multiple stainable bands with, however, no overlap. The most prominent prolactin band had an R, value of 0.83-0.85. Three strongly staining bands were seen with the growth hormone at R, values of 0.19, 0.30, and 0.38. The major amino terminus of turkey prolactin appears to be leucine, but traces of alanine, tyrosine, and lysine were also found. The amino acid composition of turkey prolactin is shown in Table 2. It is rich in aspartic acid, glutamic acid, and leucine and low in methionine and half-cystine content. Spectrophotometric determinations showed the presence of 2.9% tyrosine and 1.46% tryptophan. The latter value is consistent with the presence of two residues of tryptophan in the molecule. If two
from
DEAE-cellulose
on Sephadex
G-100;
2.5 x
BURKE AND PAPKOFF
302
residues of tryptophan are present and if one assumes 15% moisture and ash in the preparations, a molecular weight of 2 1,700 is calculated. The molecular weight as determined by SDS-polyacrylamide electrophoresis was 26,000; in addition a trace component of 12,000 MW was seen. In the same experiment ovine and turkey GH gave values of 20,500-22,800 MW. Bioassays
FIG. 2. Disc electrophoretic pattern of turkey prolactin, turkey GH, and the 0.2-0.5 SAS precipitate containing both prolactin and GH. A 7.5% gel, pH 8.3; the origin is toward the top of the figure. The arrow indicates the marker dye.
TABLE 2 AMINOACIDCOMPOSITION OFTURKEY PROLACTIN Turkey prolactin” LYS His Arg Asp Thr Ser Glu PI.0
GUY Ala MCys Val Met Ile Leu W Phe TV’
11.0 6.5 10.1 22.0 7.0 14.1 33.7 9.7 14.3 13.2 4.1 8.1 3.1 8.5 20.5 4.6 6.9 2.0
Ovine PRL* 9 8 11 22 9 15 22 11 11 9 6 10 7 11 23 7 6 2
n Residues/l97 residues, average of two determinations. b Structural analysis of Li ef al. (1970) and Li (1976). p Spectrophotometric measurement.
The final prolactin preparation was assayed in the pigeon crop assay a number of times. A typical result is seen in Table 3. Two micrograms of the turkey prolactin produced a significant increase in mucosal dry weight over the solvent-injected control. A linear dose-response curve was obtained between 2 and 50 pg. Ovine prolactin was repeatedly more potent than turkey prolactin, but the lack of parallelism between the ovine and turkey dose-response curves precluded accurate potency estimates. However, the turkey prolactin is about 20% as potent as ovine prolactin. Turkey growth hormone was free of significant crop sac-stimulating activity at a dose up to 400 pg. Prolactin Radioimmunoassay
Only 3-5% of the radiolabeled prolactin which eluted from the Bio-Gel P-60 column was bound by the antiserum at a 1:7500 dilution. Purification of this material on Sephadex G-100 yielded major peaks at TABLE 3 CROPSAC-STIMULATING ACTIVITY OFTURKEY PROLACTIN,TURKEY GH, AND OVINE PROLACTIN Treatment Saline Turkey prolactin Turkey GH Ovine prolactin
Dose 64) 2 10 50 80 400 2 10
Mucosal dry weight (mg; 2 f SE) 15.5 21.3 32.4 41.2 14.9 18.6 37.8 42.1
+ 1.2 t 0.6 lr 2.1 k 2.5 + 2.5 z!z2.3 2 1.1 f 1.6
TURKEY
PROLACTIN
PURIFICATION
Ve/Vo ratios of 1.35, 1.79, and 3.0. Material in the first and third peaks was not bindable by antiserum, while about 20% of the material in the middle peak was bound at an antiserum dilution of 1:7500. When the sample, tracer, and antiserum were added to tubes at the same time and incubated 24 hr before addition of second antibody, the sensitivity of the prolactin RIA, defined as 2SD below the 100% bound point, averaged 0.42 + 0.13 ng (x + SE) in six assays. The 50% bound point was 3.8 f 0.32 ng. The 50% bound point can be decreased about lo-fold by delaying addition of the radioligand for 48 hr after the antiserum and unlabeled hormone are mixed. The within-assay coefficient of variation, based on differences between duplicate potency estimates of samples in the midrange of the assay (45-65% bound), was 7.9%. The between-assay CV, based on the average potency of duplicate assay tubes from a pool of serum from broody turkey hens run in seven assays, was 8.9%. The agreement between immuno- and bioactivity (pigeon crop-sac assay) was excellent. In all comparisons made, bioactivity estimates and immunoactivity potency estimates of partially purified prolactin
TABLE
AND
303
RIA
fractions have been in perfect rank order. An example of these comparisons is shown in Table 4. Comparison of the prolactin potency of pituitaries from broody and laying turkeys has been made several times (Cherms et al., 1%2; Cogger and Burke, unpublished). In both these earlier studies broody turkey pituitary glands were 3 - 5 times as active in stimulating pigeon crop-sac development as glands from layers. The RIA comparison (Table 5) also shows tissue and sera from broody hens to contain more prolactin than that from laying hens. Cephalic lobe tissue from five broody hens had about 2.5 times as much prolactin, on a weight basis, as comparable tissue from layers. Total cephalic lobe prolactin content was about 2.4 times as great in broody hens. The caudal lobes of both groups had relatively little prolactin and there was no difference between groups. Immunoassay of cephalic lobe tissue from the 10 hens at 2.5, 0.25, and 0.025 pg yielded a dose-response curve which did not differ significantly from that of the prolactin standard (b = - 1.97 for pituitary tissue and -2.11 for the standard). Rat pituitary extract did not inhibit binding of radioligand when tested over the same
4
RELATIONSHIP BETWEEN BIOACTIVITY AND IMMUNOACTIVITY OF TURKEYPROLACTIN PREPARATIONSATVARIOUSSTAGES OF PURIFICATION Crop-sac assay Preparation
Dose tested w3)
Saline B88A” B88B B88D B9OC B88C B84C B151B
200 150 150 100 150 100 50
Mucosal dry weight (mg) 14.6 15.2 16.3 22.0 23.4 33.9 37.8 42.4
? k f k 2 k + k
0.9 3.1 2.1 2.8 1.4 3.2 7.4 5.0
RIA potency (X B15lB)
a B88A, B, C, and D are fractions from the DEAE-cellulose column eluted sequentially by buffers shown in Table 1. Preparation B9OC is a fraction precipitating from the MP at 0.2-0.5 SAS. Fraction B84C is a preparation derived from B9OC and the Bl51B is the final purified prolactin (Fig. 1).
BURKE
304
AND
PAPKOFF
TABLE PITUITARY
AND
SERUM
PROLACTIN
5
LEVELS
OF BROODY
Cephalic lobe CLg/mg Laying turkeys Broody turkeys
9.5 k 1.8* 24.6 k 3.6
AND
LAYING
TURKEYS
Caudal lobe l*gimg
230.8 -c 55.1 544.8 k 59.2
1.6 2 1.0 1.2 k 0.2
dose range as the turkey tissue (0.025 to-25 /49. Serum from a broody turkey, assayed in duplicate at six doses ranging from 50 to 1.56 ~1, gave a dose-response curve parallel to the standard prolactin (Fig. 3). Sera from two hypophysectomized hens at doses up to 50 ~1 depressed binding slightly. The dose-response characteristics of these sera were erratic and did not parallel the standard. Many sera from somatically mature, sexually immature female turkeys housed under short photoperiods (6L:18D) have been assayed and found not to inhibit binding of the tracer at doses up to 100 ~1
Serum (n&4
/.&gland
I.cdgland
15.5 * 9.6 4.9 t 1.2
360.6 2 185.5 1602.3 2 244.8
(the highest tested). The same is true of sera from sexually immature male turkeys between 14 and 20 weeks old. Highly purified turkey GH, LH, and FSH and highly purified ovine and murine prolactins showed no inhibition of binding at doses up to 1000 ng. Four hundred microliters of undiluted anti-prolactin serum completely neutralized the crop sac-stimulating activity of 50 Fg of partially purified prolactin and partially reduced the activity of 150 pg of the hormone (Table 6). In each of six pigeons the hemicrop receiving hormone in the antiserum was less stimulated than the contralateral
Hypophyseltomlmd Turkey Sea
Bmdy
\\
.L
Turkey Sera
(b--2.61))
Turkey Prolaclinl 81518 ) ( b- -2.63 I
1
I
I
12.5 .lB 1.56 3.12 6.25 LOG DOSE OF HORMONE( nglOR SERAluII
1
25
50
FIG. 3. Radioimmunoassay dose-response curves of turkey prolactin, a serum pool from broody turkeys and sera from two hypophysectomized turkeys. Three-day assay with sample, antisera, and tracer added on Day 1, second antibody on Day 2, and assay termination on Day 3. The calculated least detectable dose of hormone (i.e., a dose which would depress counts 2 SD deviations below Bo) was 0.26 ng of turkey prolactin (B151B).
TURKEY
PROLACTIN
TABLE 6 EFFECTOFPROLACTIN ANTISERA ON THE CROPSACSTIMULATINGACTIVITY OF TURKEY PROLACTIN~ Assay No.
Prolactin (pg)
Solvent
MDWb (mkd
1 1 1 1
0 50 50 50
NRS NRS AS NH,HCO,
18.3 24.0 17.2 22.5
t zt 2 k
1.4 5.0 1.0 1.4
2 2 2 2
0 150 150 150
NRS NRS AS NHIHC03
19.2 32.9 28.7 31.2
t f k k
1.2 0.6 0.9 2.0
u N = 3 hemicropsltreatment; the comparison of antiserum-treated and normal serum-treated prolactin was made on a within-pigeon basis. b MDW, mucosal dry weight, x f SE: AS, antiprolactin sera; NRS, normal rabbit serum.
side which received hormone in normal serum. The difference between crop-sac muoscal dry weights of pigeons receiving antiserum-treated prolactin and those receiving prolactin incubated in normal rabbit serum was significant (P < 0.05) by paired r test. DISCUSSION
As part of a broad program of studies on the biochemistry and physiology of nonmammalian pituitary hormones, we have evolved purification procedures which allow the purification of as many of the pituitary hormones as possible from the same batch of glands (Licht et al., 1977; Farmer and Papkoff, in press). The procedures employed for the purification of turkey prolactin are largely derived from these studies. It is of interest that the prolactin and GH exhibited very similar fractionation behavior in most of the steps employed herein. Complete separation was achieved, however, by the DEAE chromatography as described. Previous studies with GH and prolactin from a teleost fish (Farmer et al., 1976b, 1977) were similar in this regard. The physicochemical characterization
PURIFICATION
AND RIA
305
studies show that the turkey prolactin, while not identical, shares many properties with prolactin from other species.. These include gel filtration behavior, molecular weight, disc gel electrophoretic patterns, and amino acid analysis. The gel filtration experiments (Ve/Vo=2.0), spectrophotometric studies, and the SDS-gel electrophoretic analysis indicate a molecular weight of 22-26,000 for turkey prolactin, a value consistent with that determined for ovine prolactin (Li, 1974). The amino acid content of turkey prolactin is characterized by large amounts of leucine, aspartic acid, glutamic acid, and two tryptophan residues, all properties of the more extensively studied mammalian prolactins. The low half-cystine content in turkey prolactin, suggestive of two disultide bridges, should be regarded with caution insofar as this amino acid is diflicult to accurately determine. It may be noted that mammalian prolactins possess three disulfide bonds (Li et al., 1970; Li, 1976). The purified turkey prolactin was found to be essentially free of other pituitary hormones , including growth hormone. Turkey prolactin is readily bioassayed in the pigeion crop-sac assay, but displays different dose-response characteristics than ovine prolactin and as a result appears to be somewhat less potent. Species variation in hormonal response has been well documented for gonadotropins (Licht et al., 1977) as well as for growth hormones (Farmer et al., 1976a; Farmer and Papkoff, in press), and it can be anticipated that various species of prolactin will also behave differently in a given assay. The prolactin radioimmunoassay described is free from cross-reactions with other turkey pituitary hormones. RIA measurements agree well with the bioactivity of partially purified pituitary preparations as assessed by the crop-sac assay and with previous bioassay studies with regard to the potency of pituitary glands from broody and laying turkeys.
306
BURKE
AND
Quantitation of unknown serum samples against purified prolactin is possible since parallelism is obtained. The assay is sensitive enough to detect prolactin in as little as 2-5 ~1 of sera from broody turkeys while as much as 50-100 ~1 from certain sexually quiescent birds has too little prolactin to quantitate. The failure of these relatively high levels of sera to inhibit binding of tracer supports the absence of nonspecific serum effects in the assay. The sera from hypophysectomized turkeys tested in this RIA appeared to depress binding of labeled prolactin to a minor degree. The hypophysectomies appeared complete by visual examination, but microscopic examinations of the sella turcica were not made. The presence of remnants of pituitary tissue cannot be excluded. A homologous avian (chicken) prolactin RIA has been reported (Scanes et al., 1976) and a heterologous assay has been employed for measurement of turkey prolactin (McNeilly et al., 1978). No direct comparison of these assays with our own have been made. However, both of these reports appear to agree with this study in that serum from sexually immature turkeys has less prolactin than serum from laying females. There appears, however, to be a major discrepancy between our findings and those of McNeilly et al. (1978) and Etches et al. (1979). In our experience, sera from broody turkeys have always contained substantially more prolactin than sera from laying hens, whereas their report shows no difference. However, unpublished studies from our laboratory do show that changes in a broody bird’s environment can cause a decrease in prolactin from the very high levels of the broody hen to levels in the range of laying birds in 24 hr or less. Consequently, care must be exercised to avoid this possibly confounding effect. Even though the prolactin levels are labile over a 24-hr period, we have not seen an elevation or depression in levels as a result of handling and repeated venipuncture over periods of 1-2 hr. Further data will
PAPKOFF
have to accrue before critical comparison these three RIAs can be made.
of
ACKNOWLEDGMENTS The authors wish to express their sincere appreciation to Dr. C. H. Li for his interest, support, and advice during the course of these studies. Our thanks are also expressed to Dr. S. W. Farmer for her advice and assistance; to Dr. T. Hayashida, E. Rowley, and J. Walker for assaying many samples for GH, and to Dr. B. B. Aggarwal, M. Davenport, P. Dennison, K. Hoey, and J. D. Nelson for splendid technical assistance. We wish to thank Dr. Paul Licht for his aid in the assessment of prolactin antisera used in these studies and Dr. John Proudman for the gift of antiturkey GH sera. Special thanks are due to the following students, staff, and faculty of the University of California and the University of Minnesota for assistance in the tedious job of collecting the turkey pituitary glands used in this work: E. Daniels, J. DeMichael, M. El Halawani, L. Fuqua, J. Hagman, L. Hallacher, P. Martin, J. Millam, F. X. Ogasawara, L. Ogren, S. Pallas, W. Rainey, T. Siopes, M. Vodian, L. Wolcott, and W. Yu. Thanks are given to Dr. Howard Opel, USDA, ARS, Beltsville, Maryland for the gift of sera from hypophysectomized turkeys. We thank the National Institutes of Arthritis, Metabolism and Digestive Diseases of the National Institutes of Health for the gift of murine and ovine prolactin. The studies were supported by grants from the Minnesota Turkey Growers Association and Merck, Inc. to W. H. Burke and by NSF Grant PCM 78-12470 to Harold Papkoff and a Rockefeller Center Grant.
REFERENCES Beavan, G. H., and Holiday, E. R. (1952). Ultraviolet absorption spectra of proteins and amino acids. Advan. Protein Chem. 7, 319-386. Blakesley, R. W., and Boezi, J. A. (1977). A new staining technique for proteins in polyacrylamide gels using Coomassie brilliant blue G250. Anal. Biochem.
82, 580-582.
Burke, W. H., Licht, P., Papkoff, H., and Bona Gallo, A. (1979). Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris
gallopavo).
Gen.
Comp.
Endocrinol.
37,
508-520. Cherms, F. L., Jr., Herrick, R. B., McShan, W. H., and Hymer, W. C. (1%2). Prolactin content of the anterior pituitary gland of turkey hens in different reproductive stages. Endocrinology 71,288-292. Etches, R. I., McNeilly, A. S., and Duke, C. E. (1979). Plasma concentrations of prolactin during the reproductive cycle of the domestic turkey (Meleagris
gallopavo).
Poultry
Sci. 58, %3-970.
TURKEY
PROLACTIN
Farmer, S. W., and Papkoff, H. In “Hormones and Evolution” (E. J. W. Barrington, ed.). Academic Press, London (in press). Farmer, S. W., Papkoff, H., Bewley, T. A., Hayashida, T., Nishioka, R. S., Bern, H. A., and Li, C. H. (1977). Purification and properties of teleost prolactin. Gen. Comp. Endocrinol. 31, 60-71. Farmer, S. W., Papkoff, H., and Hayashida, T. (1976a). Purification and properties of reptilian and amphibian growth hormones. EmZocrinoZogy 99,692-700. Farmer, S. W., Papkoff, H., Hayashida, T., Bewley, T. A., Bern, H. A., and Li, C. H. (1976b). Purification and properties of teleost growth hormone. Cm. Comp. Endocrinol. 30, 91- 100. Goldfine, I. D., Amir, S. M., Peterson, A. W., and Ingbar, S. H. (1974). Preparation of biologically active ?-TSH. Endocrinology 95, 1228- 1233. Gray, W. R. (1%7). Sequential degradation plus dansylation. In “Methods in Enzymology” (C. H. W. Hirs, ed.), vol. 11, pp. 469-475. Academic Press, New York/London. Hayashida, T., Farmer, S. W., and Papkoff, H. (1975). Pituitary growth hormones: Further evidence for evolutionary conservatism based on immunochemical studies. Proc. Nut. Acad. Sci. USA 72, 4322-4326.
Li, C. H. (1957). Studies on the pituitary lactogenic hormone. XVI. Oxidation of the ovine hormone with performic acid. J. Biol. Chem. 229, 157- 163. Li, C. H. (1974). In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.), Sec. 7, Vol. IV, Pt. 2, pp. 103- 110. Amer. Physiol. Sot., Wash., D.C.
Li, C. H. (1976). Studies on pituitary lactogenic hormone. The primary structure of the porcine hormone. Znr. J. Peptide Protein Res. 8, 205-224. Li, C. H., Dixon, J. S., Lo, T-B., Schmidt, K. D., and Pankov, Y. A. (1970). Studies on pituitary lactogenic hormone. XXX. The primary structure of the sheep hormone. Arch. Biochem. Biophys. 141,705-737.
PURIFICATION
AND
RIA
307
Licht, P., Papkoff, H., Farmer, S. W., MulIer, C. H., Tsui, H. W., and Crews, D. (1977). Evolution in gonadotropin structure and function. Rec. Progr. Horm.
Res. 33, 169-248.
McNeilly, A. S., Etches, R. J., and Friesen, H. G. (1978). A heterologous radioimmunoassay for avian prolactin:Application to the measurement of prolactin in the turkey. Acta Endocrinol. 89, 60-69. Nicoll, C. S. (1%7). Bio-assay of prolactin. Analysis of the pigeon crop-sac response to local injection by an objective and quantitative method. Endocrinology 80, 64-655. Nicoll, C. S. (1974). Physiological actions of prolactin. In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.), Vol. IV, Pt. 2. Amer. Physiol. Sot., Washington, D.C. Nicoll, C. S., and Nichols, C. W., Jr. (1971). Evolutionary biology of prolactins and somatotrophins. I. Electrophoretic comparisons of tetrapod prolactins. Gen. Comp. Endocrinol. 17, 300-310. Proudman, J. A., and Wentworth, B. C. (1978). Radioimmunoassay for turkey growth hormone. Gen. Comp. Endocrinol. 36, 194-200. Scanes, C. G., Bolton: N. J., and Chadwick, A. (1975). Purification and properties of an avian prolactin. Gen. Comp. Endocrinol. 27, 371-379. Scanes, C. G., Chadwick, A., and Bolton, N. J. (1976). Radioimmunoassay of prolactin in the plasma of domestic fowl. Gen. Comp. Endocrinol. 30, 12-20. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Automatic recording apparatus for use in chromatography of amino acids. Anal. Chem. 30, 1190- 1206. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T. (1972). A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. Metabol. 33, 988-991. Woods, K. R., and Wang, K. T. (1%7). Separation of dansyl-amino acids by polyamide layer chromatography. Biochim. Biophys. Acta 133, 369-370.
AND
COMPARATIVE
ENDOCRINOLOGY
40, 297-307 (1980)
Purification of Turkey Prolactin and the Development of a Homologous Radioimmunoassay for Its Measurement W.H.
BURKE* AND HAROLDPAPKOFF~
*Department of Animal Science, University of Minnesota St. Paul, Minnesota Research Laboratory and Reproductive Endocrinology Center, University San Francisco, California 94143
55108 and tHormone of California,
Accepted September 24, 1979 Prolactin has been purified from pituitary glands of the turkey (Meleagris gallopavo) and a radioimmunoassay has been developed for its measurement. The prolactin is a molecule of about 26,000 MW. It is high in glutamic and aspartic acids and in leucine, while it contains few methionine and half-cystine residues. Turkey prolactin elutes from Sephadex G-100 with a Ve/Vo ratio of 2.0. It displays multiple bands in disc electrophoresis, when run at pH 8.3. The most prominent prolactin band is at a RI value of 0.83-0.85. Prolactin is active in stimulating pigeon crop-sac development, but does not give a parallel dose-response to ovine prolactin. An antiserum was developed against turkey prolactin and was used to develop a homologous RIA using 1251-labeled turkey prolactin as the radioligand. The antiserum binds about 20% of the radioligand at a dilution of 1:7500. The least detectable dose of prolactin was 0.42 * 0.13 ng when tracer and sample were simultaneously mixed with antiserum. The 50% bound point averaged 3.8 f 0.32 ng. Considerably greater sensitivity was achieved by incubating the sample with the antiserum for 1 or 2 days prior to addition of tracer. The within-assay coefficient of variation was 7.9% for samples in the 45-65% bound range. Between-assay coefficient of variation was 8.9%. Binding is inhibited by sera from turkeys in some, but not all, stages of growth and reproductive development. Serial dilutions of sera and pituitary extracts yield dose-response curves which do not differ in slope from the purified turkey prolactin standard. Potency estimates of pituitary preparations by cropsac assay and by the prolactin RIA are in excellent agreement. Rat pituitary extract, rat prolactin, ovine prolactin, turkey FSH, LH, and GH all show no inhibition of binding even at very high doses. The antiserum neutralizes crop sac-stimulating activity of turkey prolactin.
A number of indirect studies implicate prolactin in a wide variety of physiological functions in avian species i.e., an involvement in incubation and the associated gonadal regression in Galliformes, the regulation of nasal gland secretion in Anseriformes, fat deposition in Galliformes and Passerines, growth promotion in Columbiformes, and stimulation of crop-sac development in this same group (see Nicoll, 1974 for a review). The general unavailability of avian prolactins has made the development of homologous radioimmunoassays for their detection impossible. Prolactin from the chicken has recently been purified by Scanes et al. (1975) and with it they developed a radioimmunoassay (RIA) for its measurement (Scanes et al.,
1976). McNeilly ef al. (1978) have reported on a mammalian prolactin RIA which was useful for measurement of turkey prolactin. It was the purpose of these studies to obtain highly purified turkey prolactin suitable for the development of a homologous radioimmunoassay. The purification of turkey prolactin, its chemical characteristics and the development of a radioimmunoassay for its measurement are described. MATERIALS AND METHODS Pituitaries and Fractionation Pituitary glands used in these studies were described Burke et al. (1979) as were the initial stages of purification. In brief, glands were homogenized in a Waring Blendor with ice-cold water and the homogenate was adjusted to pH 9.5 with Ca(OH),. After removal of the residual tissue by centrifugation, the suby
297 0016~6480/80/030297-11$01.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
298
BURKE
AND
pernatant was adjusted to 0.15 M (NH&SO, and the pH was reduced to 4.0 by the addition of metaphosphoric acid. The precipitate which formed was removed by centrifugation. It was taken up in H,O, dialyzed against water, and lyophilized. This fraction, termed MP, was extracted for 1 hr with pH 9.50.1 N NaCl and then centrifuged. The supernatant, which contained the GH and prolactin, was brought to a concentration of 0.2 saturated ammonium sulfate (SAS) and adjusted to pH 5.1. The precipitate which formed was removed by centrifugation and the supernatant was then adjusted to 0.5 SAS. The precipitate which formed at 0.5 SAS contained the bulk of the GH and prolactin and was removed by centrifugation. The supernatant was adjusted to 0.8 saturated ammonium sulfate and the precipitate that formed was also collected. The precipitated materials were dialyzed against water and lyophilized. The fraction precipitating between 0.2 and 0.5 SAS was further fractionated using a DEAE-cellulose column. The DEAE-cellulose was equilibrated with 0.03 M Tris-HCI, pH 7.4, and the hormone-containing fraction was dissolved in the same buffer and applied to the column. After the first protein peak was eluted with this buffer, the column was eluted with the same buffer adjusted to 0.07 M NaCl. The column was then eluted with 1 M NH,HCO,. The prolactin-rich fraction that eluted from the DEAE column with 1 M NH,HCO, was dialyzed against water, lyophilized, and then reapplied to a DEAE column as described previously. The column was again eluted with 0.03 M Tri-HCl pH 7.4, then with 0.03 M Tri-HCl pH 7.4 adjusted to 0.07 M NaCl, and finally with the same Tris buffer adjusted to 0.17 M NaCl. The protein emerging with the latter buffer had most of the prolactin activity and it was dialyzed against water and lyophilized. It was then taken up in 0.05 M NH,HCO, and chromatographed on a 2.5 x 90-cm Sephadex G-100 column equilibrated with the same buffer. The prolactin emerged with a Ve/Vo ratio of 2.0. A yield of 185 mg/kg wet wt of pituitaries was obtained.
Assays Fractions obtained during purification were monitored for prolactin by the local pigeon crop-sac assay, using the procedure of Nicoll(1%7) and for GH by the anti-snapping turtle GH radioimmunoassay (RIA) described by Hayashida et al. (1975). They were also examined by polyacrylamide gel disc electrophoresis (DE) in 7.5% gels using Tris-glycine buffer at pH 8.3. The gels were stained with Coomassie brilliant blue G250 according to the procedure of Blakesly and BoeLi (1977). Distribution of GH was also confirmed by the homologous turkey GH RIA of Proudman and Wentworth (1978). The amino acid composition of the purified preparation was determined as described by Spackman et al.
PAPKOFF
(1958) on a Beckman Model 119C amino acid analyzer following oxidation in performic acid (Li, 1957) and hydrolysis in constant boiling HCl (20 hr at 105”). Tryptophan and tyrosine determinations were made by the spectroscopic method of Beaven and Holiday (1952). Amino terminal amino acid analyses were done according to the method of Gray (1967) and Woods and Wang (1%7). Late in the course of these studies an RIA was developed for the measurement of turkey prolactin (see below). It was then used to examine precursor fractions and purified preparations.
Prolactin Antisera Production During the course of the studies a fraction with substantial crop sac-stimulating activity and low GH contamination was obtained and used to immunize several rabbits. Before use as an antigen, 200 pg of the preparation was applied to each of five polyacrylamide gel columns prepared as described above. After electrophoresis, one gel was stained and a prominent band was seen at an Rf value of 0.83, similar to the R, of chicken prolactin (Nicoll and Nichols, 1971: Scanes et al., 1975) and other avian prolactins (Nicoll and Nichols, 1971). A 6-mm region centered on an R, of 0.83 was cut from each of the unstained gels. Each gel piece was forced through a l-ml syringe into 1 ml of saline. The gel-saline suspensions were stored frozen until used for immunization. At immunization a suspension was thawed and emulsified with Freund’s complete adjuvant. Immunizations were done using the procedure of Vaitukaitus et al. (1972). After the first two immunizations, further boosters were given using highly puritied turkey prolactin, which was then available, without prior electrophoresis.
Prolactin RIA: Iodination Turkey prolactin (B15lB) which resulted from these studies was iodinated using a slight modification of the method of Goldfine et al. (1974). Five micrograms of the hormone was dissolved in 5 ~1 of 0.03 M NH,HCO, and diluted to 30 ~1 with 0.5 M phosphate buffer, pH 7.4. lp51, 0.5 mCi, was added to the vessel, followed by 10 ~1 of chloramine-T (0.4-0.8 pg/lO ~1). The reaction was monitored as described by Goldfine et al. (1974) and terminated when approximately 60% of the lz51 was precipitatable by trichloroacetic acid. The free iodine was separated from bound by chromatography on a disposable Bio-Gel P-60 column (0.7 x 18 cm) previously equilibrated with 0.05 M phosphate buffer-0.25% bovine serum albumin, pH 7.4. The column was eluted with the same buffer and 0.4 ml fractions were collected. Protein-bound iodine peaked in fraction 8. The ability of contents of this tube and others on each side of it to bind to antisera was quite low. Rechromatography of tubes on the descending limb of the peak (tubes 9 and 10) on a Sephadex G-100 column (1.5 x 50 cm) resulted in the resolution of a large peak with a Ve/Vo ratio of 1.35 and a second
TURKEY
PROLACTIN
PURIFICATION
smaller peak with a Ve/Vo ratio of 1.79. A third peak with a VelVoratio of about 3.0 was also obtained. Only the second peak showed good binding to the antisera. It was diluted to about 15,000 cpml50 ~1 with a pH 7.4 0.1 M phosphate-O.15 M NaCl-0.05 M EDTA buffer containing 0.6% normal rabbit serum.
Prolactin
RIA Protocol
Assay tubes were set up containing a maximum of 100 ~1 of sample or sample diluted in pH 7.4, 0.1 M phosphate-O. 15 M NaCl buffer containing 0.1% bovine serum albumin. To this was added 100 ~1 of a 1:3000 dilution (final dilution 1:7500) of the antiserum in 0.1 M phosphate-O.15 M NaCl-0.05 M EDTA buffer adjusted to pH 7.4 with NaOH. Fifty microliters of the radioligand was added to the assay tubes immediately before the antiserum or following a 24- or 48-hr delay. Twenty-four hours after addition of the radioligand, 100 ~1 of a 1:30 dilution of sheep anti-rabbit y-globulin was added. The tubes were allowed to incubate overnight and were then diluted with 1 ml of the phosphate-buffered saline described above and centrifuged at 24OOgfor 30 min. Supematants were poured off; the tubes were allowed to drain and the radioactivity in the pellets was determined. Samples were kept at 5”, except for brief intervals when reagents were added. In each assay triplicate sets of tubes containing tracer, tracer plus buffer (NSB), and tracer plus buffer plus antiserum (Bo) were run. The mean NSB counts were subtracted from the Bo tubes and from all standard or unknown samples. All standards or unknowns were expressed as a percentage of the Bo tubes (B/B0 x 100) and dose-response curves were obtained by calculating the linear regression of the logit percentage B vs the log of the hormone dose. Potency of unknowns were obtained by comparison with the turkey prolactin standard (Bl5lB). The standard was run in triplicate in six to eight serial dilutions, while unknowns were run in duplicate at multiple doses.
Prolactin
RIA Validation
Hormone-neutralizing activity of the antiserum was tested by incubating partially purified prolactin (150 pg or 50 pg) in 0.4-ml aliquots of undiluted antiserum or in normal rabbit serum for 24 hr at 5”. Six pigeons received antiserum-hormone mixtures over one hemicrop and the normal serum-hormone mixture over the other hemicrop. Injections were done and responses were determined as described by Nicoll (1%7). Control pigeons received injections of normal rabbit serum. Responses obtained from the two hemicrops of the hormone-injected pigeons were compared and neutralization effects were then assessed on a withinpigeon basis. Pituitary tissue was obtained from five laying turkeys and from five which had been nesting for several weeks (i.e., broody turkeys). A blood sample was ob-
AND
RIA
299
tamed from the hens and they were then killed by cervical dislocation. The anterior pituitary glands were quickly removed, separated into cephalic and caudal lobes, weighed, and quick frozen on dry ice. The glands were later individually homogenized in pH 7.4 phosphate-buffered saline with a Brinkmann Polytron Model PCU-2. The homogenates were kept at 5” for 2 hr and then centrifuged at 11,OOOgfor 30 min. The supematant fluids were diluted in assay buffer and frozen until used for assay. To control for nonspecific effects of tissue extracts, several rat pituitary glands were comparably processed and assayed. Sera from five broody and five laying hens were stored frozen until assayed. During the course of purification many samples were assayed in the crop-sac bioassay, but few of these were retained. When the prolactin RIA was developed, fractions of widely different bioactivity which had been assayed in the same crop-sac assay were assayed in a single immunoassay to determine the relationship between immuno- and bioactivity. Many other fractions which had been bioassayed in different crop-sac assays, and were known to differ vastly in bioactivity, were assayed by RIA and the responses were compared. Many fractions which had been monitored by DE and found either to have prolactin bands or be free of them were run in the RIA and the results were compared to expectations based on DE. Specificity of the assay was tested by assaying available turkey pituitary hormones. Turkey LH, FSH, and GH were tested in the prolactin RIA at doses 40- to 50-fold higher than the maximum assayable dose of turkey prolactin. Likewise ovine prolactin (NIH P-S15) and rat prolactin (NIH RP-1) were also tested at doses up to 1000 ng. Sera from turkeys of a variety of ages, sexes, and reproductive states were assayed at multiple doses to determine if serum per se would inhibit binding of radioligand and to determine parallelism of serum samples with the purified hormone. Sera from two surgically hypophysectomized turkeys were kindly donated by Dr. Howard Opel and were tested at multiple doses in this assay.
RESULTS
As determined by crop-sac assay (Table 1) all of the prolactin was precipitated by metaphosphoric acid at pH 4. From this MP material the most enriched prolactin was obtained at SAS concentrations between 0.2 and 0.5 and a slight amount above 0.5 SAS. Prolactin was absorbed onto the DEAE-Tris column at 0.03 M Tris pH 7.4 and was eluted. by the Tris-0.17 M NaCl buffer or by 1 M NH,HCO,. The prolactin RIA (see validation below) strongly sup-
DEAE-cellulose 1.05 1.13 2.34 1.52 Sephadex G- 100 2.12 2.30
200 150 150 150 100 20
13.8 mg 17.0 mg 22.8 mg 7.6 mg
21.8 mg 24.0 mg
0.03 M Tris 0.03 M Tris-0.07 M NaCl 0.03 M T&-0.17 M NaCl 1 M NHIHCO,
Fraction A Fraction B
(NH&SO,
fractionation 1.12 2.60 1.72
0.17 1.0
0.04 0.17 0.02
0.05 0.003
Potency
13.4 86.6
%.4 3.5
-
26.9 69.2 3.8
97.6 2.3
Percentage
0.15 0.04 0.009 0.006
0.03 0.44 0.02
0.11 0.04
Potency
69.2 22.7 6.7 1.3
6.1 89.8 4.1
87.4 12.6
Percentage
Not assayed Not assayed
GH RIA
a Examples of the distribution of hormones in the various purification steps. b Mucosal dry weight of test substance divided by mucosal dry weight of control. Three to four hemicrops per treatment. c Percentage of the activity in each fraction. The potency of each fraction in a given purification procedure was multiplied by the mass of that fraction to obtain the units of activity in each fraction. The units of activity in a given fraction were divided by the total units of activity obtained in that procedure and the quotient was multiplied by 100.
50 50 100
3.6 g 2.1 g 0.8 g
acid precipitation 1.51 1 .Ol
O-O.2 SAS 0.2-0.5 SAS 0.5-0.8 SAS
Metaphosphoric 100 100
12.0 g 4.8 g
MP MS
Mucosal dry weight*
Dose (IL@
Yield
Prolactin RIA
TABLE 1 GROWTH HORMONE IN VARIOUS PURIFICATION STEPS
Crop-sac assay
AND
Fraction
DISTRIBUTION” OF PROLACTIN
TURKEY
PROLACTIN
PURIFICATION
ported the interpretation obtained by the bioassays which in turn was supported by disc gel electrophoresis. The RIA indicated that nearly 98% of the prolactin was precipitated by metaphosphoric acid and nearly 70% of that went into the 0.2-0.5 SAS fraction. Over 96% of the immunoreactive prolactin absorbed to the DEAE-Tris column and was eluted with the Tris buffer adjusted to 0.17 M NaCl. Disc electrophoresis clearly showed the presence of GH in the MP fraction with lit-. tle apparent in the MS. The homologous GH RIA indicated that the bulk of the GH did in fact go into the MP fraction (Table 1). About 90% of the GH precipitated between 0.2 and 0.5 SAS as assessed by GH RIA. Complete separation of the prolactin and GH was achieved by the chromatography on DEAE-cellulose. This was confirmed by both disc electrophoresis and the homologous turkey GH RIA (Table 1). The most potent turkey prolactins consistently showed a cross-reaction in the heterologous GH RIA (Hayashida et al., 1975), exhibiting potencies of 0.02-0.04 times the rat standard and also in the turkey GH RIA with a potency of about 0.02 times the turkey GH. These preparations, when run in DE at 100 /.&gel, showed no stain-
FIG.
90-cm
1. Gel filtration of the 0.17 M NaCl column; 3.6 ml collected per tube.
fraction
AND
301
RIA
able bands in the region in which GH migrates. Biochemical
Characterization
The gel filtration elution pattern of prolactin (Fig. 1) shows a major peak, designated B, with a Ve/Vo ratio of 2.0 and an additional larger molecular component designated A. Both were active in the pigeon crop assay. The disc electrophoretic pattern of turkey prolactin is seen in Fig. 2 and compared with turkey GH. Characteristically, both display multiple stainable bands with, however, no overlap. The most prominent prolactin band had an R, value of 0.83-0.85. Three strongly staining bands were seen with the growth hormone at R, values of 0.19, 0.30, and 0.38. The major amino terminus of turkey prolactin appears to be leucine, but traces of alanine, tyrosine, and lysine were also found. The amino acid composition of turkey prolactin is shown in Table 2. It is rich in aspartic acid, glutamic acid, and leucine and low in methionine and half-cystine content. Spectrophotometric determinations showed the presence of 2.9% tyrosine and 1.46% tryptophan. The latter value is consistent with the presence of two residues of tryptophan in the molecule. If two
from
DEAE-cellulose
on Sephadex
G-100;
2.5 x
BURKE AND PAPKOFF
302
residues of tryptophan are present and if one assumes 15% moisture and ash in the preparations, a molecular weight of 2 1,700 is calculated. The molecular weight as determined by SDS-polyacrylamide electrophoresis was 26,000; in addition a trace component of 12,000 MW was seen. In the same experiment ovine and turkey GH gave values of 20,500-22,800 MW. Bioassays
FIG. 2. Disc electrophoretic pattern of turkey prolactin, turkey GH, and the 0.2-0.5 SAS precipitate containing both prolactin and GH. A 7.5% gel, pH 8.3; the origin is toward the top of the figure. The arrow indicates the marker dye.
TABLE 2 AMINOACIDCOMPOSITION OFTURKEY PROLACTIN Turkey prolactin” LYS His Arg Asp Thr Ser Glu PI.0
GUY Ala MCys Val Met Ile Leu W Phe TV’
11.0 6.5 10.1 22.0 7.0 14.1 33.7 9.7 14.3 13.2 4.1 8.1 3.1 8.5 20.5 4.6 6.9 2.0
Ovine PRL* 9 8 11 22 9 15 22 11 11 9 6 10 7 11 23 7 6 2
n Residues/l97 residues, average of two determinations. b Structural analysis of Li ef al. (1970) and Li (1976). p Spectrophotometric measurement.
The final prolactin preparation was assayed in the pigeon crop assay a number of times. A typical result is seen in Table 3. Two micrograms of the turkey prolactin produced a significant increase in mucosal dry weight over the solvent-injected control. A linear dose-response curve was obtained between 2 and 50 pg. Ovine prolactin was repeatedly more potent than turkey prolactin, but the lack of parallelism between the ovine and turkey dose-response curves precluded accurate potency estimates. However, the turkey prolactin is about 20% as potent as ovine prolactin. Turkey growth hormone was free of significant crop sac-stimulating activity at a dose up to 400 pg. Prolactin Radioimmunoassay
Only 3-5% of the radiolabeled prolactin which eluted from the Bio-Gel P-60 column was bound by the antiserum at a 1:7500 dilution. Purification of this material on Sephadex G-100 yielded major peaks at TABLE 3 CROPSAC-STIMULATING ACTIVITY OFTURKEY PROLACTIN,TURKEY GH, AND OVINE PROLACTIN Treatment Saline Turkey prolactin Turkey GH Ovine prolactin
Dose 64) 2 10 50 80 400 2 10
Mucosal dry weight (mg; 2 f SE) 15.5 21.3 32.4 41.2 14.9 18.6 37.8 42.1
+ 1.2 t 0.6 lr 2.1 k 2.5 + 2.5 z!z2.3 2 1.1 f 1.6
TURKEY
PROLACTIN
PURIFICATION
Ve/Vo ratios of 1.35, 1.79, and 3.0. Material in the first and third peaks was not bindable by antiserum, while about 20% of the material in the middle peak was bound at an antiserum dilution of 1:7500. When the sample, tracer, and antiserum were added to tubes at the same time and incubated 24 hr before addition of second antibody, the sensitivity of the prolactin RIA, defined as 2SD below the 100% bound point, averaged 0.42 + 0.13 ng (x + SE) in six assays. The 50% bound point was 3.8 f 0.32 ng. The 50% bound point can be decreased about lo-fold by delaying addition of the radioligand for 48 hr after the antiserum and unlabeled hormone are mixed. The within-assay coefficient of variation, based on differences between duplicate potency estimates of samples in the midrange of the assay (45-65% bound), was 7.9%. The between-assay CV, based on the average potency of duplicate assay tubes from a pool of serum from broody turkey hens run in seven assays, was 8.9%. The agreement between immuno- and bioactivity (pigeon crop-sac assay) was excellent. In all comparisons made, bioactivity estimates and immunoactivity potency estimates of partially purified prolactin
TABLE
AND
303
RIA
fractions have been in perfect rank order. An example of these comparisons is shown in Table 4. Comparison of the prolactin potency of pituitaries from broody and laying turkeys has been made several times (Cherms et al., 1%2; Cogger and Burke, unpublished). In both these earlier studies broody turkey pituitary glands were 3 - 5 times as active in stimulating pigeon crop-sac development as glands from layers. The RIA comparison (Table 5) also shows tissue and sera from broody hens to contain more prolactin than that from laying hens. Cephalic lobe tissue from five broody hens had about 2.5 times as much prolactin, on a weight basis, as comparable tissue from layers. Total cephalic lobe prolactin content was about 2.4 times as great in broody hens. The caudal lobes of both groups had relatively little prolactin and there was no difference between groups. Immunoassay of cephalic lobe tissue from the 10 hens at 2.5, 0.25, and 0.025 pg yielded a dose-response curve which did not differ significantly from that of the prolactin standard (b = - 1.97 for pituitary tissue and -2.11 for the standard). Rat pituitary extract did not inhibit binding of radioligand when tested over the same
4
RELATIONSHIP BETWEEN BIOACTIVITY AND IMMUNOACTIVITY OF TURKEYPROLACTIN PREPARATIONSATVARIOUSSTAGES OF PURIFICATION Crop-sac assay Preparation
Dose tested w3)
Saline B88A” B88B B88D B9OC B88C B84C B151B
200 150 150 100 150 100 50
Mucosal dry weight (mg) 14.6 15.2 16.3 22.0 23.4 33.9 37.8 42.4
? k f k 2 k + k
0.9 3.1 2.1 2.8 1.4 3.2 7.4 5.0
RIA potency (X B15lB)
a B88A, B, C, and D are fractions from the DEAE-cellulose column eluted sequentially by buffers shown in Table 1. Preparation B9OC is a fraction precipitating from the MP at 0.2-0.5 SAS. Fraction B84C is a preparation derived from B9OC and the Bl51B is the final purified prolactin (Fig. 1).
BURKE
304
AND
PAPKOFF
TABLE PITUITARY
AND
SERUM
PROLACTIN
5
LEVELS
OF BROODY
Cephalic lobe CLg/mg Laying turkeys Broody turkeys
9.5 k 1.8* 24.6 k 3.6
AND
LAYING
TURKEYS
Caudal lobe l*gimg
230.8 -c 55.1 544.8 k 59.2
1.6 2 1.0 1.2 k 0.2
dose range as the turkey tissue (0.025 to-25 /49. Serum from a broody turkey, assayed in duplicate at six doses ranging from 50 to 1.56 ~1, gave a dose-response curve parallel to the standard prolactin (Fig. 3). Sera from two hypophysectomized hens at doses up to 50 ~1 depressed binding slightly. The dose-response characteristics of these sera were erratic and did not parallel the standard. Many sera from somatically mature, sexually immature female turkeys housed under short photoperiods (6L:18D) have been assayed and found not to inhibit binding of the tracer at doses up to 100 ~1
Serum (n&4
/.&gland
I.cdgland
15.5 * 9.6 4.9 t 1.2
360.6 2 185.5 1602.3 2 244.8
(the highest tested). The same is true of sera from sexually immature male turkeys between 14 and 20 weeks old. Highly purified turkey GH, LH, and FSH and highly purified ovine and murine prolactins showed no inhibition of binding at doses up to 1000 ng. Four hundred microliters of undiluted anti-prolactin serum completely neutralized the crop sac-stimulating activity of 50 Fg of partially purified prolactin and partially reduced the activity of 150 pg of the hormone (Table 6). In each of six pigeons the hemicrop receiving hormone in the antiserum was less stimulated than the contralateral
Hypophyseltomlmd Turkey Sea
Bmdy
\\
.L
Turkey Sera
(b--2.61))
Turkey Prolaclinl 81518 ) ( b- -2.63 I
1
I
I
12.5 .lB 1.56 3.12 6.25 LOG DOSE OF HORMONE( nglOR SERAluII
1
25
50
FIG. 3. Radioimmunoassay dose-response curves of turkey prolactin, a serum pool from broody turkeys and sera from two hypophysectomized turkeys. Three-day assay with sample, antisera, and tracer added on Day 1, second antibody on Day 2, and assay termination on Day 3. The calculated least detectable dose of hormone (i.e., a dose which would depress counts 2 SD deviations below Bo) was 0.26 ng of turkey prolactin (B151B).
TURKEY
PROLACTIN
TABLE 6 EFFECTOFPROLACTIN ANTISERA ON THE CROPSACSTIMULATINGACTIVITY OF TURKEY PROLACTIN~ Assay No.
Prolactin (pg)
Solvent
MDWb (mkd
1 1 1 1
0 50 50 50
NRS NRS AS NH,HCO,
18.3 24.0 17.2 22.5
t zt 2 k
1.4 5.0 1.0 1.4
2 2 2 2
0 150 150 150
NRS NRS AS NHIHC03
19.2 32.9 28.7 31.2
t f k k
1.2 0.6 0.9 2.0
u N = 3 hemicropsltreatment; the comparison of antiserum-treated and normal serum-treated prolactin was made on a within-pigeon basis. b MDW, mucosal dry weight, x f SE: AS, antiprolactin sera; NRS, normal rabbit serum.
side which received hormone in normal serum. The difference between crop-sac muoscal dry weights of pigeons receiving antiserum-treated prolactin and those receiving prolactin incubated in normal rabbit serum was significant (P < 0.05) by paired r test. DISCUSSION
As part of a broad program of studies on the biochemistry and physiology of nonmammalian pituitary hormones, we have evolved purification procedures which allow the purification of as many of the pituitary hormones as possible from the same batch of glands (Licht et al., 1977; Farmer and Papkoff, in press). The procedures employed for the purification of turkey prolactin are largely derived from these studies. It is of interest that the prolactin and GH exhibited very similar fractionation behavior in most of the steps employed herein. Complete separation was achieved, however, by the DEAE chromatography as described. Previous studies with GH and prolactin from a teleost fish (Farmer et al., 1976b, 1977) were similar in this regard. The physicochemical characterization
PURIFICATION
AND RIA
305
studies show that the turkey prolactin, while not identical, shares many properties with prolactin from other species.. These include gel filtration behavior, molecular weight, disc gel electrophoretic patterns, and amino acid analysis. The gel filtration experiments (Ve/Vo=2.0), spectrophotometric studies, and the SDS-gel electrophoretic analysis indicate a molecular weight of 22-26,000 for turkey prolactin, a value consistent with that determined for ovine prolactin (Li, 1974). The amino acid content of turkey prolactin is characterized by large amounts of leucine, aspartic acid, glutamic acid, and two tryptophan residues, all properties of the more extensively studied mammalian prolactins. The low half-cystine content in turkey prolactin, suggestive of two disultide bridges, should be regarded with caution insofar as this amino acid is diflicult to accurately determine. It may be noted that mammalian prolactins possess three disulfide bonds (Li et al., 1970; Li, 1976). The purified turkey prolactin was found to be essentially free of other pituitary hormones , including growth hormone. Turkey prolactin is readily bioassayed in the pigeion crop-sac assay, but displays different dose-response characteristics than ovine prolactin and as a result appears to be somewhat less potent. Species variation in hormonal response has been well documented for gonadotropins (Licht et al., 1977) as well as for growth hormones (Farmer et al., 1976a; Farmer and Papkoff, in press), and it can be anticipated that various species of prolactin will also behave differently in a given assay. The prolactin radioimmunoassay described is free from cross-reactions with other turkey pituitary hormones. RIA measurements agree well with the bioactivity of partially purified pituitary preparations as assessed by the crop-sac assay and with previous bioassay studies with regard to the potency of pituitary glands from broody and laying turkeys.
306
BURKE
AND
Quantitation of unknown serum samples against purified prolactin is possible since parallelism is obtained. The assay is sensitive enough to detect prolactin in as little as 2-5 ~1 of sera from broody turkeys while as much as 50-100 ~1 from certain sexually quiescent birds has too little prolactin to quantitate. The failure of these relatively high levels of sera to inhibit binding of tracer supports the absence of nonspecific serum effects in the assay. The sera from hypophysectomized turkeys tested in this RIA appeared to depress binding of labeled prolactin to a minor degree. The hypophysectomies appeared complete by visual examination, but microscopic examinations of the sella turcica were not made. The presence of remnants of pituitary tissue cannot be excluded. A homologous avian (chicken) prolactin RIA has been reported (Scanes et al., 1976) and a heterologous assay has been employed for measurement of turkey prolactin (McNeilly et al., 1978). No direct comparison of these assays with our own have been made. However, both of these reports appear to agree with this study in that serum from sexually immature turkeys has less prolactin than serum from laying females. There appears, however, to be a major discrepancy between our findings and those of McNeilly et al. (1978) and Etches et al. (1979). In our experience, sera from broody turkeys have always contained substantially more prolactin than sera from laying hens, whereas their report shows no difference. However, unpublished studies from our laboratory do show that changes in a broody bird’s environment can cause a decrease in prolactin from the very high levels of the broody hen to levels in the range of laying birds in 24 hr or less. Consequently, care must be exercised to avoid this possibly confounding effect. Even though the prolactin levels are labile over a 24-hr period, we have not seen an elevation or depression in levels as a result of handling and repeated venipuncture over periods of 1-2 hr. Further data will
PAPKOFF
have to accrue before critical comparison these three RIAs can be made.
of
ACKNOWLEDGMENTS The authors wish to express their sincere appreciation to Dr. C. H. Li for his interest, support, and advice during the course of these studies. Our thanks are also expressed to Dr. S. W. Farmer for her advice and assistance; to Dr. T. Hayashida, E. Rowley, and J. Walker for assaying many samples for GH, and to Dr. B. B. Aggarwal, M. Davenport, P. Dennison, K. Hoey, and J. D. Nelson for splendid technical assistance. We wish to thank Dr. Paul Licht for his aid in the assessment of prolactin antisera used in these studies and Dr. John Proudman for the gift of antiturkey GH sera. Special thanks are due to the following students, staff, and faculty of the University of California and the University of Minnesota for assistance in the tedious job of collecting the turkey pituitary glands used in this work: E. Daniels, J. DeMichael, M. El Halawani, L. Fuqua, J. Hagman, L. Hallacher, P. Martin, J. Millam, F. X. Ogasawara, L. Ogren, S. Pallas, W. Rainey, T. Siopes, M. Vodian, L. Wolcott, and W. Yu. Thanks are given to Dr. Howard Opel, USDA, ARS, Beltsville, Maryland for the gift of sera from hypophysectomized turkeys. We thank the National Institutes of Arthritis, Metabolism and Digestive Diseases of the National Institutes of Health for the gift of murine and ovine prolactin. The studies were supported by grants from the Minnesota Turkey Growers Association and Merck, Inc. to W. H. Burke and by NSF Grant PCM 78-12470 to Harold Papkoff and a Rockefeller Center Grant.
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82, 580-582.
Burke, W. H., Licht, P., Papkoff, H., and Bona Gallo, A. (1979). Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris
gallopavo).
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Comp.
Endocrinol.
37,
508-520. Cherms, F. L., Jr., Herrick, R. B., McShan, W. H., and Hymer, W. C. (1%2). Prolactin content of the anterior pituitary gland of turkey hens in different reproductive stages. Endocrinology 71,288-292. Etches, R. I., McNeilly, A. S., and Duke, C. E. (1979). Plasma concentrations of prolactin during the reproductive cycle of the domestic turkey (Meleagris
gallopavo).
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Sci. 58, %3-970.
TURKEY
PROLACTIN
Farmer, S. W., and Papkoff, H. In “Hormones and Evolution” (E. J. W. Barrington, ed.). Academic Press, London (in press). Farmer, S. W., Papkoff, H., Bewley, T. A., Hayashida, T., Nishioka, R. S., Bern, H. A., and Li, C. H. (1977). Purification and properties of teleost prolactin. Gen. Comp. Endocrinol. 31, 60-71. Farmer, S. W., Papkoff, H., and Hayashida, T. (1976a). Purification and properties of reptilian and amphibian growth hormones. EmZocrinoZogy 99,692-700. Farmer, S. W., Papkoff, H., Hayashida, T., Bewley, T. A., Bern, H. A., and Li, C. H. (1976b). Purification and properties of teleost growth hormone. Cm. Comp. Endocrinol. 30, 91- 100. Goldfine, I. D., Amir, S. M., Peterson, A. W., and Ingbar, S. H. (1974). Preparation of biologically active ?-TSH. Endocrinology 95, 1228- 1233. Gray, W. R. (1%7). Sequential degradation plus dansylation. In “Methods in Enzymology” (C. H. W. Hirs, ed.), vol. 11, pp. 469-475. Academic Press, New York/London. Hayashida, T., Farmer, S. W., and Papkoff, H. (1975). Pituitary growth hormones: Further evidence for evolutionary conservatism based on immunochemical studies. Proc. Nut. Acad. Sci. USA 72, 4322-4326.
Li, C. H. (1957). Studies on the pituitary lactogenic hormone. XVI. Oxidation of the ovine hormone with performic acid. J. Biol. Chem. 229, 157- 163. Li, C. H. (1974). In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.), Sec. 7, Vol. IV, Pt. 2, pp. 103- 110. Amer. Physiol. Sot., Wash., D.C.
Li, C. H. (1976). Studies on pituitary lactogenic hormone. The primary structure of the porcine hormone. Znr. J. Peptide Protein Res. 8, 205-224. Li, C. H., Dixon, J. S., Lo, T-B., Schmidt, K. D., and Pankov, Y. A. (1970). Studies on pituitary lactogenic hormone. XXX. The primary structure of the sheep hormone. Arch. Biochem. Biophys. 141,705-737.
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AND
RIA
307
Licht, P., Papkoff, H., Farmer, S. W., MulIer, C. H., Tsui, H. W., and Crews, D. (1977). Evolution in gonadotropin structure and function. Rec. Progr. Horm.
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McNeilly, A. S., Etches, R. J., and Friesen, H. G. (1978). A heterologous radioimmunoassay for avian prolactin:Application to the measurement of prolactin in the turkey. Acta Endocrinol. 89, 60-69. Nicoll, C. S. (1%7). Bio-assay of prolactin. Analysis of the pigeon crop-sac response to local injection by an objective and quantitative method. Endocrinology 80, 64-655. Nicoll, C. S. (1974). Physiological actions of prolactin. In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.), Vol. IV, Pt. 2. Amer. Physiol. Sot., Washington, D.C. Nicoll, C. S., and Nichols, C. W., Jr. (1971). Evolutionary biology of prolactins and somatotrophins. I. Electrophoretic comparisons of tetrapod prolactins. Gen. Comp. Endocrinol. 17, 300-310. Proudman, J. A., and Wentworth, B. C. (1978). Radioimmunoassay for turkey growth hormone. Gen. Comp. Endocrinol. 36, 194-200. Scanes, C. G., Bolton: N. J., and Chadwick, A. (1975). Purification and properties of an avian prolactin. Gen. Comp. Endocrinol. 27, 371-379. Scanes, C. G., Chadwick, A., and Bolton, N. J. (1976). Radioimmunoassay of prolactin in the plasma of domestic fowl. Gen. Comp. Endocrinol. 30, 12-20. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Automatic recording apparatus for use in chromatography of amino acids. Anal. Chem. 30, 1190- 1206. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T. (1972). A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. Metabol. 33, 988-991. Woods, K. R., and Wang, K. T. (1%7). Separation of dansyl-amino acids by polyamide layer chromatography. Biochim. Biophys. Acta 133, 369-370.