GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
38,421-440
(1979)
Studies on Two Types of Gonadotropins Carp Pituitaries’ DAVID Marine
Sciences
Research
Laboratory,
from both Salmon and
R. IDLER AND T. BUN Nc Memorial University of Newfoundland, Canada, AIC SS7
St. John’s,
Newfoundland,
Accepted February 26, 1979 Two molecular weight forms of salmon vitellogenic Con AI (unadsorbed on concanavalin A-Sepharose) hormone and the salmon maturational Con AI1 (adsorbed on concanavalin A-Sepharose) hormone were able to stimulate vitellogenesis in the female winter flounder (Pseudopleuronectes americanus). Both the salmon vitellogenic Con AI 45,000~dalton hormone and the maturational Con AI1 hormone were able to enhance in vivo phosphate and leucine uptake by the flounder testis and incorporation into testicular proteins. Only the salmon maturational Con AI1 hormone was able to evoke spermiation and ovulation and it was much more potent than the vitellogenic Con AI hormones in stimulating testicular steroidogenesis. The vitellogenic and maturational activities of the salmon Con AI1 gonadotropin were partially destroyed on desialylation while the vitellogenic activity of the Con AI gonadotropins was not affected. The immunologic activity of the maturational Con AI1 hormone was retained after desialylation, but was abolished upon carboxamidomethylation. The immunologic potencies of the vitellogenic Con AI hormones were preserved after desialylation. The two salmon vitellogenic Con AI hormones displayed numerous prominent differences in amino acid and carbohydrate compositions from, and were immunologically remote from, the salmon maturational Con AI1 hormone. There was a marked dissimilarity in amino acid composition between the immunologically distinct salmon vitellogenic Con AI hormones. Two gonadotropins (vitellogenic Con AI and maturational Con AII), with an identical molecular weight of 30,000 daltons, were prepared from the pituitaries of the carp Cyprinus carpio. The distribution of biological activities was in general similar to that observed between the salmon hormones. The carp vitellogenic Con AI hormone could also be distinguished from the maturational Con AI1 hormone in the composition of its protein and carbohydrate moieties.
Until recently data from chemical fractionation studies and bioassays have been conducive to the concept that the teleost pituitary elaborates a solitary gonadotropin (see Fontaine, 1975). Presence of vitellogenic activity in a fraction of the American plaice (Hippoglossoides platessoides) pituitary extract not retained on concanavalin A -Sepharose was first reported by Campbell and Idler (1976), but this vitellogenic fraction also contained an appreciable amount of maturational and ovulatory activities (Campbell and Idler, 1977) which could be removed by rechromatography on the immobilized lectin (Ng and Idler, 1978a). The latter authors estab’ M.S.R.L.
Contribution
lished the existence of two different types of plaice gonadotropins, vitellogenic Con AI2 and maturational Con AI1 hormones, and in addition found “big” and “little” forms of hormones. In the chum salmon (Oncorhynchus keta) Ng and Idler (1978b) discovered vitellogenic Con AI hormones which were immunologically distinct from the salmon maturational Con AI1 hormone and exhibited size heterogeneity. Vitel-
Number 332.
* The term Con AI hormone is used to designate a preparation which is unadsorbed on concanavalin A-Sepharose and which has been purified by processes such as gel filtration and ion-exchange chromatography. The term Con AI1 hormone is employed for a hormone adsorbed on the immobilized lectin and purified by procedures similar to those used for the Con AI hormone.
421 00166480/79/080421-20$01.00/O Copy@@ @ 1979 by Academic Rcss. Inc. All ri@tts of rcploduction in any form rererved.
IDLER
422
logenic activity was also observed in the entire Con AI fraction of a pituitary extract of the chinook salmon Oncorhynchus tshawytscha (Campbell, 1978). Chum salmon maturational Con AI1 hormone had been isolated by Idler et al. (1975a-c) using the chick testicular radiophosphate uptake and trout gonadal CAMP production assays, but detailed studies on the physiologic functions and chemical compositions of the hormone had not been conducted. The present investigation was undertaken to purify the two molecular forms of chum salmon vitellogenic Con AI hormones. Comparison of the biochemical and biological properties of the salmon vitellogenic Con AI hormones with those of the maturational Con AI1 gonadotropin (Idler et al., 1975a,b) would hopefully lend further credence to their separate chemical identities and thereby consolidate the concept that more than one type of gonadotropin is elaborated by the salmon pituitary. Since Teleostei comprises an assemblage of diverse piscine species, it would also be informative to ascertain if the dualistic concept can be extended to the carp Cyprinus carpio. MATERIALS
AND METHODS
Preparation of vitellogenic Con AI and maturational Con AII gonadotropins. Chum salmon pituitaries were procured as described by Idler et a/. (1975a); acetone dried carp (Cyprinus carpio) pituitary powder was purchased from Staller Fisheries, Arkansas. The scheme of hormone isolation was as that described by Ng and Idler (1978 a,b). In short, the salmon or the carp pituitary extract was fractionated on Con ASepharose into an unadsorbed (Con AI) fraction and an adsorbed (Con AII) fraction. Rechromatography of the two fractions on Con A-Sepharose (in the case of the Con AI1 fraction, a-methyl-o-glucoside, which was used to elute the fraction, was eliminated by dialysis) was performed to ensure there was no contamination by the other fraction. Further purification of the Con AI and Con AI1 gonadotropic fractions was achieved by gel filtration on Sephacryl S-208 Superfine (Pharmacia) and/or Ultrogel AcA 44 (LKB) until single sharp peaks were obtained. Subfractions of the salmon and carp Con AI fractions with appropriate molecular weight were pooled, ultrafiltered, and extensively dialyzed against 5 mM NH,HCO,, pH 9, over a period
AND
NG
of 3 days before being chromatographed on DEAE Bio-Gel A (Bio-Rad). The ion exchanger was fully regenerated with a saturated solution of NH,HCO,, followed by 1 M NH,HCO,, and then equilibrated with 5 mu NH,HCO, over a period of several days before use. Unadsorbed proteins were eluted by 5 mM NH,HCO,. Adsorbed proteins were eluted by a linear gradient of 50 to 150 mM NH,HCO, (mixing volume 250 ml : 250 ml) followed by a second gradient of 150 to 600 mM NH,HCO, (mixing volume 250 ml : 250 ml). Strongly adsorbed proteins were eluted by IM NH,HCO,. All fractions were collected at 8 mI!hr from a 0.9 x 27 cm column. The vitellogenic Con AI fractions from the DEAE ion exchanger were further processed, after extensive dialysis against 3 n&J NH,OAc at pH 6, through a column of CM Bio-Gel A (Bio-Rad) equilibrated with the same buffer. Unadsorbed proteins were eluted with the starting buffer. Adsorbed proteins were eluted with 1 M NH,OAc (pH 6). The fraction from salmon Con AI 45,000-dalton peak which was adsorbed on DEAE Bio-Gel A (designated 45 DEIII) and subsequently unadsorbed on CM BioGel A was put through a column of sulfopropyl Sephadex C-50 (Pharmacia) equilibrated with 5 n&f NH,HCOS, pH 7.3. Unadsorbed proteins were eluted with the initial buffer. Adsorbed proteins were eluted with 2 M NH,HCO,, pH 9. The salmon maturational Con AI1 hormone was prepared as described by Idler et al. (1975a). Fractions corresponding to the ascending half of the biologically active peak (MW 40,000 daltons) were pooled to form Con AI1 G-75 Fraction II as described by Idler et al. (1975a) and used for radioimmunological and chemical characterization studies without further purification on the DEAE ion exchanger due to high biological activity and minimal contamination with extraneous proteins (Idler et al.. 1975a,b), while the whole peak designated Con AI1 MW 40,000 fraction was used for bioassays. The carp Con AI1 fraction was filtered through Ultrogel Ac44, and the ascending portion of the biologically active peak (MW 30,000 daltons), designated carp Con AI1 AcA 44 Fraction II, was analyzed in the chick testicular phosphate uptake assay and immature carp ovarian CAMP production assay. The hormone preparations from both salmon and carp pituitaries were also chromatographed on lentil lectin-Sepharose 4B (Pharmacia) which has a carbohydrate specificity similar to Con A-Sepharose (Allen et al.. 1976) but a lower affinity (Stein et al., 1971). The column was eluted in the same manner as for Con A-Sepharose. The piscine maturational Con AI1 hormones were adsorbed on the immobilized lectin whereas the vitellogenic Con AI hormones were not. Chromatography of salmon and carp vitellogenic Con AI hormones on wheat germ lectin-Sepharose
FISH
PITUITARY
6MB (Pharmacia) and Helix pomatia lectin Sepharose 6MB (Pharmacia) was also performed. The hormones were unadsorbed on both types of immobilized lectins equilibrated with Buffer B (Ng and Idler, 1978a). No adsorbed material was found subsequently when the wheat germ lectin-Sepharose column was eluted with Buffer B containing N-acetylglucosamine (100 mghnl) and when the Helix pomatia lectin - Sepharose column was eluted with Buffer B containing N-acetylgalactosamine (10 m&ml). Reduction and S-carboxamidomethylation of salmon maturational Con AII hormone.Twenty-five microliters of an aqueous solution of 30 pg hormone was allowed to react overnight at room temperature with $1 2-mercaptoethanol and 100 ~1 10 M urea in Tris buffer (pH 8.5). Iodoacetamide (30 mg) was added and reaction was allowed to proceed in the dark for 20 min. The solution was then serially diluted with barbital buffer (pH 8.6) to yield a range of hormone concentrations to be assayed for immunological potency. The reaction medium was diluted and tested likewise in order to ascertain whether it had any effect on the antigen-antibody reaction. The double-antibody technique (Crim et a[., 1975) was used for radioimmunoassay. Fraction II from G-75 (Idler er al., 1975a) was labeled with ‘“I (New England Nuclear) using the chloramine-T method of Greenwood et al. (1963) and cleaned up on a column of Bio-Gel P60 (Bio-Rad). The antiserum used was raised against SG-GlOO (Donaldson et al., 1972) by Crim et al. (1973). A standard curve was set up using Con AI1 G-75 Fraction II as the standard. Treatment of hormones with neuraminidase. To 25 ~1 of an aqueous solution of 30 pg hormone was added 60 pg Cl. perfringens neuraminidase (Type V, Sigma) in 0.1 M sodium acetate buffer pH 5.7 containing 5mM CaClp. The enzymic reaction was allowed to proceed overnight at room temperature. The solution was then serially diluted with barbital buffer to yield a range of hormone concentrations for RIA. The reaction medium was likewise diluted to find out whether the antigen-antibody reaction was affected by the presence of the medium. Hormones used for bioassays were treated with neuraminidase under the same conditions except for the reduction of the enzyme : sub strate ratio to 1 : 20. The efficacy of the enzyme preparation was checked by the detection of free sialic acid in the reaction mixture at the end of the enzymic reaction by the Warren method (Warren, 1959). Testing of the ability of the salmon vitellogenic Con AI hormones to bind to antisera raised against SGGlOO and Con AII G-75 Fraction II. An antiserum was raised against Con AI1 G-75 Fraction II in a New Zealand white rabbit by giving the animal a primary intradermal injection3 of 200 pg hormone followed 2 months later with monthly booster intramuscular injections of 1 mg. An antiserum obtained after two
GONADOTROPIN&
II
423
booster injections3 which showed 50% binding to iodinated Con AH G-75 Fraction II at 1 : 80,000 dilution was used for the study. Antiserum to SG-GIOO was raised by Grim et al. (1973). Five micrograms each of the purified salmon vitellogenic Con AI MW 25,080 and 45,000 hormones (DE III fraction) was labeled with 2 mCi of 13’1and cleaned up on Ultrogel AcA 44. Labeled hormone, 200 ~1, containing 10,000 cpm, was added to 200 ~1 of a serial dilution of the antiserum and incubated with 100 ~1 of normal rabbit serum (1 : 40 dilution) at 4C for 72 hr before addition of 100 ~1 of goat anti-rabbit globulin (1 : 20 dilution). Incubation at 4” was continued for a further 24 hr before centrifugation and counting of the precipitate. Testing for cross-reaction from Con AII G-75 Fraction II in radioimmunoassays for the salmon vitellogerric Con AI hormones. An antiserum was raised in a New Zealand white rabbit using a similar immunization schedule as described above. At the primary injection the rabbit received an intradermal injection of 1 mg Con AI (Salmon) 45,000-dalton fraction at multiple sites on the back. Two months later an intramuscular injection of 1 mg of the fraction was administered into the hind legs. This was followed by five monthly booster injections of the same fraction. This series of booster injections had been interrupted by an injection of 3 mg of the entire Con AI fraction, with the result that the antiserum contained antibodies to both the 25,000-dalton and the 45,~dalton fractions. However, when the antiserum was used at a dilution of 1 : 6,400 with the MW 25,000 DE III gonadotropin (see Results) as label, a radioimmunoassay for this gonadotropin could be set up. On the other hand, when the antiserum was used at a dilution of 1 : 6400 with the MW 45,000 DE III gonadotropin (see Results) as label, an assay for this hormone could also be developed. Details of the radioimmunoassays were the same as those described in the paragraph above, and tests for cross-reaction were carried out as described by Ng and Idler (1978b). Testing of the ability of salmon Con AII G-75 Fraction II to bind to antisera raised against the salmon vitellogenic Con AI hormones. Five micrograms of Con AI1 G-75 Fraction II was labeled with 2 mCi of 1311 and cleaned up on Bio-Gel P-60. Conditions of the radioimmunoassay were the same as described above, and a serial dilution of the antiserum (to both salmon Con AI MW 25,000 and MW 45,000 fractions) was tested. Antiserum was also raised in rabbits, in a similar fashion as mentioned above, to either the salmon Con AI 25,000-dalton fraction or the 45,000-dalton fraction 3 All hormones used for immunizations were homogenized in Freund’s complete adjuvant (Difco, Detroit).
424
IDLER AND NG
only. An antiserum which showed 50% binding at a dilution of 1 : 5000 to the MW 25,000 DE III gonadotropin and another antiserum which showed 50% binding at a dilution of 1 : 3000 to the MW 45,000 DE III gonadotropin were tested for binding to labeled G-75 Fraction 11. Testing for cross-reaction from salmon vitellogenic Con AI hormones in a radioimmunoassay for the salmon maturational Con All hormone. The purified Con AI (salmon) MW 25,000 and 45,000 hormones (DE III fraction) were tested as described by Ng and Idler (1978b). Testing for viteliogenic and maturational activities in hormone preparation. Assays were conducted as detailed by Ng and Idler (1978 a,b). Assay for testicular uptake of radiophosphate and tritiated leucine and isotopic incorporation into proteins. Mature male flounder collected in January were injected 2 weeks after hypophysectomy with H,33P04 and [3H]leucine (New England Nuclear), 20 &i of each isotope per kilogram body weight, together with the hormone preparation or buffer. Hormone administration was continued for another 3 days, and the fish were autopsied 48 hr after the last injection. Radioactivity in Protosol digests of the entire testicular tissue and of the trichloracetic acid-precipitable portion of the testis was determined as described in Ng and Idler (1978a), to assess the incorporation of the radioisotopes into the whole testis and testicular proteins respectively. Assay for spermiation. Male winter flounder collected in March-April with large testes but showing no signs of spermiation were selected for the experiment, They were injected, on the sixth day after hypophysectomy, with either buffer or the salmon Con AI fraction (10 mglkg) or Con AI1 (salmon) MW 40,000 fraction (0.1 mgikg). Spermiation was checked starting from 48 hr postinjection by applying a slight pressure on the abdominal region. A positive response was detected by the free flow of milt from the genital pore. Assay for steroidogenic activities in the mature hypophysectomized male winter flounder. In April mature male flounder were hypophysectomized. Beginning on the 16th day after hypophysectomy, they were injected once daily with hormone preparations or buffer for 4 consecutive days. They were bled 5 hr after the last injection. 1 I-Ketotestosterone and total androgens in plasma were quantified by radioimmunoassay. The antibody to tl-ketotestosterone was raised in a New Zealand white rabbit. 1I-Ketotestosterone-(3-0carboxymethyl)-oxime-bovine serum albumin co@gate (100 M) was injected intradermally at multiple sites at the primary injection. Two months later a booster injection of 2 mg was given intramuscularly into the hindhmbs, followed by a booster every fortnight. An antiserum which showed 50% binding to tritiated 11-ketotestosterone at 1 : 30,000 dilution
was utilized for radioimmunoassay. Flounder plasma (10 ~1) was extracted with 2 ml of dichloromethane, washed with 0.5 ml of water, and the dried-down extract was then utilized for radioimmunoassay. Tritiated 1 I-ketotestosterone was used as the tracer; for separating free from bound steroid dextran charcoal was employed. There was little cross-reaction from either testosterone or 1 I-hydroxytestosterone over the range of the assay (unpublished data). The essential details for preparation of the antibody had been described (Simpson and Wright, 1977). The radioimmunoassay for total androgens was performed with the Micromedic Kit (Micromedic Kit for testosterone. Since 1 lInc., Philadelphia) ketotestosterone and 11-hydroxytestosterone competed with testosterone at about equimolar ratios (unpublished data), the assay was used as an estimate of total androgens because 11-ketotestosterone and testosterone were both prominent in male flounder in April when this experiment was done (Campbell et al., 1976). Fiiy microliters of flounder plasma was extracted with 4 ml of dichloromethane, washed with 1 ml of water, and the dried-down extract was then utilized for radioimmunoassay instead of following the extraction procedure outlined in the Micromedic testosterone RIA instruction manual. The doubleantibody procedure and lZsI-labeled testosterone tyrosine methyl ester tracer were used in accordance with the manual. Chick testicular radiophosphate uptake assay. Carp Con AI1 AcA 44 Fraction II was assayed as described by Idler et a/. (1975a). Ovine LH, NlH-LHSl8, was used as reference standard. Gonadotropin activity was expressed in SG units where 1 SG unit = 1 pg NlHLH-Sl8; this unit was adopted since ovine LH was not active in some assays using fish (Idler et a/., 1975~). Immature carp ovarian CAMP production assay. Carp Con AI1 AcA 44 Fraction II was assayed as described by Idler et al. (1975~) using year-old grass carp [Ctenopharynogodon idelius). The sensitivity of the assay was increased IO-fold by acetylation of CAMP prior to quantification by radioimmunoassay (Harper et al., 1975). Amino acid and carbohydrate analyses. Amino acid analyses were performed on protein samples which had been hydrolyzed in 6 N HCI at 110” for 24 hr, following the method of Spackman et al. (1958) in a Beckman automatic amino acid analyzer. Tryptophan was determined after 24 hr hydrolysis in 3 N ptoluenesulfonic acid containing 0.2% tryptamine (Liu and Chang, 1971). Ammo sugars were quantitated on the amino acid analyzer, after hydrolysis in 4 N HCI at 100” for 8 hr, with a IS-cm BioRad Aminex A-5 resin bed and a standard pH 5.28 citrate buffer for elution (Black et a/., 1970). Hexose contents were estimated with the anthrone procedure (Roe, 1955). Optical density was read at 585 nm, an isosbestic point at which the absorbance by hexoses is independent of the
FISH
PITUITARY
amount of tryptophan. An equimolar mixture of mannose and galactose was used as the standard. S&k acid was measured with the Warren method (Warren, 1959) in samples which had been incubated in 0.1 N H$O, at 80” for 1 hr to release the bound carbohydrate. Electrophoresis. Analytical polyacrylamide gel disc electrophoresis was carried out as described by Davis (1964) at pH 8.3. Gels were stained with 0.02% Coomassie blue, 5% trichloroacetic acid, 5% sut fosaiicylic acid, 18% methanol, and destained by diffusion in 7.5% acetic acid.
RESULTS Salmon Gonadotropins Chromatographic behavior. Chromatography of the chum salmon pituitary Con AI fraction on Ultrogel AcA 44 to yield two vitellogenic subfractions with molecular weights 45,000 and 25,000 daltons had been reported earlier (Ng and Idler, 1978b). When the MW 45,000 fraction was ion exchanged on DEAE Bio-Gel A four main peaks were discerned in addition to two minor ones (Fig. la). The largest one (45 DE I) was eluted by 5 m&Y N&HC03. Two peaks of smaller size were eluted adjacent to each other when a gradient of SOto 150 mM NH,HCO, was applied, the molarity of elution being 54 and 66 mM, respectively. Both peaks, designated 45 DE II and 45 DE III, were active in the vitellogenesis assay. The minor peaks and 45 DE I possessed no vitellogenic activity when tested at an equal dosage (Table 1). Upon ion-exchange chromatography on DEAE Bio-Gel A the MW 25,000 fraction was resolved into four peaks (Fig. lb). Only the third one (designated 25 DE III) passessed activity in the vitellogenesis assay (Table 1). It was eluted at a molarity of 68 mM. The other peaks tested at equal dosage were not active. The yields from 1 g of pituitary of 25 DE III and G-75 Fraction II were, respectively, 0.2 and 1.2 mg. The maturational Con AIL gonadotropin content of chum salmon pituitaries used in these studies was 3.18 + 0.17 (SEM) mg/g of pituitary. This result was based on the radioimmunoassay of extracts from 10 sep-
GONADOTROPIN!&
II
425
arate pituitary collections averaging 130 g each made in September and October. The rich pituitary content accounted in part for the high yield. Chromatography of salmon 45 DE II and 45 DE III on Ultrogel AcA 54 yielded a single peak at 45,000 daltons in each case. Salmon 25 DE III, salmon 45 DE III, and Con AI (carp) MW 30,000 DE II were largely unadsorbed on CM Bio-Gel A equilibrated with 3 mM ammonium acetate buffer at pH 6. Salmon 45 DE III hormone was unadsorbed on SP-Sephadex equilibrated with 5 mM NH,HCO, at pH 7.3. Biological activities. Both the 45 DE III and the Con AI1 MW 40,000 hormones stimulated the winter flounder testis to take up tritiated leucine and radiophosphate with a concomitant increased incorporation into testicular proteins. However, interestingly only the 45 DE III hormone was able to elicit an increase in testis weight (Table 2). The vitellogenic activity of Con AI1 MW 40,000 hormone was manifested in its ability to stimulate an increase in the incorporation of [Y-IJleucine and H,33POd into the yolk fraction of the ovaries of both estrogenized and nonestrogenized fish, similar to the action of the Con AI hormones. Other parameters of gonadal activities, including uptake of the radioisotopes and their incorporation into ovarian proteins, and ovarian weight, were also stimulated (Table 1). Despite the overlap in the biological activities outlined above, the actions of saImon Con AI1 MW 40,000 hormone in effecting ovulation were not paralleled by the salmon Con AI hormones (Table 3). Seven fish injected with the salmon Con AI fraction did not spermiate, while seven other fish treated with buffer also showed no change, in contrast to six fish injected with Con AI1 MW 40,000 which all spermiated. The Con AI1 MW 40,000 hormone was more potent in enhancing testicular steroidogenesis (Table 4).
IDLER
426 r,
5mM
_.
AND
NG
50 to 150 mM
_ _
150 to6OOmM
)
a
ISOto6OOmM.
FRACTION
.600mM
NUMBER
FIG. 1. (a) Profile of elution of 100 mg salmon Con AI MW 45,000 fraction from DEAE Bio-Gel A. Yields of DE I, II, III, IV, and V were respectively 20, 10, 28, 5.3, and 2.5 mg. Fraction size was 7 ml for DE I and 3.6 ml for all the other fractions. (b) Profile of elution of 44 mg salmon Con AI MW 25,000 fraction from DEAE Bio-Gel A. Yields of DE I, II, III, and IV were 1.5, 10.5, 9.9, and 13.2 mg, respectively. Fraction size was 3.6 ml. (c) Profile of elution of 39 mg of carp Con AI MW 30,000 fraction from DEAE Bio-Gel A. Yields of DE I, IIa, and IIb were l.%, 7.8, and 18 mg, respectively. Fraction sizewas 3.6 ml.
The vitellogenic activity of the salmon vitellogenic Con AI hormones was not diminished by neuraminidase treatment. The maturational activity of the Con AI1 MW 40,000 hormone showed a slight decline but
inactivation was incomplete (Table 3), the same was true of its vitellogenic activity. Immunologic activities. The immunologic potencies of the salmon vitellogenic Con AI and maturational Con AI1 hor-
FISH
PITUITARY
GONADOTROPIN&
mones were unaltered after treatment with neuraminidase (e.g., Fig. 2a). However, subsequent to reduction by 2-mercaptoethanol and blocking of the sulfhydryl groups by iodoacetamide, the ability of the modified Con AI1 G-75 Fraction II hormone to displace the labeled native hormone was lost (Fig. 2a). The validity of the above conclusions was demonstrated by the lack of any effect of, NaOAc, CaCl,, neuraminidase, urea, and mercaptoethanol and iodoacetamide, on the binding of the antigen to the antibody. The binding of iodinated preparations of salmon 25 DE III and 45 DE III to the Con AI1 G-75 Fraction II and SG-GlOO antisera was meager (
AND
COMPARATIVE
ENDOCRINOLOGY
38,421-440
(1979)
Studies on Two Types of Gonadotropins Carp Pituitaries’ DAVID Marine
Sciences
Research
Laboratory,
from both Salmon and
R. IDLER AND T. BUN Nc Memorial University of Newfoundland, Canada, AIC SS7
St. John’s,
Newfoundland,
Accepted February 26, 1979 Two molecular weight forms of salmon vitellogenic Con AI (unadsorbed on concanavalin A-Sepharose) hormone and the salmon maturational Con AI1 (adsorbed on concanavalin A-Sepharose) hormone were able to stimulate vitellogenesis in the female winter flounder (Pseudopleuronectes americanus). Both the salmon vitellogenic Con AI 45,000~dalton hormone and the maturational Con AI1 hormone were able to enhance in vivo phosphate and leucine uptake by the flounder testis and incorporation into testicular proteins. Only the salmon maturational Con AI1 hormone was able to evoke spermiation and ovulation and it was much more potent than the vitellogenic Con AI hormones in stimulating testicular steroidogenesis. The vitellogenic and maturational activities of the salmon Con AI1 gonadotropin were partially destroyed on desialylation while the vitellogenic activity of the Con AI gonadotropins was not affected. The immunologic activity of the maturational Con AI1 hormone was retained after desialylation, but was abolished upon carboxamidomethylation. The immunologic potencies of the vitellogenic Con AI hormones were preserved after desialylation. The two salmon vitellogenic Con AI hormones displayed numerous prominent differences in amino acid and carbohydrate compositions from, and were immunologically remote from, the salmon maturational Con AI1 hormone. There was a marked dissimilarity in amino acid composition between the immunologically distinct salmon vitellogenic Con AI hormones. Two gonadotropins (vitellogenic Con AI and maturational Con AII), with an identical molecular weight of 30,000 daltons, were prepared from the pituitaries of the carp Cyprinus carpio. The distribution of biological activities was in general similar to that observed between the salmon hormones. The carp vitellogenic Con AI hormone could also be distinguished from the maturational Con AI1 hormone in the composition of its protein and carbohydrate moieties.
Until recently data from chemical fractionation studies and bioassays have been conducive to the concept that the teleost pituitary elaborates a solitary gonadotropin (see Fontaine, 1975). Presence of vitellogenic activity in a fraction of the American plaice (Hippoglossoides platessoides) pituitary extract not retained on concanavalin A -Sepharose was first reported by Campbell and Idler (1976), but this vitellogenic fraction also contained an appreciable amount of maturational and ovulatory activities (Campbell and Idler, 1977) which could be removed by rechromatography on the immobilized lectin (Ng and Idler, 1978a). The latter authors estab’ M.S.R.L.
Contribution
lished the existence of two different types of plaice gonadotropins, vitellogenic Con AI2 and maturational Con AI1 hormones, and in addition found “big” and “little” forms of hormones. In the chum salmon (Oncorhynchus keta) Ng and Idler (1978b) discovered vitellogenic Con AI hormones which were immunologically distinct from the salmon maturational Con AI1 hormone and exhibited size heterogeneity. Vitel-
Number 332.
* The term Con AI hormone is used to designate a preparation which is unadsorbed on concanavalin A-Sepharose and which has been purified by processes such as gel filtration and ion-exchange chromatography. The term Con AI1 hormone is employed for a hormone adsorbed on the immobilized lectin and purified by procedures similar to those used for the Con AI hormone.
421 00166480/79/080421-20$01.00/O Copy@@ @ 1979 by Academic Rcss. Inc. All ri@tts of rcploduction in any form rererved.
IDLER
422
logenic activity was also observed in the entire Con AI fraction of a pituitary extract of the chinook salmon Oncorhynchus tshawytscha (Campbell, 1978). Chum salmon maturational Con AI1 hormone had been isolated by Idler et al. (1975a-c) using the chick testicular radiophosphate uptake and trout gonadal CAMP production assays, but detailed studies on the physiologic functions and chemical compositions of the hormone had not been conducted. The present investigation was undertaken to purify the two molecular forms of chum salmon vitellogenic Con AI hormones. Comparison of the biochemical and biological properties of the salmon vitellogenic Con AI hormones with those of the maturational Con AI1 gonadotropin (Idler et al., 1975a,b) would hopefully lend further credence to their separate chemical identities and thereby consolidate the concept that more than one type of gonadotropin is elaborated by the salmon pituitary. Since Teleostei comprises an assemblage of diverse piscine species, it would also be informative to ascertain if the dualistic concept can be extended to the carp Cyprinus carpio. MATERIALS
AND METHODS
Preparation of vitellogenic Con AI and maturational Con AII gonadotropins. Chum salmon pituitaries were procured as described by Idler et a/. (1975a); acetone dried carp (Cyprinus carpio) pituitary powder was purchased from Staller Fisheries, Arkansas. The scheme of hormone isolation was as that described by Ng and Idler (1978 a,b). In short, the salmon or the carp pituitary extract was fractionated on Con ASepharose into an unadsorbed (Con AI) fraction and an adsorbed (Con AII) fraction. Rechromatography of the two fractions on Con A-Sepharose (in the case of the Con AI1 fraction, a-methyl-o-glucoside, which was used to elute the fraction, was eliminated by dialysis) was performed to ensure there was no contamination by the other fraction. Further purification of the Con AI and Con AI1 gonadotropic fractions was achieved by gel filtration on Sephacryl S-208 Superfine (Pharmacia) and/or Ultrogel AcA 44 (LKB) until single sharp peaks were obtained. Subfractions of the salmon and carp Con AI fractions with appropriate molecular weight were pooled, ultrafiltered, and extensively dialyzed against 5 mM NH,HCO,, pH 9, over a period
AND
NG
of 3 days before being chromatographed on DEAE Bio-Gel A (Bio-Rad). The ion exchanger was fully regenerated with a saturated solution of NH,HCO,, followed by 1 M NH,HCO,, and then equilibrated with 5 mu NH,HCO, over a period of several days before use. Unadsorbed proteins were eluted by 5 mM NH,HCO,. Adsorbed proteins were eluted by a linear gradient of 50 to 150 mM NH,HCO, (mixing volume 250 ml : 250 ml) followed by a second gradient of 150 to 600 mM NH,HCO, (mixing volume 250 ml : 250 ml). Strongly adsorbed proteins were eluted by IM NH,HCO,. All fractions were collected at 8 mI!hr from a 0.9 x 27 cm column. The vitellogenic Con AI fractions from the DEAE ion exchanger were further processed, after extensive dialysis against 3 n&J NH,OAc at pH 6, through a column of CM Bio-Gel A (Bio-Rad) equilibrated with the same buffer. Unadsorbed proteins were eluted with the starting buffer. Adsorbed proteins were eluted with 1 M NH,OAc (pH 6). The fraction from salmon Con AI 45,000-dalton peak which was adsorbed on DEAE Bio-Gel A (designated 45 DEIII) and subsequently unadsorbed on CM BioGel A was put through a column of sulfopropyl Sephadex C-50 (Pharmacia) equilibrated with 5 n&f NH,HCOS, pH 7.3. Unadsorbed proteins were eluted with the initial buffer. Adsorbed proteins were eluted with 2 M NH,HCO,, pH 9. The salmon maturational Con AI1 hormone was prepared as described by Idler et al. (1975a). Fractions corresponding to the ascending half of the biologically active peak (MW 40,000 daltons) were pooled to form Con AI1 G-75 Fraction II as described by Idler et al. (1975a) and used for radioimmunological and chemical characterization studies without further purification on the DEAE ion exchanger due to high biological activity and minimal contamination with extraneous proteins (Idler et al.. 1975a,b), while the whole peak designated Con AI1 MW 40,000 fraction was used for bioassays. The carp Con AI1 fraction was filtered through Ultrogel Ac44, and the ascending portion of the biologically active peak (MW 30,000 daltons), designated carp Con AI1 AcA 44 Fraction II, was analyzed in the chick testicular phosphate uptake assay and immature carp ovarian CAMP production assay. The hormone preparations from both salmon and carp pituitaries were also chromatographed on lentil lectin-Sepharose 4B (Pharmacia) which has a carbohydrate specificity similar to Con A-Sepharose (Allen et al.. 1976) but a lower affinity (Stein et al., 1971). The column was eluted in the same manner as for Con A-Sepharose. The piscine maturational Con AI1 hormones were adsorbed on the immobilized lectin whereas the vitellogenic Con AI hormones were not. Chromatography of salmon and carp vitellogenic Con AI hormones on wheat germ lectin-Sepharose
FISH
PITUITARY
6MB (Pharmacia) and Helix pomatia lectin Sepharose 6MB (Pharmacia) was also performed. The hormones were unadsorbed on both types of immobilized lectins equilibrated with Buffer B (Ng and Idler, 1978a). No adsorbed material was found subsequently when the wheat germ lectin-Sepharose column was eluted with Buffer B containing N-acetylglucosamine (100 mghnl) and when the Helix pomatia lectin - Sepharose column was eluted with Buffer B containing N-acetylgalactosamine (10 m&ml). Reduction and S-carboxamidomethylation of salmon maturational Con AII hormone.Twenty-five microliters of an aqueous solution of 30 pg hormone was allowed to react overnight at room temperature with $1 2-mercaptoethanol and 100 ~1 10 M urea in Tris buffer (pH 8.5). Iodoacetamide (30 mg) was added and reaction was allowed to proceed in the dark for 20 min. The solution was then serially diluted with barbital buffer (pH 8.6) to yield a range of hormone concentrations to be assayed for immunological potency. The reaction medium was diluted and tested likewise in order to ascertain whether it had any effect on the antigen-antibody reaction. The double-antibody technique (Crim et a[., 1975) was used for radioimmunoassay. Fraction II from G-75 (Idler er al., 1975a) was labeled with ‘“I (New England Nuclear) using the chloramine-T method of Greenwood et al. (1963) and cleaned up on a column of Bio-Gel P60 (Bio-Rad). The antiserum used was raised against SG-GlOO (Donaldson et al., 1972) by Crim et al. (1973). A standard curve was set up using Con AI1 G-75 Fraction II as the standard. Treatment of hormones with neuraminidase. To 25 ~1 of an aqueous solution of 30 pg hormone was added 60 pg Cl. perfringens neuraminidase (Type V, Sigma) in 0.1 M sodium acetate buffer pH 5.7 containing 5mM CaClp. The enzymic reaction was allowed to proceed overnight at room temperature. The solution was then serially diluted with barbital buffer to yield a range of hormone concentrations for RIA. The reaction medium was likewise diluted to find out whether the antigen-antibody reaction was affected by the presence of the medium. Hormones used for bioassays were treated with neuraminidase under the same conditions except for the reduction of the enzyme : sub strate ratio to 1 : 20. The efficacy of the enzyme preparation was checked by the detection of free sialic acid in the reaction mixture at the end of the enzymic reaction by the Warren method (Warren, 1959). Testing of the ability of the salmon vitellogenic Con AI hormones to bind to antisera raised against SGGlOO and Con AII G-75 Fraction II. An antiserum was raised against Con AI1 G-75 Fraction II in a New Zealand white rabbit by giving the animal a primary intradermal injection3 of 200 pg hormone followed 2 months later with monthly booster intramuscular injections of 1 mg. An antiserum obtained after two
GONADOTROPIN&
II
423
booster injections3 which showed 50% binding to iodinated Con AH G-75 Fraction II at 1 : 80,000 dilution was used for the study. Antiserum to SG-GIOO was raised by Grim et al. (1973). Five micrograms each of the purified salmon vitellogenic Con AI MW 25,080 and 45,000 hormones (DE III fraction) was labeled with 2 mCi of 13’1and cleaned up on Ultrogel AcA 44. Labeled hormone, 200 ~1, containing 10,000 cpm, was added to 200 ~1 of a serial dilution of the antiserum and incubated with 100 ~1 of normal rabbit serum (1 : 40 dilution) at 4C for 72 hr before addition of 100 ~1 of goat anti-rabbit globulin (1 : 20 dilution). Incubation at 4” was continued for a further 24 hr before centrifugation and counting of the precipitate. Testing for cross-reaction from Con AII G-75 Fraction II in radioimmunoassays for the salmon vitellogerric Con AI hormones. An antiserum was raised in a New Zealand white rabbit using a similar immunization schedule as described above. At the primary injection the rabbit received an intradermal injection of 1 mg Con AI (Salmon) 45,000-dalton fraction at multiple sites on the back. Two months later an intramuscular injection of 1 mg of the fraction was administered into the hind legs. This was followed by five monthly booster injections of the same fraction. This series of booster injections had been interrupted by an injection of 3 mg of the entire Con AI fraction, with the result that the antiserum contained antibodies to both the 25,000-dalton and the 45,~dalton fractions. However, when the antiserum was used at a dilution of 1 : 6,400 with the MW 25,000 DE III gonadotropin (see Results) as label, a radioimmunoassay for this gonadotropin could be set up. On the other hand, when the antiserum was used at a dilution of 1 : 6400 with the MW 45,000 DE III gonadotropin (see Results) as label, an assay for this hormone could also be developed. Details of the radioimmunoassays were the same as those described in the paragraph above, and tests for cross-reaction were carried out as described by Ng and Idler (1978b). Testing of the ability of salmon Con AII G-75 Fraction II to bind to antisera raised against the salmon vitellogenic Con AI hormones. Five micrograms of Con AI1 G-75 Fraction II was labeled with 2 mCi of 1311 and cleaned up on Bio-Gel P-60. Conditions of the radioimmunoassay were the same as described above, and a serial dilution of the antiserum (to both salmon Con AI MW 25,000 and MW 45,000 fractions) was tested. Antiserum was also raised in rabbits, in a similar fashion as mentioned above, to either the salmon Con AI 25,000-dalton fraction or the 45,000-dalton fraction 3 All hormones used for immunizations were homogenized in Freund’s complete adjuvant (Difco, Detroit).
424
IDLER AND NG
only. An antiserum which showed 50% binding at a dilution of 1 : 5000 to the MW 25,000 DE III gonadotropin and another antiserum which showed 50% binding at a dilution of 1 : 3000 to the MW 45,000 DE III gonadotropin were tested for binding to labeled G-75 Fraction 11. Testing for cross-reaction from salmon vitellogenic Con AI hormones in a radioimmunoassay for the salmon maturational Con All hormone. The purified Con AI (salmon) MW 25,000 and 45,000 hormones (DE III fraction) were tested as described by Ng and Idler (1978b). Testing for viteliogenic and maturational activities in hormone preparation. Assays were conducted as detailed by Ng and Idler (1978 a,b). Assay for testicular uptake of radiophosphate and tritiated leucine and isotopic incorporation into proteins. Mature male flounder collected in January were injected 2 weeks after hypophysectomy with H,33P04 and [3H]leucine (New England Nuclear), 20 &i of each isotope per kilogram body weight, together with the hormone preparation or buffer. Hormone administration was continued for another 3 days, and the fish were autopsied 48 hr after the last injection. Radioactivity in Protosol digests of the entire testicular tissue and of the trichloracetic acid-precipitable portion of the testis was determined as described in Ng and Idler (1978a), to assess the incorporation of the radioisotopes into the whole testis and testicular proteins respectively. Assay for spermiation. Male winter flounder collected in March-April with large testes but showing no signs of spermiation were selected for the experiment, They were injected, on the sixth day after hypophysectomy, with either buffer or the salmon Con AI fraction (10 mglkg) or Con AI1 (salmon) MW 40,000 fraction (0.1 mgikg). Spermiation was checked starting from 48 hr postinjection by applying a slight pressure on the abdominal region. A positive response was detected by the free flow of milt from the genital pore. Assay for steroidogenic activities in the mature hypophysectomized male winter flounder. In April mature male flounder were hypophysectomized. Beginning on the 16th day after hypophysectomy, they were injected once daily with hormone preparations or buffer for 4 consecutive days. They were bled 5 hr after the last injection. 1 I-Ketotestosterone and total androgens in plasma were quantified by radioimmunoassay. The antibody to tl-ketotestosterone was raised in a New Zealand white rabbit. 1I-Ketotestosterone-(3-0carboxymethyl)-oxime-bovine serum albumin co@gate (100 M) was injected intradermally at multiple sites at the primary injection. Two months later a booster injection of 2 mg was given intramuscularly into the hindhmbs, followed by a booster every fortnight. An antiserum which showed 50% binding to tritiated 11-ketotestosterone at 1 : 30,000 dilution
was utilized for radioimmunoassay. Flounder plasma (10 ~1) was extracted with 2 ml of dichloromethane, washed with 0.5 ml of water, and the dried-down extract was then utilized for radioimmunoassay. Tritiated 1 I-ketotestosterone was used as the tracer; for separating free from bound steroid dextran charcoal was employed. There was little cross-reaction from either testosterone or 1 I-hydroxytestosterone over the range of the assay (unpublished data). The essential details for preparation of the antibody had been described (Simpson and Wright, 1977). The radioimmunoassay for total androgens was performed with the Micromedic Kit (Micromedic Kit for testosterone. Since 1 lInc., Philadelphia) ketotestosterone and 11-hydroxytestosterone competed with testosterone at about equimolar ratios (unpublished data), the assay was used as an estimate of total androgens because 11-ketotestosterone and testosterone were both prominent in male flounder in April when this experiment was done (Campbell et al., 1976). Fiiy microliters of flounder plasma was extracted with 4 ml of dichloromethane, washed with 1 ml of water, and the dried-down extract was then utilized for radioimmunoassay instead of following the extraction procedure outlined in the Micromedic testosterone RIA instruction manual. The doubleantibody procedure and lZsI-labeled testosterone tyrosine methyl ester tracer were used in accordance with the manual. Chick testicular radiophosphate uptake assay. Carp Con AI1 AcA 44 Fraction II was assayed as described by Idler et a/. (1975a). Ovine LH, NlH-LHSl8, was used as reference standard. Gonadotropin activity was expressed in SG units where 1 SG unit = 1 pg NlHLH-Sl8; this unit was adopted since ovine LH was not active in some assays using fish (Idler et a/., 1975~). Immature carp ovarian CAMP production assay. Carp Con AI1 AcA 44 Fraction II was assayed as described by Idler et al. (1975~) using year-old grass carp [Ctenopharynogodon idelius). The sensitivity of the assay was increased IO-fold by acetylation of CAMP prior to quantification by radioimmunoassay (Harper et al., 1975). Amino acid and carbohydrate analyses. Amino acid analyses were performed on protein samples which had been hydrolyzed in 6 N HCI at 110” for 24 hr, following the method of Spackman et al. (1958) in a Beckman automatic amino acid analyzer. Tryptophan was determined after 24 hr hydrolysis in 3 N ptoluenesulfonic acid containing 0.2% tryptamine (Liu and Chang, 1971). Ammo sugars were quantitated on the amino acid analyzer, after hydrolysis in 4 N HCI at 100” for 8 hr, with a IS-cm BioRad Aminex A-5 resin bed and a standard pH 5.28 citrate buffer for elution (Black et a/., 1970). Hexose contents were estimated with the anthrone procedure (Roe, 1955). Optical density was read at 585 nm, an isosbestic point at which the absorbance by hexoses is independent of the
FISH
PITUITARY
amount of tryptophan. An equimolar mixture of mannose and galactose was used as the standard. S&k acid was measured with the Warren method (Warren, 1959) in samples which had been incubated in 0.1 N H$O, at 80” for 1 hr to release the bound carbohydrate. Electrophoresis. Analytical polyacrylamide gel disc electrophoresis was carried out as described by Davis (1964) at pH 8.3. Gels were stained with 0.02% Coomassie blue, 5% trichloroacetic acid, 5% sut fosaiicylic acid, 18% methanol, and destained by diffusion in 7.5% acetic acid.
RESULTS Salmon Gonadotropins Chromatographic behavior. Chromatography of the chum salmon pituitary Con AI fraction on Ultrogel AcA 44 to yield two vitellogenic subfractions with molecular weights 45,000 and 25,000 daltons had been reported earlier (Ng and Idler, 1978b). When the MW 45,000 fraction was ion exchanged on DEAE Bio-Gel A four main peaks were discerned in addition to two minor ones (Fig. la). The largest one (45 DE I) was eluted by 5 m&Y N&HC03. Two peaks of smaller size were eluted adjacent to each other when a gradient of SOto 150 mM NH,HCO, was applied, the molarity of elution being 54 and 66 mM, respectively. Both peaks, designated 45 DE II and 45 DE III, were active in the vitellogenesis assay. The minor peaks and 45 DE I possessed no vitellogenic activity when tested at an equal dosage (Table 1). Upon ion-exchange chromatography on DEAE Bio-Gel A the MW 25,000 fraction was resolved into four peaks (Fig. lb). Only the third one (designated 25 DE III) passessed activity in the vitellogenesis assay (Table 1). It was eluted at a molarity of 68 mM. The other peaks tested at equal dosage were not active. The yields from 1 g of pituitary of 25 DE III and G-75 Fraction II were, respectively, 0.2 and 1.2 mg. The maturational Con AIL gonadotropin content of chum salmon pituitaries used in these studies was 3.18 + 0.17 (SEM) mg/g of pituitary. This result was based on the radioimmunoassay of extracts from 10 sep-
GONADOTROPIN!&
II
425
arate pituitary collections averaging 130 g each made in September and October. The rich pituitary content accounted in part for the high yield. Chromatography of salmon 45 DE II and 45 DE III on Ultrogel AcA 54 yielded a single peak at 45,000 daltons in each case. Salmon 25 DE III, salmon 45 DE III, and Con AI (carp) MW 30,000 DE II were largely unadsorbed on CM Bio-Gel A equilibrated with 3 mM ammonium acetate buffer at pH 6. Salmon 45 DE III hormone was unadsorbed on SP-Sephadex equilibrated with 5 mM NH,HCO, at pH 7.3. Biological activities. Both the 45 DE III and the Con AI1 MW 40,000 hormones stimulated the winter flounder testis to take up tritiated leucine and radiophosphate with a concomitant increased incorporation into testicular proteins. However, interestingly only the 45 DE III hormone was able to elicit an increase in testis weight (Table 2). The vitellogenic activity of Con AI1 MW 40,000 hormone was manifested in its ability to stimulate an increase in the incorporation of [Y-IJleucine and H,33POd into the yolk fraction of the ovaries of both estrogenized and nonestrogenized fish, similar to the action of the Con AI hormones. Other parameters of gonadal activities, including uptake of the radioisotopes and their incorporation into ovarian proteins, and ovarian weight, were also stimulated (Table 1). Despite the overlap in the biological activities outlined above, the actions of saImon Con AI1 MW 40,000 hormone in effecting ovulation were not paralleled by the salmon Con AI hormones (Table 3). Seven fish injected with the salmon Con AI fraction did not spermiate, while seven other fish treated with buffer also showed no change, in contrast to six fish injected with Con AI1 MW 40,000 which all spermiated. The Con AI1 MW 40,000 hormone was more potent in enhancing testicular steroidogenesis (Table 4).
IDLER
426 r,
5mM
_.
AND
NG
50 to 150 mM
_ _
150 to6OOmM
)
a
ISOto6OOmM.
FRACTION
.600mM
NUMBER
FIG. 1. (a) Profile of elution of 100 mg salmon Con AI MW 45,000 fraction from DEAE Bio-Gel A. Yields of DE I, II, III, IV, and V were respectively 20, 10, 28, 5.3, and 2.5 mg. Fraction size was 7 ml for DE I and 3.6 ml for all the other fractions. (b) Profile of elution of 44 mg salmon Con AI MW 25,000 fraction from DEAE Bio-Gel A. Yields of DE I, II, III, and IV were 1.5, 10.5, 9.9, and 13.2 mg, respectively. Fraction size was 3.6 ml. (c) Profile of elution of 39 mg of carp Con AI MW 30,000 fraction from DEAE Bio-Gel A. Yields of DE I, IIa, and IIb were l.%, 7.8, and 18 mg, respectively. Fraction sizewas 3.6 ml.
The vitellogenic activity of the salmon vitellogenic Con AI hormones was not diminished by neuraminidase treatment. The maturational activity of the Con AI1 MW 40,000 hormone showed a slight decline but
inactivation was incomplete (Table 3), the same was true of its vitellogenic activity. Immunologic activities. The immunologic potencies of the salmon vitellogenic Con AI and maturational Con AI1 hor-
FISH
PITUITARY
GONADOTROPIN&
mones were unaltered after treatment with neuraminidase (e.g., Fig. 2a). However, subsequent to reduction by 2-mercaptoethanol and blocking of the sulfhydryl groups by iodoacetamide, the ability of the modified Con AI1 G-75 Fraction II hormone to displace the labeled native hormone was lost (Fig. 2a). The validity of the above conclusions was demonstrated by the lack of any effect of, NaOAc, CaCl,, neuraminidase, urea, and mercaptoethanol and iodoacetamide, on the binding of the antigen to the antibody. The binding of iodinated preparations of salmon 25 DE III and 45 DE III to the Con AI1 G-75 Fraction II and SG-GlOO antisera was meager (
