GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

88, 298-306

(1992)

identification of Growth Hormone Molecular Variants in Chicken Serum Jo& LUIS MONTIEL,* LucR. BERGHMAN,~ ANDCARLOSAR,~MBURO*-' *Departamento Mixico,

de Fisiologia, Institute de Investigaciones BiomL;dicas. Universidad Mkxico, D.F. 04510, Mkxico; and fLaboratory for Neuroendocrinology Biotechnology, Naamsrstraat 59, B 3ooO Leuven, Belgium

National Autdnoma and Immunological

de

Accepted April 8, 1992 It has been described that pituitary growth hormone shows molecular and functional heterogeneity. In birds, size and charge variants of chicken growth hormone (cGH) have been shown in the chicken pituitary @and and in purified preparations of the hormone. Here we demonstrate the existence of cGH molecular isoforms in chicken serum, thus suggesting that they are secreted from the gland. The isolation of total cGH present in sera was performed by immunoaffinity chromatography employing a specific monoclonal antibody against cGH. Different analytical electrophoretic methods (SDS-polyacryIamide gel electrophoresis, isoelectric focusing, bidimensional polyacrylamide gel electrophoresis) followed by Western blot and immunostaining were employed to characterize the serum cGH isoforms, and compared to those present in a fresh pituitary extract. Several identical immunoreactive bands corn&rated in both serum and the gland extract in the different systems (SDS-PAGE, MW 16, 22, 26, 29, 52, 62, 66 kDa; IEF, pls 8.1, 7.5, 7.1, 6.8, 6.2), thus revealing a high correspondence of molecular isoforms of the hormone in the two tissues. Additionally, a glycosylated variant of chicken growth hormone (G-cGH) was also revealed in the serum after concanavahn A-Sepharose chromatography. o 1992 Academic Press. Inc.

Pituitary growth hormone(GH) is heterogeneous,both structurally andfunctionally. In mammals, a complex array of mass and charge variants of GH coexist in the pituitary (Lewis et al., 1980;Lewis, 1984;Hart et al., 1984;Baumann, 1988).In addition to its somatotrophic action, GH exhibits in viva lipolytic, diabetogenic, insulin-like, and, in the case of human GH, lactogenic activities. It also inhibits insulin-inducedlipogenesisand epinephrine-inducedlipolysis. Molecular heterogeneityof GH has also been demonstrated in other vertebrates. Growth hormone isoforms have been described in the eel (Kishida et al., 19&7), chum salmon (Kawauchi et al., 198% atlantic salmon (Skibelli et al., 1990),andthe bullfrog (Kobayashi et al., 1989). ’ To whom correspondence

should be addressed. 298

0016-6480192

$4.00

Copyright 0 1992 by Academic Press, Inc. AU rights of reproduction in any form reserved.

In birds, mass and charge isoforms of chicken growth hormone (cGH) have been reportedin the pituitary (Houstonand Goddard, 1988;Aramburo et al., 199Oa)and in purified hormone preparations(Ar&mburo et al., 1989a).Some of the structural differences between isoforms appear to be derived from post-translationalmodifications. Thus, a glycosylated variant of cGH (Berghmanet al., 1987)whichproved to be heterogenous(Aramhuro et al., 1991)has been described.Additionally, a significant proportion of the hormone appears to be phosphorylated(Aramburo et al., 1989x+, and chicken pituitary cells in primary culture can incorporateradioactivephosphate into a cGH-like immunoreactive band whose releaseis stimulated by somatocrinin (GRF) (Aramburo et al., 199Qb).Highmolecular-weight(MW) forms of the hormone probably correspondingto disulfide

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linked oligomers (dimer, trimer, etc.) have 1 mM HCl, pH 3.0, during 15 min in a lO-ml syringe an “end-over-end” shaker, and then also been reported in the gland (Houston employing washed with 100 ml 1 mM HCl to eliminate preservaand Goddard, 1988; Aramburo et al., tives. The swollen gel was then equilibrated in cou199Oa),as well as a proteolytically cleaved pling buffer (50 mM phosphate, 0.5 M NaCl, pH 7.6). “two-chain” form which gives rise to a Five milligrams of purified monoclonal antibodies =16-kDa fragment upon reduction (Arhm- (Mab) anti-cGH (Be&man et al., 1987) was resusin coupling buffer and added to the Sepharose. buro and Scanes,1991).Although Houston pended The mixture was shaken “end-over-end” during 4 hr et al. (1990)have reported that the single at room temperature. Coupling of the antibody to the most abundant charge isomer of cGH is resin was higher than 90% and the remaining active likely to contain all the biological activities sites in the resin were blocked by adding 1 Methanolascribedto the hormone, other studies sug- amine, pH 8.0, at 4” overnight. Finally the resin was washed alternately with the loading and elution buffers gestthat the cGH variants may have differ- (see below). ent bioactivities (Ar&nburo et al., 1989b). Two of the monomeric charge variants show a similar growth promoting activity in Zmmunoaffinity Chromatography dwarf mice @caneset al., PM), but oppo- Total cGH present in the circulation was isolated by site effects on chicken adipose tissue ex- immunoaffinity chromatography at room temperature plants: one is mainly lipolytic whereasthe following the method by Berghman et al. (1988) with other shows antilipolytic activity (Arzim- slight modifications. Chicken serum (lO-ml aliquots) was loaded into the monoclonal immunoabsorbent buro et al., 199Oa). equilibrated with 50 mM phosphate, 0.9% To acquirephysiological significance,the previously NaCl, 0.01% Tween-20, pH 7.6 (loading buffer), and hormone isoforms must enter into the recirculated in the column at a flow rate of 12 mlihr bloodstreamto reachtheir targettissues.In during 2.5 hr. The column was subsequently washed the present study, the pattern of heteroge- with 50 mM phosphate, 1 M NaCl, 0.01% Tween-20, neity previously described for chicken pH 7.6, to discard unspecifically retained proteins in resin, until the baseline was close to zero. Aftergrowth hormone (in the pituitary or in pu- the ward, specifically bound protein (cGH) was eluted rified preparations of the hormone) was from the column with 50 mM Gly, 0.9% NaCl, 0.01% searchedfor in sera, to test if the variants Tween-20, pH 3 (elution buffer). Fractions (1 ml) were collected in tubes containing 500 p,l of 100 mM Trisare normally releasedfrom the gland. MATERIAL

AND METHODS

Animals, Monoclonal Antibodies, Polyclonal Antiserum

and

Blood was collected from 7-week-old broilers (Pilch) after decapitation and serum was obtained and kept frozen at -20” until used. Monoclonal antibodies (Mab) anti-chicken growth hormone (a-cGH) (Berghman er al., 1987) were purified from ascites fluid by protein A-Sepharose affinity chromatography, dialyzed in coupling solution (see below), lyophilized, and stored at - 20” until employed. Polyclonai antiserum against cGH (Arfimburo et al., 1989~) was used for immunostaining the Western blots.

Synthesis of Immunoaflnity

Column

A monoclonal antibody affinity column was synthesized as described by Berghman et al. (1988). Briefly, activated CNBr-Sepharose 4B (350 mg) was swollen in

HCl, pH 7.5. Absorbance of all fractions was monitored at 280 nm. Peak fractions were pooled, dialyzed against distilled deionized water (DDW), lyophilized, and stored at - 20” until analyzed.

Affinity Chromatography A-Sepharose

with Con

To determine if the glycosylated variant of cGH was present in the circulation, the total cGH fraction isolated from serum by immunotinity was chromatographed in concanavalin A-Sepharose (Sigma) following the method previously described (Arhmburo et al., 1991). Briefly, the cGH fraction obtained from serum as mentioned above was resuspended in equilibrium buffer (20 mM Tris-HCl, 0.15 M NaCl, 1 m&f MnCl,, 1 mM CaCl,, pH 7.0) and applied onto the previously equilibrated column, at 4”. The retained fraction was eluted with 10 and 100 mM a-methylmannoside in equilibrium buffer. Peak fractions were pooled, dialyzed against distilled deionized water (DDW), lye-’ philized, and stored at - 20” until analyzed.

300

MONTIEL,

Electrophoretic Characterization cGH Variants

BERGHMAN,

qf‘ Serum

The fraction specifically retained to the immunoabsorbent was analyzed by different electrophoretic methods followed by Western blot and the immunoreactive cGH-like bands were developed employing a polyclonal antiserum (a-cGH) raised in rabbits (ArAmburo ei al., 1989~). A fresh chicken pituitary extract prepared as previously described (ArBmburo et ul., 1990) was analyzed simultaneously in the same electrophoretic systems as a control. Ocassionally a purified preparation of cGH (fraction B-DE-l, prepared as described by Aramburo et al., 1989a) was also employed. When necessary, the blots were stained with concanavalin A conjugated to horseradish peroxidase (HRP), to reveal the glycoproteins. (A) Denaturing electrophoresis (SDS-PAGE). An aliquot was analyzed in one-dimensional SDS-PAGE using the buffer system of Laemmli (1970) in the MiniProtean II Cell (Bio-Rad). Acrylamide concentration was 12.5% in the separating gel and 4% in the stacking gel. The samples were run under reducing conditions in presence of 5% 2-mercaptoethanol, and also under nonreducing conditions. (B) Analytical isoelectrofocusing (IEE). This was performed in thin 4% polyacrilamide slab gels (100 x 125 x 0.6 mm), employing 2% ampholines (pH 3.5-10: 6-8, 4: 1, v/v). Cahode buffer: 2 M ethylendiamine, 20 mA4 Lys, 20 mM Arg. Anode buffer: 20 m&f Glu, 20 mM Asp. The run was performed at 4” and 4000 Vihr was applied. (C)

Bidimensional

elecrrophoresis

(ZD-PAGE).

Two-dimensional analysis was performed according to O’Farrell(1975) and Hochstrasser et al. (1988).

Western Blot Analysis After the electrophoretic runs (SDS-PAGE, IEF and 2D-PAGE), corresponding gels were equilibrated in transfer solution (25 mAl Tris, 192 mM Gly, 20% methanol) for 30 min, and proteins were electrotransferred to nitrocellulose sheets at 200 mA during 30 min, and then immunostained as previously described (ArBmburo et al., 1990a). Briefly, the membranes were blocked (3% gelatin in PBS for antibody staining, and 3% BSA in PBS for Con-A HRP staining) overnight, and after washing, they were incubated either with a-cGH antiserum (1:2,000) for 2-3 hr, and then incubated with a second antibody (goat anti-rabbit IgG conjugated to HRP, Bio-Rad) during 1 hr, or alternatively, the membranes were incubated with Con-A HRP (Sigma, 4 p&ml) for 3-4 hr. Immunoreactive or glycosylated cGH bands were revealed after incubating the membranes in developing solution (0.05% 4-chloronaphtho1, 0.015% H202, 16.6% methanol, in PBS) for 30 min.

AND

ARAMBURO

Determination

of Proteins

Total protein was determined by the Bio-Ra.d microassay method, using bovine serum albumin (BSA) as standard.

RESULTS

An affinity immunosorbent column was prepared by coupling CNBr-activated Sepharose 4B with a monoclonal antibody directed against chicken growth hormone. Coupling of the antibody to the resin was higher than 95%. This column allowed the isolation of total cGH present in the blood. Ten milliliter chicken serum batches were chromatographed each time. Figure 1 shows a typical chromatogram. Initially a huge amount of protein washed off the column with the loading buffer, as expected. There was, however, some protein which interacted nonspecifically with the column and this was eliminated by eluting with a

I .c1 -

E

0.0

c 0 03 N D d

0.6

0.4

0.2

NC.

Fraction

FIG. 1. Isolation of circulating &H by chromatography of chicken serum (10 ml) in an immunoaginity coIumn (a-cGH Mat, coupled to Scpharose43; 3 ml). (A) Sa@pIe was i&ally I@& nmd &@d with 50 m&f phosphate, 0.9% NaC1, 0.01% Tweeti-20, pH 7.6 (loading buffer); @) the protein w&i& i@~&& nonspecificalIy with the cohmm was eltlted with SQn# phosphate, 1 M NaCl, 0.01% Tween-20, pH 7.6; (C) the specifcaily boundprotcin was &u&I with 50 &Gly, 0.9% NaCl, O.Oi% Tween-20, pH 3.0; (D) reequitibration with loa&ngbu&r. Wow rate, 12 mvhr; vd~ of fraction, 1 ml. Fra&oas were e&ectcd in tubes containing 500 ~1 of 100 m&Z Tris-Hdl, pH 7.5.

CHICKEN

GROWTH

HORMONE

high ionic strength buffer. Finally, after the baseline absorbance was close to zero, the low pH elution buffer was applied and the protein which interacted specifically with the column was recovered in a small peak. The material in these fractions was dialyzed exhaustively against water and lyophilized. This constituted the total cGH fraction that was further analyzed by different electrophoresis systems combined with Western ,Blot. Figure 2 shows the results obtained when the cGH-like immunoreactivity obtained from the monoclonal immunosorbent was analyzed by SDS-PAGE and Western blot employing a polyclonal antibody, and compared with a fresh pituitary homogenate. Several immunoreactive cGH-like bands were detected in the serum sample under nonreducing (NR) conditions, with the following apparent molecular weights (MW): 22, 45, 50, 80, 87, and two above 110 kDa. The pituitary extract showed bands at 18, 22, 29, 45, 62, and 80 kDa. Under reducing (R) conditions immunoreactive bands of 16, 22, 26, 51, and 64 kDa were found in the serum fraction, whereas bands of 16, 22, 26,5 1, and 56 kDa appeared to be present in the gland homogenate. As shown some bands are more abundant than others.

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301

These results revealed the size heterogeneity of cGH in chicken blood, and at least for several bands the correspondence with their pituitary counterparts was unequivocal. Charge heterogeneity of cGH in chicken sera was analyzed with isoelectric focusing and Western blot (Fig. 3, lane 3; pl 5.45, 6.2, 6.8,7.1,7.5, and 8.1), and compared to that present in the pituitary homogenate (Fig. 3, lane 2) and in a purified hormone preparation (Fig. 3, lane 1). Several bands present in the gland extract coincide with those found in the circulation (~1 6.2, 6.8, 7.1, 7.5, and 8.1) and in the pure preparation. At least two further immunoreactive bands, more acidic than those in the pituitary, were detected in serum, though in relatively small amounts. This could imply some sort of modification that is not readily present in the gland. Bidimensional analysis confirmed the charge heterogeneity of serum cGH (Fig. 4). Results of 2D-PAGE showed a number of immunoreactive bands with different pls (from 6.2 to 8.1) but similar molecular weight (26 kDa). In addition, an immunore-

6-

(Kj

97.4 66.2

97.4

45.0

66.2 NW MW 45 o

PH

7-

31.0 21.5 14.4

31.0 21.5

9-

14.4 9-

WR)

(RI

2. Analysis of serum and pituitary chicken growth hormone size variants by SDS-PAGE under nonreducing (NR) and reducing (R) conditions, followed by Western blot. (Lanes A and C) Fresh pituitary extract; (lanes B and D) serum fraction containing total cGH isolated by monoclonal immunoa&ity chromatography. The immunostaining was performed with a specific polyclonal antibody a-cGH. FIG.

”

3. Analysis of serum and pituitary chicken growth hormone charge variants by IEF and Western blot. (Lane A) Puritied pituitary cGH (fraction B-DE1, as in Aramburo et al., 1989a); (lane B) fresh pituitary extract; (lane C) serum fraction containing total cGH isolated by monoclonal immunoaffinity chromatography. The immunostaining was performed with a specific polyclonal antibody a-cGH. FIG.

302

MONTIEL,

BERGHMAN,

AND

ARAMBURO

PH

FIG. 4. Analysis of circulating cGH charge heterogeneity by 2D-PAGE and Western blot. The chicken serum fraction containing total cGH isolated by monoclonal immunoaffinity chromatography was separated by charge in the first dimension (IEF) and by size in the second dimension (SDS-PAGE under reducing conditions), and after electrotransferring the bands to immobilon membrane, immunostaining was performed with a specific polyclonal antibody a-cGH.

active spot of -21.5 kDa with a pl = 5.3 was also observed. This resembles previous findings with either fresh pituitary extracts or purified hormone (Aramburo et al., 1990a). An aliquot of the total serum cGH fraction was chromatographed in a column of concanavalin A-Sepharose, to test if the glycosylated variant of cGH was also present in the circulation. Figure 5 shows that most of the protein loaded in the column was not retained by the resin; however, a small amount of protein was eluted with 10 and 100 mM of cx-methylmannoside. Analysis of the material present in both peaks with SDS-PAGE showed them to be similar, and revealed an enriched immunoreactive band of approximately 29 kDa MW, which could correspond to the glycosylated chicken growth hormone (Fig. 6, lane E). This 29-kDa band was absent from the material not bound by the lectin column (Fig. 6, lane D), but was apparent in the fresh pituitary extract when an overloaded sample was analyzed by SDS-PAGE (Fig. 6, lane C). The 29-kDa band was also slightly stained by Con A-HRP (Fig. 6, lane G), thus confirming the presence of carbohydrates in that fraction. DISCUSSION

Growth hormone isoforms have been detected by chromatographic and electropho-

FIG. 5. Isolation of the glycosylated variant of cGH (G-cGH) by affinity chromatography in Con A-Sepharose. The total cGH fraction isolated from c&ken serum by monoclonal immunoaffiity was chromatographed in a Con A-Sepharose cohrmn (2.5 ml). The sample was loaded and nonretained material was eluted with 20 mN Tris-HCl, O-15 M NaCl, I mM MnC12, 1 m&4 CaCl,, pH 7.@(equilibrium b&er). Specificahy bound protein was eluted with (A) 10 mM a-methylmannoside and (B) 100 mM u-methylmannoside in equilibrium buffer. FIow rate, 0.2 ml/mm; vol of fraction, 1 ml.

retie techniques in hormone preparations of et al., various vertebrates (Chrambach 1973; Hart et al., 1984; Kishida et al., 1987). Heterogeneity was initially attributed to the rather harsh conditions of extraction, the presence of impurities, or laboratory artifacts (Kaplan and Grumbach, 1962; Lewis, 1%2). Later on, improved experimental conditions allowed the purification of a less altered hormone. Analysis of fresh pituitary extracts has shown that the molecular heterogeneity of GH is not generated during purification. The heterogeneous nature of pituitary growth hormone is presently well established (Baumann, 1991) althuugh the physiological significance of this heterogeneity is yet not clearly understood. Some authors have ascribed the well known functional diversity of this hormone to its mo;tewufirr hcterogeneity (Lewis, 1984; BaunBnn, 1

CHICKEN

GROWTH

HORMONE

ABCDEFG

FIG. 6. Analysis of glycosylated cGH variant obtained by affinity chromatography. The total cGH fraction isolated from serum by monoclonal immunoaffinity was chromatographed in Con A-Sepharose column and the fractions obtained were analyzed by SDS-PAGE and Western blot. (Lane A) MW markers (Bio-Rad); (lane B) purified pituitary cGH (fraction B-DE-l, as in Aramburo et al., 1989a); (lane C) fresh chicken pituitary extract; (lanes D and F) nonretained fraction on Con A-Sepharose chromatography; (lanes E and G) retained fraction in Con A-Sepharose chromatography. (Lanes B-E) Immunostained with a polyclonal antibody a-cGH; (lanes F-G) stained with Con A coupled to horseradish peroxidase. The migration of monomeric G-cGH is indicated by the arrowhead.

thermore, if growth hormone variants are to exert an effect on target tissues, they should be present in the circulation. Several isoforms of growth hormone (including mass and charge variants) have been detected in mammalian plasma under different physiological conditions (Chawla ef al., 1983). “Big” and “big big” forms have been shown to circulate in human blood (Stolar et al., 1984). Also, the glycosylated variant of GH has been measured in plasma and shown to vary according to physiological fluctuations (Sinha and Jacobsen 1987). Data from other vertebrates are scarce. In chicken, a heterogeneous pattern of GH has been shown in pituitary extracts and in purified hormone preparations (Houston and Goddard, 1988; Aramburo et al., 1989a, 1990a). At least two cGH charge variants revealed differential effects on lipid metabolism in vitro (Aramburo et al., 199Oa). It was relevant to investigate if the

VARIANTS

IN SERUM

303

same pattern of GH heterogeneity is present in the blood of these birds, which would suggest physiological significance. The fraction containing cGH was enriched by immunoaffinity chromatography, since the direct study of GH variants in plasma or serum with electrophoretic systems is readily obstructed by the more abundant blood proteins, and also because the circulating concentrations of the minor GH isoforms were expected to be low. This procedure served on the one hand to eliminate interfering proteins and on the other to increase the relative amount of the cGH variants. The method proved to be specific, fast, easy, and reliable. Sufficient amounts of total cGH were thus obtained from batches of lo-ml serum samples to study their variant composition. The combination of electrophoretic techniques with Western blot and immunostaining allowed identification of mass and charge variants present in chicken serum. The GH-like nature of the analyzed bands was doubly confirmed. They were recognized by two different sets of antibodies: monoclonals in the affinity column and polyclonals in the immunodetection after the Western blot. Different epitopes were probably detected by the different antibodies, but all corresponding to GH. Crossreaction of a non-GH protein with both antibodies is very unlikely, since these are highly specific. Electrophoretic comparison of mass variants from chicken sera and pituitary gland homogenates revealed several immunoreactive bands which were apparently similar. Thus, bands of MW 22,45, and 80 kDa (probably corresponding to monomeric, dimeric, and tetrameric forms, respectively) under nonreducing conditions were found in both samples, and bands of MW 16, 26, 29, 51, and 66 dKa (probably corresponding to a proteolytic fragment, a monomer, a glycosylated form, a reduction resistant dimer, and a higher MW oligomer) under reducing conditions were also found in

304

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BERGHMAN,

both samples. Additionally, a 22-kDa band was found under reducing conditions which could correspond either to an incompletely reduced monomeric form or to a processed form of the monomer. Two further bands were observed in chicken serum under nonreducing conditions, of MW 50 and 87 kDa. These could correspond to complexes of cGH with growth hormone-binding proteins (BP), copuritied during the affinity chromatography step, supposing the recognized hormone epitope had not been hindered by the binding protein. Growth hormone-binding proteins have been reported to occur in chicken plasma (Jones er al., 1990). In the case of hGH-BP, this complex can be fully immunoreactive and is recognized by antihGH antibodies (Baumann et al., 1988, 1990). On the other hand, the observed bands could represent circulating hormone aggregates . Regarding the cGH charge variants, at least five bands from pituitary gland homogenates and serum samples comigrated. When analyzed with IEF, the pls were 8.1, 7.5, 7.1, 6.8, and 6.2. Bidimensional analysis confirmed that cGH in serum consists of several charge variants which have a MW of 26 kDa under reducing conditions. Similar results have been reported for the pituitary gland, when analyzed with the same method (Aramburo et al., 199Oa). Analysis of serum cGH with Con A-Sepharose affinity chromatography showed enrichment of a 29-kDa band, immunoreactive to cGH, which was also slightly stained by Con A-HRP after the Western blot. This band apparently corresponds to the glycosylated variant of cGH (G-cGH), previously reported to occur in the chicken pituitary (Berghman et al., 1987; Aramburo et al., 1991). The difference in staining intensity of this band might be due to a higher affinity of the antibodies for the hormone than of the lectin for the carbohydrate moeity . The high MW band which showed intense lectin staining may

AND

AtiMBURO

correspond to an aggregate of the glycosylated variant, or to a G-cGH-BP complex, since it is recognized by the specific antibody. The possibility that it could be a contaminant from the monoclonal immunoabsorbent is unlikely, since a pure monoclonal antibody control which was run on a separate gel disclosed a different migration pattern (data not shown). The presence of chicken growth hormone variants in circulation suggests that they are released from the pituitary gland, which supports the possibility of their physiological significance. Whether there is differential control over their rate of synthesis, release, and turnover remains to be determined. As to this, some studies have shown that the metabolic clearance rate of the glycosylated variant of porcine prolactin (PRL) is higher than that of the nonglycosylated hormone when injected in rats (Sinha et al., 1991), suggesting that glycosylation may be a modulation mechanism of PRL biological actions at tissue level. Further studies are needed to elucidate the functional significance of growth hormone variants secreted into the bloodflow. ACKNOWLEDGMENTS The authors express their gratitude to Mr. Guillermo Le6n (Procesadora de Aves de Morelos) for his continuous support. We thank Mr. Pedro Medina for his efficient help in the lab and Mr. Jose Avil& for the photographic work. We are grateful to Isabel PerezMonfort for revising and editing the manuscript. J.L.M. had a postgraduate studentship from DGAPA, UNAM. This work was partially supported by Grant Dill-903823 from CONACYT (Mexico).

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Aldmburo, C., Sanchez, R., Fenton, B., Perera, G., and Valverde-R., C. (1989~). Desarrollo de un radioinmunoensayo hom6logo y especifico para la determinaci6n de hormona de crecimiento de poll0 (cGH). Vet Mex 20, 397-405. Ammburo, C., Montiel, J. L., Perera, G., Navarrete, S., and Sanchez, R. (199Oa). Molecular isoforms of chicken growth hormone (cGH). Different bioactivities of cGH charge variants. Gen. Camp. Endocrinol.

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plex of proteins. Recent

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