GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

rowth Hormone

86, ii l-118 (1992)

Binding Sites in Tilapia (Ore mossambicus) Liver

T . B . NG,*'" T. C. LEUNG, *J C.H.K. Departments

of *Biochemistry

GHENG,* ANDN.U.

and tBiology, The Chinese Shatin, N.T., Hong Kong

University

S.

ofHong

Kong,

Accepted July 18, 1991 ‘2SI-labeled bovine and tilapia growth hormones were used to assess the presence of growth hormone receptors in membranes prepared from tissues of the tilapia Oreochromis mossambicus. The highest level of specific binding was detected in liver membranes from animals of both sexes and the binding was protein-dependent. Tilapia growth hormone, bovine growth hormone, and ovine prolactin, but not tilapia prolactin, potently inhibited the hepatic binding of ‘*‘I-labeled bovine growth hormone. Scatchard analysis of the iZ51-labeled bovine growth hormone binding data revealed a B,,, (maximum binding) value of 180 fmol/mg protein and a Kd (dissociation constant) value of 13 nM. Tilapia growth hormone potently inhibited hepatic binding of ‘251-labeled tilapia growth hormone. Scatcbard analysis revealed a single class of binding sites with B,,, and Kd values of 390 fmol/mg protein and 2.5 nM, respectively. Bovine growth hormone and ovine prolactin were less potent while tilapia prolactin was inactive in inhibiting hepatic iZ51-labeled tilapia growth hormone binding. 0 1992 Academic Press, Inc.

The mammalian liver is a major target for growth hormone (GH) action. GH binding sites are also found elsewhere (Hughes and Friesen, 1985; Isaksson et al., 1985), and the effect of GH on cartilage (Isaksson et al., 1982; Lindahl et al., 1986) has been demonstrated. There have been few studies of GH binding sites in fishes. Hepatic binding sites for bovine growth hormone (bGH) have been identified in Gillichthys mirabilis and Acipenser transmontanus but not in tilapia (Tarpey and Nicoll, 1985). Highly purified tilapia growth hormone (tGH) (Farmer et al., 1976) binds hepatic GH receptors in several fish species (Gillichthys, Salmo, Oncorhynchus, and tilapia) (Fryer, 1979a,b). The results of Tarpey and Nicoll (1985) and Fryer (1979a) regarding the presi To whom correspondence should be addressed. ’ Present address: Department of Biological Sciences, Clemson University, Clemson, South Carolina, 29634.

ence of hepatic GH binding sites Hn t~~a~~~ are not in accord, and only Fryer (197%) has examined gills and kidneys for G binding ability. Diverse tillapia tissues examined for their capaci-

D MET Animals. Tilapia (Oreochromis mossambicus) weighing about 100 g and reared in freshwater, were used. Preparation of membranes tilapia. Preparation of tilapia

from

various

organs

of

liver membranes, as well as kidney, muscle, intestine, stomach, gill ~~a~e~~, and heart membranes, was based on the original method for pregnant rabbit liver membranes (Tsushima and Friesen, 1913) as modified by Cheng et al. (1984). All steps were performed at ice-cold temperature. Tilapia tissue (collected from 30 lish) was rinsed in 0.3 M sucrose, blotted dry, and weighed. The tissue was minced and homogenized in about 4 vol of 0.3 M sucrose solution. The homogenate was centrifuged at 10,OOOgfor 10 min, and the resulting supe~at~t was ultracentrifuged at 105,OOOgfor 60 min. The supernatant from the ultracentrifugation step discarded Tris-HC1 and the pellet was rehomogenized in 24 111 00166480192 $1.50 Copyright All rights

0 1992 by Academic Press, Inc. of reprodwtion in any form reserved.

112

NC

ET

buffer (pH 7.2). The protein concentration of the membrane preparation was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. The membrane preparation was stored at - 70” until it was used for bindng studies. Hormones. Tilapia prolactin (tPRL) and tGH were prepared as described by Farmer et al. (1976, 1977). Bovine prolactin (oPRL) and bGH were gifts from the late Professor Choh Hao Li. Radioiodination of growth hormone. The iodination of tGH and bGH was done at room temperature by the lactoperoxidase method (Thorell and Johansson, 1971). For iodination, 5 pg GH was added to 1 mCi Na’*‘I (10 pl) (Amersham), 20 p10.4 M sodium acetate buffer (pH 5.6), and 5 pg lactoperoxidase (Calbiothem) in 10 ~1 acetate buffer. To initiate the iodination, 150 pg H,02 (10 pl) was added with constant flicking. After 1 min, the reaction was stopped by addition of 100 (~1transfer buffer (0.01 M sodium phosphate, 1% potassium iodide, 16% sucrose, 0.1% sodium azide, pH 7.5), followed by 50 ~1 of 5% BSA. A lo-p1 aliquot was used to determine total and specific activities. The iodinated GH was separated from free iz51 by gel filtration on a 1 x 50-cm Sephadex G-100 (Pharmacia) column using an eluting buffer containing 0.01 M sodium phosphate and 0.15 M sodium chloride (pH 7.6). The specific radioactivity of the labeled hormone was determined by the method of trichloroacetic

AL.

acid precipitation as described by Shiu and Friesen (1974). Radioreceptor assays. The lZ51-bGH and lz41-tGH binding studies were done in triplicate following Shiu and Friesen (1974) as modified by Cheng et al. (1984). Membrane preparation containing 1 mg protein (unless otherwise stated) in 100 nl assay buffer (25 mM TrisHCl buffer, pH 7.2, containing 20 mM CaCI, and 0.4% BSA) was incubated with 100,000 cpm iz51-GH (100 ~1) with or without unlabeled GH in a final volume of 500 pl for 16 hr at room temperature (20”). The binding reaction was terminated by dilution with 3 ml ice-cold 25 mM Tris-HCl buffer (pH 7.2) containing 20 m&4 CaCl, and 0.4% BSA. Separation of the free and bound hormone was achieved by centrifugation at 5OOOgfor 60 min at 4”. The supernatant was decanted and the “‘1-GH bound to the membrane-pellet was counted in a Beckmann Gamma~counter.

RESULTS

Tissue Distribution of Specific ‘251-bGH Binding Table 1 shows the specific and nonspecific binding of ‘251-bGH to tissue membranes of male and female tilapia. Among

TABLE BINDING

OF

lz51-bGH

TO TILAPIA

1 TISSUE

MEMBRANES

Male

Organ/tissue

Membrane protein per tube hid

Specific binding (%I

Testis ovary Gill filament Muscle Gas bladder Anterior gut Posterior gut Spleen Kidney Liver Stomach Heart

1000 1000 850* 470* 1000 350* 400* 670* 1000 1000 1000 1000

0.92 f 0.16 0.58 + 0.18 0.11 * 0.13 0.09 + 0.25 0.27 2 0.33 0.56 * 0.11 0.40 k 0.24 0.71 +- 0.04 3.56 rt 0.11 0.99 -c 0.29 0.43 f 0.18

Female Nonspecific binding (“ro) 3.84 f 0.06 3.09 2 0.01 1.78 f 0.28 4.42 Y!Z0.01 1.70 f 0.01 1.52 + 0.02 2.61 f 0.18 4.22 -+ 0.04 10.7 + 0.08 7.50 f 0.05 3.04 k 0.14

Specific binding (%I 0.60 + 0.08 0.27 f 0.21 0.09 k 0.15 0.15 4 0.32 0.65 k 0.35 0.55 k 0.26 0.76 k 0.08 0.81 k 0.17 3.45 2 0.10 0.76 k 0.13 0.18 jl 0.25

Nonspecific binding 6%

3.03 4.09 1.52 4.55 0.99 3.76 2.86 4.61 5.68 7.38 3.83

+ 0.06 +- 0.13 +- 0.02 2 0.13 +- 0.09 k 0.03 + 0.19 f 0.22 2 0.14 + 0.05 k 0.17

Note. The specific and nonspecific binding of lz51-bGH (expressed as percentage of the total radioactivity of ‘*‘I-bGH added per tube) to tilapia tissue membranes was determined as described under Materials and Methods. Less than 1000 pg protein was used in samples marked with asterisks (*) because of the lower protein concentrations of the membrane preparations from these tissues. One milligram protein was used in other samples because of the low binding. One-half to two milligrams protein was used in similar studies on amphibian tissues (White and Nicoll, 1979; White, 1981). Values shown were the means of triplicate determinations.

GH BINDING

SITE IN TILAPIA

FK 1. Effect of membrane protein concentration on binding of IzSI-bGH to tilapia liver membranes. Different amounts of membrane protein were incubated with 100,000 cpm ‘?-bGH at 20” for 16 hr. Specific (SB) and nonspecific (NSB) binding were determined as described under Materials and Methods. Values shown are the means 2 SEM of triplicate detenninations and are expressed as the percentage of the radioactivity of ‘*‘I-bGH added. SEM is not shown in cases where it is smaller than 0.2%.

the tissues examined, liver membranes showed the highest degree of specific binding (around 3.5%) and this was true in both sexes, although nonspecific binding was high (5-10%). Low levels (below 1%) of specific binding were found in membranes prepared from other organs/tissues, again associated with extensive nonspecific bindThe effect of membrane protein concentration on ‘251-bGH binding to tilapia liver membranes is shown in Fig. 1. As the membrane protein concentration increased, the binding of ‘251-bGH to liver membranes increased. The specific binding increased linearly whereas the nonspecific binding increased but with a decreasing slope. Characteristics of 12’1-bGH Binding Tilapia Liver Membranes

to

Addition of increasing amounts of unlabeled bGH resulted in a gradual decrease in binding of ‘251-bGH to tilapia liver membranes, with 50% inhibition at approximately 30 ng unlabeled bGH. oPRL also competed with ‘251-bGH for binding to the

LIVER

liver membranes with a potency sirn~~~~ to that of bGH. tGH showed a tion curve but with a slightly Powe tency, with 50% inhibition at about tGH. tPRL was not potent in i bGH binding even at coracentra 400 ng per tube. Thus bGH, oPRL were potent inhibitors binding to tilapia liver membranes tPRL was a poor inhibitor (F pare the potency of bGH in bGH binding to tilapia liver membranes and to rat liver membranes, an assay of jz5BbGH binding to rat liver membranes was conducted (Fig. 3). Fifty percent i~~ibiti~~ was observed at a much lower level of about 7 ng bGH, whereas oPRL was snl slightly potent, with 50% inhibi above 100 ng oPRL. Scatchard (Scatchard, 11949) of the data of binding to tilapia liver membrane that the maximum binding (B, fmol/mg protein and the d~sso~iati~~ constant (&) was 13 nM (Fig. 4), wbeseas the ‘251-bGH binding sites in rat liver mem-

0.1

I hmunt

10 of cold hormone

Km

(ngjtube)

FIG. 2. Specificity of binding of ‘251-bGH to tilapia liver membranes. One thousand micrograms of membrane protein was incubated with 100,000 cpm of “‘IbGH and increasing amounts of unlabeled bGH, tGH, tPRL, and oPRlL. One hundred percent specific binding represents binding of 3.9% of the radioactivity of ‘251-bGH added in the absence of any unlabeled hormone. Nonspecific binding was estimated as 10% of the radioactivity of lz51-bGH added in the presence of 1000 ng unlabeled bGH. Values represent means t SEM of triplicate determinations. SEM is not shown in cases where it is smaller than I%.

114

NG ET AL.

0

50

Bound Amount

of cold hormone

(ng / Nk)

3. Specificity of binding of lz51-bGH to rat liver membranes. Inhibition of lZSI-bGH binding by increasing amounts of unlabeled bGH and oPRL. One hundred percent specific binding represents binding of 11.2% of the radioactivity of iz51-bGH added in the absence of any unlabeled hormone when 300 pg membrane proteins was used. Nonspecific binding was estimated as 1.7% of the radioactivity of I*?-bGH added in the presence of 1000 ng unlabeled bGH. Values represent means + SEM of triplicate determinations. SEM is not shown in cases where it is smaller than 1%. FIG.

branes had a B,,, value of 600 fmol/mg protein and a Kd value of 2.5 nM (Fig. 5). Characteristics of ‘251-tGH Binding to Tilapia Liver Membranes

1M (fmole/mg

FIG. 5. Scatchard plot of the binding of 1251-bGH to rat liver membranes based on the data in Fig. 3. The Kd and B,, values were, respectively, 2.5 r&f and 600 fmohmg protein.

binding but with a much lower potency when compared to tGH, with 50% inhibition at about 110 ng bGH. oPRL was even less potent, with 50% inhibition well above 1000 ng oPRL. tPRL was practically ineffective in inhibiting the binding of ‘251-tGH. tGH and to a lesser extent bGH were potent inhibitors of ‘251-tGH binding to tilapia liver membranes, whereas oPRL and tPRL were poor inhibitors (Fig. 6). The specific binding of ‘251-tGH to tilapia

Addition of increasing amounts of unlabeled tGH resulted in a gradual decrease in binding of ‘251-tGH. Fifty percent inhibition was observed at approximately 15 ng of unlabeled tGH. bGH also inhibited ‘251-tGH

Y = (2.1 I lO”‘)X

. tGH

+ 0.124 0

Bound (fmole/mg

protein)

4. Scatchard plot of the binding of iz51-bGH to tilapia liver membranes based on the data in Fig. 2. The Kd and B,, values were, respectively, 13 n&4 and 180 fmol/mg protein. FIG.

20

ISO protein)

0.1

. . . ....‘I

1 Amount

. . ..‘.a

10

of cold hormone

100

lo90

@g/tube)

FIG. 6. Specificity of binding of 1251-tGH to tilapia liver membranes. Inhibition of ‘*?-tGH binding by increasing amounts of unlabeled tGH, tPRL, oPRL, and bGH. One hundred percent specific binding represents binding of 9.5% of the radioactivity of ‘*-?-tGH added in the absence of any unlabeled hormone when 300 ug membrane protein was used. Nonspecific binding was estimated as 12.1% of the radioactivity of rz51-tGH added in the presence of 1000 ng unlabeled tGH. Values represent means 5 SEM of triplicate determinations. SEM is not shown in cases where it is smaller than 1%.

GH BINDING

SITE IN TILAPIA

liver membranes (300 p,g protein) was 9.8% of the added radioactivity. When lz51-bGH was used as the ligand, the specific binding was about 3.5%. Scatchard analysis suggested a single class of ‘251-tGH binding sites on tilapia liver membranes. The apparent dissociation constant (&) and the maximum binding (B,,,) were 2.5 nA4 and 390 fmol/mg protein, respectively (Fig. 7). DISCUSSION

Two forms of growth hormone and prolactin have been isolated from chum salmon pituitaries (Kawauchi et al., 1986; Yasuda et al., 1986), and two forms of growth hormone have been purified from eel pituitaries (Kishida et al., 1987). The two forms of chum salmon growth hormone have identical molecular weights but exhibit differences in isoelectric points, N-terminal amino acid residues, and amino acid composition, indicating that they are genetic variants coded on two separate genes (Kawauchi et al., 1986). The two forms of eel growth hormone have different isoelectric points but similar molecular weights, amino acid compositions, and biological potencies. The N-terminal amino acid residues l-36 of one form are identical to 4-39 of the other form (Kishida et al., 1987). The two forms of salmon prolactin differ in only 4 of 187 amino acid residues (Yasuda et d., 0.12,

I Y = (2.4 x 10-4)X + 0.092

.,I

0

50

100 Bound

250 150 200 (fmole/mg protein)

350

400

FIG. 7. Scatchard plot of the binding of ‘*‘I-tGH to tilapia liver membranes based on data in Fig. 6. The Kd and B,, values were, respectively, 2.5 nM and 390 fmolkng protein.

115

s of 1986). In tilapia pituitaries two prolactin (Specker et al., 19SS) but one form of growth hormone (Farmer et al., 1976; Specker et al., 1985) are el The two salmon prolactins differ i tric points, molecular weights, and 5 of t first N-terminal amino acid resi both are biologically active (Spec 1985). Whether the different isofo hormone demonstrate difference ing to various tissues is not known theless, the esent investigation is not complicated the presence of isofor tilapia growth hormone. In both female and male tilapia, t cific ‘2SI-bGH binding in vario was very low (below 1%) e (3.5%). The data suggest that bi for bGH were absent from the alimentary canal, gill filaments, gas bladder, muscle, spleen, that the liver was the major t liver membranes exhibited ‘251-tGH binding and that kidney membranes showed

(Leung et al., 1991) such and reduction of hepatic appeared to be due to an mone on the liver. The somatom acts on the liver to produ which mediate the biologi tilapia

3cQ

LIVER

remains to be elucidated.

membranes was only about 3.5% of the added radioactivity, much lower t nonspecific binding which was as 3.610.7%. This may be due t nonhomologous hormone (b tGH as the ligand in the radi since a specific binding of appr~xirn~~~~ 10% was achieved by using ‘251-~ab~~~ tGH instead, In view of the low ~~~~i~~~ 1 mg tissue membrane protein per assay tube

116

NG

was used in radioreceptor assays in the present study. The same amount of protein was used in studies on mammalian (Marshall et al., 1978) and amphibian (White and Nicoll, 1979; White, 1981; White et al., 1981) tissues. In both sexes of tilapia the liver was the major 1251-bGH binding tissue. There was no significant difference in the distribution of GH binding sites between male and female tilapia. Results of the present investigation are in contrast to those of Tarpey and Nicoll (1985), who demonstrated that tilapia liver membranes did not bind 1251-bGH. The reason for the discrepancy is unknown. However, the observation of “‘1-bGH binding sites is consistent with the demonstration of 1251-tGH binding sites in tilapia liver in the present study as well as in the study of Fryer (1979a). The binding site for 1251-bGH on tilapia liver membranes had a B,,, of 180 fmol/mg protein and a Kd of 13 I%, whereas the binding site for 1251-bGH on rat liver membranes had a B,, of 600 fmol/mg protein and a Kd of 2.5 IS. The binding characteristics of GH receptors on rat liver membranes were comparable to those of labeled human growth hormone (hGH) binding to rabbit liver membranes with a B,,, of 1300 fmol/mg and Kd of 1.1 I&! (Tsushima, 1980) and the binding of 1251-bGH to solubilized rabbit hepatic GH receptors with a B,,, of 219 fmol/mg and a Kd of 1.4 ti (Herington and Veith, 1977). The tilapia hepatic GH binding site thus appeared to have a relatively low affinity and binding capacity for radioiodinated mammalian GH compared with rat and rabbit liver membranes. It was most probably due to the heterologous nature of the mammalian GH ligand because in the case of the homologous assay system, the binding capacity of 1251-tGH to tilapia liver membranes was much higher ULX = 390 fmol/mg) and the affinity was also higher (Kd = 2.5 nM) than the corresponding data obtained by using 1251-bGH as the ligand. The B,,, value was compa-

ET AL.

rable in order of magnitude to that (125 fmol/mg) obtained by Fryer (1979a) in a similar binding study using 1251-tGH and tilapia liver membranes, but there was a difference in Kd (0.067 1144). It is important to note that when 1251bGH was used as the ligand, the rat hepatic GH receptor could distinguish bGH from oPRL readily. However, the tilapia hepatic GH receptor could not distinguish between bGH or oPRL, although it could differentiate tPRL from tGH. The potency of tGH in inhibiting 1251-bGH binding to tilapia liver membranes was slightly lower than that of bGH. Despite the fact that tPRL shares structural features with both tGH and bGH and has some activity in the rat tibia bioassay (Farmer et al., 1977), a high degree of specificity of binding of tGH to the tilapia hepatic GH receptor was observed in the present study. The seemingly anomalous behavior of oPRL may have been due to its heterologous nature since the experiment was performed twice and the results were reproducible. Edery et al. (1984) also noted that hGH was only slightly less potent than oPRL in inhibiting ‘251-labeled oPRL binding to tilapia liver membranes. When 1251-tGH was used as the ligand, tilapia liver membranes were able to differentiate bGH, oPRL, and tPRL from tGH. Although bGH could slightly inhibit 12’1tGH binding, tilapia liver membranes were able to differentiate tGH from the mammalian GH readily. Results of this specificity study were similar to those of Fryer (1979a). The present study illustrates the necessity of using a homologous label for radioreceptor assay. The affinity, capacity, and specificity of binding of the assay may be altered to a certain extent when a heterologous instead of a homologous label is used. The present data suggest the existence of separate binding sites for PRL and GH in tilapia liver, because tPRL was much less potent than tGH in inhibiting the binding of

GH BINDING

SITE IN TILAPIA

1251-bGH or 1251-tGH to tilapia liver membranes. Edery et al. (1984) observed that bGH was much less potent than tPRL and oPRL in inhibiting the binding of 12?-oPRL to tilapia liver membranes. Fryer (1979b) showed that tilapia renal but not hepatic membranes were capable of binding 1251L and that tGH was much less potent than tPRL in inhibiting the binding of ‘251tPRL to tilapia kidney membranes. These data overall are consistent with the presence of distinct PRL and GH receptors in tilapia Biver. ACKNOWLEDGMENTS

REFERENCES Cheng, C. I-I. K.: Tsim, K. W. K., Wai, M K., and Pak, R. C. K. (1984). Rat hepatic prolactin receptors: Pubertal exposure to sex steroids alters responsivity to testosterone. Znt. J. Pept. Prot. Res. 23, 521-527. Edery, M., Young, G., Bern, II. A., and Steiny, S. (1984). Prolactin receptors in tilapia (Sarotherodon mossambicus) tissues: Binding studies using “SI-oPRL ovine prolactin. Gen. Comp. Endo56, 19-23.

Farmer, S. W.. Papkoff, II., Hayashida, T., Bewley, T. A., Bern, II. A., and Li, C. H. (1976). Puritication and properties of teleost growth hormone. Gen. Comp. Endocrinol. 30, 91-100. Farmer, S. W., Papkoff, H., Bewley, T. A., Hayashida, T., Nishioka, R. S., Bern, H. A., and Li, C. II. (1977). Purification and properties of teleost prolactin. Gen. Comp. Endocrinol. 31, 60-71. Fryer, 9. N. (1979a). A radioreceptor assay for purified teleost growth hormone. Gen. Comp. Endocrinol.

39, 123-130.

Fryer, J. N. (1979b). Prolactin-binding (Sarotkerodon mossambicus) Comp. Endocrinol39, 397-403.

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Herington, A. C., and Veith, N. (1977). Solubilization of a growth hormone-specific receptor from rabbit liver. Endocrinology 101, 984-987. Hughes, J. P., and Friesen, H. G. (1985). The nature and regulation of the receptors for pituitary growth hormone. Annu. Rev. Physiol. 47, 46948%.

HE7

Isaksson, 0. G. P., Jansson, .I. G., and Gause, I. A. M. (1982). Growth hormone stimulates Eongitudinal bone growth directly. Science 216, I2371239. Isaksson, 0. G. P., Eden. S., and Jansson, J. 0. (1985). Mode of action of pituitary growth hormone on target cells. Anmr. Rev. Physiol. 4?,483499. Kawauchi. II., Moriyama, S., Yasuda, A., Yamaguchi, K., Shirahata, K., Kubota, J., and Hirano, T. (1986). Isolation and characterization of chum salmon growth hormone. Arch. Biochem. Biophys. 244, 542-555. Kishida, M., Hirano, T., Kubota, J., Hasegawa, S.: Kawauchi, I-I., Yamaguchi, K., and Shirahata, K. (1987). Isolation of two forms of growth hormone secreted from eel pituitaries in vitro. Gen. Camp. Endocrinol.

The authors are grateful to Professor Harold Papkoff of the Hormone Research Institute, University of California at San Francisco for his generous gift of tilapia growth hormone and its antiserum. We also thank him for his critical appraisal of the manuscript.

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Yasuda, A., Itoh, H., and Kawauchi, H. (1986). Primary structure of chum salmon prolactins: Occurrence of highly conserved regions. Arch. Biothem. Biophys. 244, 528-541.