Partial purification of parathyrin from the corpuscles of Stannius (PCS) of the eel (Anguilla anguilla, L.)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

76, 83-94 (1989)

Partial Purification of Parathyrin from the Corpuscles (PCS) of the Eel (Anguilla anguilla, L.) CHRISTIAN MILET,ELIZABETH Laboratoire

de Physiologic

MARTELLY,ANDEVELYAE

G&&-ale et Compart!e du MusCum National d’Histoire CNRS, 7 Rue Cuvier, 75231 Paris Cedex 05, France

of Stannius

LOPEZ Naturelle;

U.A.

90

Accepted January 12, 1989 It has been previously shown that the eel corpuscles of Stannius (CS) synthesize and secrete a substance (PCS) which is functionally and immunologically related to the mammalian parathyrin family. Purification of PCS, including anion-exchange chromatography, ODS C-18 reverse-phase HPLC, and affinity chromatography, showed that a biologically active peak, eluted in 32% acetonitrile, contains a 32- to 34-kDa protein which is 600-fold more potent than the crude extract is a test involving the hypocalcemic response in the CS-deprived eel. Specific immunoprecipitation of protein encoded by mRNA extracted from eel CS indicates that a 4S-kDa precursor is involved in PCS synthesis. The hypothetical significance of a “large” parathyrin-like molecule in fish is suggested in relation to what is known about mammalian parathyrin gene expression. 8 1989 Academic press, hc.

Surgical removal of the corpuscles of Stannius (CS) in eel leads to a very marked hypercalcemia (Fontaine, 1964). Since the first report showing that the gill may be the main target organ for the CS hormone (Fontaine et al., 1972), this result is now well established (Ma and Copp, 1978; Milet et al., 1979a), but until recently very little was known about the hormonal chemistry of the CS-active principle. First, it has been reported that a 39-kDa glycoprotein extracted from salmon CS inhibits branchial calcium uptake in juvenile rainbow trout (Wagner et al., 1986). Second, a 54-kDa glycoprotein that is released in response to increased plasma calcium levels has been identified in trout CS (Lafeber et al., 1988). Last, the nucleotide sequence of a main protein of Australian eel CS has been reported (Butkus et al., 1987). We obtained surprising data when CS extracts were injected into rat. Hypercalcemia and an osteoclastic resorption of the femoral periosteal area were observed (Milet et al., 1979b), and such a parathyrin-like effect of the CS extract on embryonic mouse bone in culture has been confirmed

(Lafeber et al., 1986). We have also demonstrated an immunological parathyrin-like substance (i-PTH) in these glands. The CS synthesize and secrete (Milet et al., 1980; 1980; Lopez et al., 1984) an i-PTH substance, detected with five different antisera, the plasma levels of which are related to the calcium metabolism status of the eel. These data were obtained by radioimmunoassay and immunocytological studies using an antiserum obtained against l-84 bPTH in guinea pig and directed mainly against the amino-terminal region of the molecule (Milet et al., 1982). Following these findings, a preliminary purification attempt using Sephadex G-50 filtration and reverse-phase ODS C-18 HPLC chromatography with a linear methanol gradient was unsuccessful, as multiple fragments with biological activity were eluted. Subsequently, we employed an acetonitrile gradient instead of a methanol gradient after prechromatography on an anion-exchange resin and incubation with anti-bPTH antiserum. Affinity chromatography was also used during the study. Translated products encoded by mRNA extracted from eel CS were studied by immu83 0016X1480/89 $1.50 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

84

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MARTELLY,

noprecipitation and compared to the labeled products synthesized during in vitro incubation of the glands in the presence of [35S]methionine. MATERIALS

AND METHODS

Preparation of Extracts for Chromatography

and Samples

Saline extracts were prepared by homogenizing fresh eel glands in 0.1 M NaCl in a potter with a Teflon pestle and centrifuged for 15 min at 5000 g. The supernatant was dialyzed using immersible CX 10 ultratilters (Millipore), and the remaining solution was lyophilized and dissolved in eluant buffer for anionexchange chromatography. Fractions eluted from Sephadex A-25 were submitted to the eel gut alkaline phosphatase (pNPPase) assay. The active peak was divided into two equal parts and freeze-dried. Both parts were chromatographed on Sephadex G-50 (85.5 x 1.6~cm column), the first without incubation with anti-bPTH antiserum and the second after an overnight incubation with anti-bPTH antiserum (l/200) in 0.05 M barbitone buffer pH 8.6. Every fraction from these two G-50 gel filtrations was analyzed by the pNPPase bioassay. The antiserum used in all work was one of the five different antisera tested for showing the iPTH in eel plasma. It was obtained against bPTH in guinea pig and directed mainly against the amino-terminal part of the molecule (Milet et al., 1982). Fractions of the biologically active peak (Nos. 8-14) from another run of CS extract on anion-exchange chromatography were lyophilized and labeled with lz51 according to the Iodogen method (Salacinsky et al., 1981); the excess iz51 was removed by Sephadex G-25 gel filtration. The resulting labeled peak was then incubated for 3 days at 4°C in 0.05 M barbitone buffer, pH 8.6, containing the antiserum (l/200) and protease inhibitor (Zymofren, Specia). The bound labeled proteins were separated from free labeled proteins by gel filtration on Sephadex G-50 (85.5 X 1.6~cm column) and incubated overnight in barbitone buffer either with 50 pg synthetic l-34 hPTH (Beckman) or with saline extract of CS corresponding to 12.5 mg of fresh glands. Displaced labeled proteins from antiserum (free) were separated from nondisplaced (bound) by G-50 Iiltration and loaded on a ODS C-18 HPLC column in water.

Chromatography Sephadex G-50 superfine was used in a 60

x

l-cm

AND

LOPEZ

column with 0.1 M formic acid, 10% isopropanol as eluant. Anion-exchange chromatography. Sephadex A-25 resin was used in a 0.8 x 25-cm column with 0.2 M acetate, pH 4.8, as buffer in isocratic conditions. HPLC ODS. A reverse-phase hydrophobic HPLC column, Cl8 octadecasilyltrichlorosilane, 5 pm (ODS C18, Spherisorb S5 ODS2, Prolabo), 0.5 x 15 cm, was used under the following conditions. In order to determine whether this chromatography step would be useful, a first run of a saline extract was performed. Five hundred milligrams of frozen glands was freezecrushed in liquid nitrogen using a dismembranator (SPEX 6700) suspended in 0.1 M NaCl and centrifuged for 15 min at 5000 rpm. The supematant was collected and the pellet submitted to another suspension and centrifugation under the same conditions. The two supematants were pooled, diluted to 100 pg/lOO ~1 of 0.1 M NaCI, and loaded on the ODS C-18 column three times. The retained material (called 1 + 2) was eluted with methanol, dried, reconstituted in 0.1 M NaCl(lO0 p&ml), and submitted to bioassays. Samples (0.1 ml) from the experiment using labeled products removed from the antiserum by 1-34 hPTH or saline CS extract were loaded twice in water (HPLC grade, Fisons, England) and the nonretained material (preeluting material PEM) was collected. Then, a 0 to 80% acetonitrile (Prolabo, HPLC grade)/water linear gradient (40 min) was applied at a flow rate of 1 mYmin (I-min fractions). The material eluted after 80% CH,CN (late eluting material, LEM) was also collected. Labeled 1-84 bPTH and 1-34 hPTH were used as internal markers. Affinity chromatography. Cyanogen bromideactivated Sepharose (Sigma) was used with antiserum obtained in guinea pig against l-84 bPTH as ligand. Saline-dialyzed extract of CS (25 mg (w/w)/200 p,I 0.1 M NaCI) was applied twice in 0.2 M carbonate bicarbonate buffer, pH 9,0.5 M NaCl. The bound material was eluted with 0.2 M acetate buffer, pH 4, 0.5 M NaCI. The material was then freeze-dried and reconstituted in loading buffer for SDS-polyacrylamide gel electrophoresis.

Polyaqlamide Gel Electrophoresis (PAGE) A 10 to 20% acrylamide/bisacrylamide gel (stacking gel 5%) was employed with 0.05 M Tris-glycine, pH 8.6, 10% SDS as electrophoresis buffer. The samples were dissolved in 0.05 M Tris pH 6.8, 12% glycerol, 12% SDS, 1.2% B-mercaptoethanol, boiled for 3 min, and cooled before application. Calibration was performed using a calibration kit (Pharmacia) containing 94-, 67-, 43-, 30-, 20.1-, and 14.4~kDa proteins. After fuation by methanol/acetic acid and ghitaraldehyde,

FISH

PTH-LIKE

HORMONE

gels were stained according to the silver method (Morrissey, 1981).

Bioassays The biological activity of the preparations was measured by their potency to stimulate eel gut alkaline phosphatase activity in vitro, by their bone-resorbing activity measured in fetal long bone calcium release in vitro (Milet et al., 1979b), or by their hypocalcemic potency measured in CS-deprived eels previously catheterized on the day of CS removal. Their content of iPTH was also determined. The alkaline phosphatase activity (orthophosphoricmonoester hydrolase, EC 3.1.3.1) from homogenate of eel gut mucosal cells, diluted l/50 in 0.1 M phosphate buffer, pH 7.4, 0.4 M sucrose, was measured with 2 n&f paranitrophenylphosphate (pNPP) in 0.2 M carbonate/bicarbonate buffer, pH 10, as substrate (Babson et nl., 1966). The photometric absorption of the hydrolysis product was measured at 400 nm (Beckman, Model 24). Standardization was performed with a Versatols (General diagnostics, France) VEN 17 IU, Ven 90 IU, 4H76 batch. Stimulation of the pNPPase activity by CS preparations was measured after the gut cell homogenates were preincubated, 100 PI/tube, for 3 hr at 15” with 100 I of the different preparations (i.e., dialyzed saline extract, fraction from the Sephadex G50 filtration and ODS eluate called 1+2) and then incubated 30 min with 0.5 ml of substrate. Catheterization of the eels involved inserting a PE50 tube in the aortic bulb, secured to tissue with Histoacryl blue (Braun, Melsungen). The pericardium, muscle, and skin were then sutured successively. This operation was suitable for injecting preparations and collecting blood samples without excessive stress for the animals. Injections of preparations (0.2 ml/ injection) began on Day 7 after the CS removal, and blood was collected every 2 hr (1000 to 1800 hr) the first day and then at 1000 hr the following days. Total plasma calcium was measured by atomic absorption spectrophotometry (Perkin-Elmer, Model 403) after dilution of samples in 0.5% La&.

Incorporation in Vitro

of Labeled Methionine

Six incubations of fresh CS (20 mg) taken from normal eels (250 2 50 g) were performed in Eagle’s MEM supplemented with amino acids and vitamins, containing 50 pCi [35S]methionine. Glands were cut in two parts and the connective tissue was removed. After a 6 hr incubation at 15” the glands were separated from the incubation medium and frozen at - 20”. They were extracted as described above and the supematant was submitted to polyacrylamide gel electrophoresis.

85

PURIFICATION

RNA Extraction, Cell-Free Translation, and Immunoprecipitation of Translational Products Fresh glands were collected from eel and immediately frozen and stored until RNA extraction in liquid nitrogen. RNA extraction was performed according to Glisin et al. (1974) using 6 M guanidine thiocyanate and ultracentrifugation (110,000g) through a 5.7 M cesium chloride cushion. The total RNA fraction was translated in the rabbit reticulocytes lysate cell-free translation system (NEN, NEK 001) with [3sS]methionine (1064 Ci/mmol) as labeled amino acid (Pelham and Jackson. 1976). The mixtures were incubated at 37” for 60 min. Immunoprecipitation of the products synthesized in the cell-free system was performed under the following conditions. Twenty microliters of lysate was incubated for 24 hr either with 50 p,l of the antiserum previously used, diluted 1110 in barbitone buffer, or with the same antiserum solution but preincubated for 2 days with 50 ug of synthetic 1-34 hPTH or with 30 p.1of puritied hormonal product (PCS ODS C-18). One hundred microliters of the second antibody [anti-guinea pig IgG obtained in rabbit (Wellcome RD 18) diluted to l/10] was added and after a further incubation of 24 hr the reaction mixture was centrifuged at 5OOOgfor 20 min using a Sigma 2 MK microcentrifuge. All incubations were performed at 4”. The pellet was dissolved in the PAGE loading buffer and processed as described. Calibration of the electrophoretic mobility was performed using the BRL 6020 SA product containing 14C-labeled proteins. Gels were impregnated with EdHance (NEN), dried with a Hoefer SE 540 slab gel dryer, and exposed to Kodak XOmat AR X-ray film.

Numerical and Statistical Analysis Because the protocols of measurement of the hypocalcemic activity of the different preparations were identical, we can compare the areas under the x axis of the curves which represent their total hypocalcemic potency. Calculated by numerical integration (Ralston and Wilf, 1967) using a Hewlett-Packard Model 20 computer, these areas are corrected according to body weight, protein content, and 1 hr unit of time, and the percentage of change obtained is expressed as calcium content change. Statistical evaluation was performed using Student’s t test; significance was accepted at PK 0.05. Mean values 2 SEM are given.

RESULTS pNPPase Activity

The pNPPase

activity

measured

in eel

86

MILET,

MARTELLY,

gut mucosal cell homogenate 1 week after CS removal was reduced and was half the control value (Table 1). The saline extract of CS in vitro strongly increased this activity, whereas several tissue extracts such as muscle, liver, and kidney (data not shown) elicited no response. The dose-response curve for the relation between the pNPPase activity and the logarithm of CS extract is shown in Fig. 1. The second part of the curve, i.e., between 1 and 10 mg of CS wet weight, was used for the bioassay. Chromatography

and Bioassays

Gel filtration on Sephadex G-50 of saline extract from 0.5 g of eel CS showed that all the three bioactivities were eluted in fractions 10, 11, and 12, that is, all gave retention coefficients (V&J,) of 1.85 (Fig. 2). Comparison of the bioactivities of the saline extract of CS with those of fraction 11 from the G-50 filtration, as a function of the protein content in each samples, showed that the mean bioactivity of fraction 11 was 50 times higher than that of the saline extract in the three bioassays, while preparation 1 + 2, from the preliminary experiment with the ODS C-18 column, exhibited bioactivities similar to those of Sephadex fraction 11 (Fig. 3). TABLE ALKALINE

1

PHOSPHATASE ACTIVITY GUT HOMOGENATES

Eel gut homogenate from Controls CS deprived (7 days) CS deprived (7 days) + saline extract of CS in vitro (2 mg/O. 1 ml)

IN EEL

pNPPase activity (u&f substrate/mitt/g gut homogenate) 2.70 2 0.55 (5)a 1.26 * 0.16 (9)b 8.04 f 1.75 (7)

Note. Effects of removal of CS (CSX) and of in virro incubation with saline extracts of eel CS (2 m&O. 1 ml). o Means k SEM are given, with number of animals in parentheses. b Statistically different from controls values. ’ Statistically different from CSX values.

AND

LOPEZ

The total activity recovered from 0.5 g of CS in fractions 10, 11, and 12 from G-50 filtration was 45 ng equivalent PTH measured by radioimmunoassay and represents a 60% yield, as the iPTH content of the saline extract of CS is 150 rig/g (Milet et al., 1982). Anion-exchange chromatography of the saline extract of CS on Sephadex A-25 revealed that bioactivity was not retained on the column and was eluted just after the void volume (Fig. 4a). Figure 4c shows that approximately 30% of the pNPPase stimulation activity was eluted in the highmolecular-weight region of the Sephadex G-50 chromatography after overnight incubation with the anti-bPTH antiserum. Fractions 8 to 14 of another run of the same amount of CS extract on the anionexchange column were lyophilized and the proteins, labeled with lz51, were incubated with anti-bPTH antiserum (see Materials and Methods). After separation of the bound labeled material from unbound by Sephadex G-50 filtration, the bound proteins were displaced from the antibody either by l-34 hPTH or by saline extract of CS. Another gel filtration on Sephadex G50 separated the displaced labeled proteins from the proteins retained on the antibody. Proteins specifically displaced by l-34 hPTH or saline extracts of CS were applied separately to the ODS HPLC column. In both cases, a radioactive peak was eluted at 18 + 2.25 ml (four different runs for each preparation), corresponding to 32.25 +1.03% acetonitrile (Figs. 5a and 5b). 1-84 bPTH and 1-34 hPTH were eluted in the system for 35 and 45% acetonitrile, respectively. The products displaced from the antibody by the saline extract of CS represented a threefold higher amount of radioactivity than the l-34 hPTH displaced products peak. The relative hypocalcemic potencies of the saline extract of CS and of preparations obtained by anion-exchange chromatography and reverse-phase HPLC are shown in

FISH PTH-LIKE )I M substrat

/min/g

HORMONE

87

PURIFICATION

gut

1

8-

8.

51, 0 0.1

02

0.4

2

1

4

10

mg CS[wwJ FIG. 1. Relationship between the stimulation of eel gut pNPPase activity and the amount of eel CS extracts during in vitro incubations. Each point represents the mean of five determinations, and three assays have been performed for each CS extract dilution.

Fig. 6. The effects of PEM and LEM are represented as control experiments that indicate the increase in plasma calcium after removal of CS in eel. The calculations of the relative hypocalcemic potency of the preparations (see Material and Methods, numerical and statistical analysis) gave, respectively 67.5 for saline extract of CS, 1857 for the anion-exchange preparation,

and 42,000 mg of Wmg protein&r for the HPLC reverse-phase preparation. Thus, the latest preparation was more than 600fold more potent than the saline starting extract. PAGE Silver

staining

of the proteinaceous

FIG. 2. Elution of bioactivities of saline extracts of CS during Sephadex G-50 (Superfine) chromatography.

MILET,

MARTELLY,

AND

LOPEZ

pp irPltt/l~o~t

pp protrin

/loopt

%I5 51 ruloosb/loopl l ECS

30.

%stimelrtira

pl?Pasr~oo

pt 'ECS

100.

1.2 fll.

I 50.

/

.d

i i

.

.

A

.

l

/

OS i

10

. ' v,To--~~ 100

mt@in

/too

pl

FIG. 3. Relationship between the bioactivities of the CS preparations and their protein content: ECS, CS crude extract; 1 + 2, pooled supematants of ODS C-18 separation; 11, fraction of Sephadex G-50 column.

bands (Fig. 7) revealed that the main purified product of successive anion-exchange chromatography and HPLC had an apparent M, of 32-34 kDa. The main protein eluted from the affinity column (i.e., the protein purified using the anti-bPTH antiserum) has the same electrophoretic mobility (Fig. 7e). Fluorography

of the Labeled Products

Labeled methionine

was incorporated

in

three main proteins as shown by autoradiography (Fig. 8b). The most abundant protein has a A4, of 45 kDa, whereas two minor bands appeared with an relative mobility of 43 and 70 kDa, respectively. Cell-Free Translation and Immunoprecipitations

RNA isolated from the CS encoded for two major proteins of M, 45 and 80 kDa (Fig. 8~). These two proteins were clearly

FISH PTH-LIKE

HORMONE

89

PURIFICATION

eDIt

234

I.

FIG. 4. Elution curve of pNPPase stimulation activity during (a) anion-exchange chromatography of CS saline extract using 0.2 M acetate, pH 4.8, as buffer under isocratic conditions, flow rate 0.2 ml/min; (b) Sephadex G-50 filtration of half the A-25 bioactive peak; (c) Sephadex G-50 permeation of the second half of the A-25 bioactive peak incubated with anti-b-PTH antiserum (- - -). Labeled synthetic 1-34 hPTH was introduced in the A-25 bioactive peak before the incubation.’

precipitated by the antiserum against bPTH (Fig. 8d) together with another protein with an apparent electrophoretic mobility of 1415 kDa. Previous incubation of the antiserum with either l-34 hPTH or purified PCS resulted in the absence of the 45kDa band (Figs. Be and 8f) and in a large decrease in amounts of both the BO-and the 15kDa protein bands.

DISCUSSION Alkaline Phosphatase @NPPase) Activify

In mammals, alkaline phosphatase is present in many tissues. In bone (Mac Partlin et al., 1978) and gut (Birge and Gilbert, 1974), its activity appears to be parathyrindependent. Parathyroidectomy in rat re-

90

MILET,

MARTELLY,

AND LOPEZ

lo.3

clution

volume

[ml]

duti0a

1011me [ml,]

FIG. 5. Elution curves, on the ODS C-18 HPLC column, of labeled CS products which have been bound to anti-bPTH antiserum and displaced (a) by saline CS extracts and (b) by the synthetic l-34 hPTH fragment (solid line), optical density at 254 nm, “‘1 radioactivity (0). Positions of elution of l-84 bPTH and l-34 hPTH are indicated by arrows.

sults in a decrease in gut Ca transport, and parathyrin injections correct both Ca transport and pNPPase activity (Birge and Gilbert, 1974). Our results for the eel show that CS deprivation induces a decrease in gut pNPPase activity and that CS extracts increase this activity in vitro. It has been

previously shown that gut Ca transport is decreased after CS removal in eel (Chat-tier et al., 1983). These results can be considered as one more example of the resemblance between the biological effects of mammalian parathyrin and the biological effects of PCS.

PEN ,/AEM ------A------/---

---4

_/---

r!trt t, 0 12 i4 1‘ 48 FIG. 6. Variations (in percentage of the basal value) of the plasma calcium level of catheterized CSX eels after injections of CS, the saline extract of CS: A-25, the active fraction from anion-exchange chromatography; ODS, the active fraction from the ODS C-18 HPLC column; PEM and LEM, preand late eluting material from the ODS C-18 HPLC column (see Figs. 5a and Sb). Each point represents the mean of values obtained with three different eels.

FISH

PTH-LIKE

HORMONE

FIG. 7. Silver staining of polyacrylamide gel electrophoresis (10 to 20% SDS) of (a) molecular weight markers (LMW Pharmacia); (b) saline extract of CS (25 mg w/w); (c) active fraction of the anion-exchange (A-25) chromatography (7.5 ug of proteins); (d) active fraction from the ODS C-18 HPLC column (5 ug of proteins); (e) products eluted from the affinity column (10 ug proteins).

Chromatography

and Bioassays

PCS, as measured by each of the three different bioassays, is eluted in the same fractions on a G-50 gel permeation column. Comparisons of the three assays show that half of the maximal response is obtained for 10 l&100 ~1 of protein of G-50 fraction 11 in B

a

be

d

of

. 001

FIG. 8. Fluorograms of the PAGE (10 to 20% SDS) of (a) molecular weight markers (BRL 6020 SA); (b) saline extracts of CS incubated with labeled methionine; (c) reticulocytes lysate (10 ~1) after translation of the total RNA extracted from the CS; (d) immunoprecipitate of the translated products, obtained with antibPTH antiserum (1110); (e)immunoprecipitate of the translated products, obtained with the anti-bPTH antiserum preincubated with 50 ug of synthetic l-34 hPTH; (f) immunoprecipitate of the translated products, obtained with the anti-bPTH antiserum preincubated with the ODS C-18 HPLC active fraction (10 pg protein).

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91

both the radioimmunoassay and the boneresorption assay. Ten milligrams of saline extract of eel CS (w/w) corresponds to 1.3 kg PTH in radioimmunoassay, close to the value of 0.7 t&IO mg of rainbow trout CS as measured by the mouse calvaria assay (Lafeber et al., 1986). The PCS, as measured by pNPPase activity stimulation, is not retained on an anion-exchange column. Incubation of PCS with anti-bPTH antiserum results in elution of the activity in the high-molecular-weight region of Sephadex G-50 chromatography, where the 1251-labeled l-34 hPTH bound to the antibody is also eluted. Hydrophobic reverse-phase chromatography of CS-labeled material bound to anti1-84 bPTH antibody and displaced either by 1.34 hPTH or by CS extract shows that a radioactive peak is eluted in 32% acetonitrile, that is, slightly before l-84 bPTH. The hydrophobic vs hydrophilic amino acid ratio of the PCS is likely to be close to those of the two parathyrin fragments. Measurement of the hypocalcemic potency of the three preparations, CS crude extract, A-25 peak, and ODS HPLC, reveals that ODS HPLC exhibits the same activity as the crude extract but with a 600fold lower protein content. This bioassay seems more directly comparable to the endocrine function of the CS originally demonstrated (i.e., hypocalcemic activity: Fontaine, 1964; Kenyon et al., 1980) than the other in vitro bioassays such as fetal bone calcium release or stimulation of eel gut pNPPase activity. Nevertheless, information given by these bioassays during the purification steps do not conflict with the in vivo tests. PAGE The electrophoretic mobility of the protein contained in the purified preparations was 32-34 kDa. This apparent molecular weight is quite different from that of teleocalcin (Ma and Copp, 1978) and mammalian

92

MILET,

parathyrin, but is in agreement data (Wendelaar Bonga et al., ner et al., 1986; Butkus et al., ber et al., 1988) on the product ostean CS. Biosynthesis

MARTELLY,

with recent 1985; Wag1987; Lafeof the tele-

of PCS

The 80-kDa product might correspond to cosecretion protein(s). A 70-kDa cosecretory protein (type I; SP I) (Morrissey et al., 1978) coexists with PTH in mammalian parathyroid secretory granules (Ravazzola et al., 1978) and is thought to bind the hormone (Cohn and MacGregor, 1981). If such a hormone-cosecretory binding protein occurs in the CS, it could explain the precipitation by anti bPTH antiserum of the complex formed by PCS and the cosecretory protein and its dissociation during SDS electrophoresis. On this interpretation, the previous binding of l-34 hPTH or purified PCS to antiserum should prevent precipitation of the complex, the result we obtained by fluorography. The report of the presence of a glycoprotein of SP I type in eel CS, detected by immunocytology and Western blotting (Tisserand-Jochem et al., 1987), is also an argument in favor of this hypothesis. The 45kDa product is present during incubations of CS with labeled methionine and is also one of the translated products of the reticulocyte lysate immunoprecipitated by the antiserum. Furthermore, it is the only product which is totally absent when the antiserum is masked by either the l-34 hPTH or the purified HPLC preparation. Finally, this molecular weight is consistent with a presumed molecular weight of a protein which may be a precursor of a 30-kDa protein. The 15kDa M, of one of the products encoded by CS RNA and immunoprecipitated by the antiserum is comparable to that of mammalian preproparathyrin ( 12.5 kDa M,) but such a product is not present either during incubations of CS with labeled me-

AND

LOPEZ

thionine or in the purified active product. At this stage we are not able to explain this result. The recent data on the amino acid sequence of the CS protein (Butkus et al., 1987; Lafeber et al., 1988) conflict with our results, since their sequence bears no homology either with parathyrin or with any other known peptide. However, it is not possible at present to say if the “main protein” of the CS analyzed by Butkus et al. is the same protein we studied, since they have not reported any bioactivity for their protein. Moreover, hypocalcin (teleocalcin), in which the first 33 N-terminal amino acids overlap with the sequence deduced from the DNA analysis reported by Butkus et al., has no effect on the plasma calcium level in normal, CS-deprived, or hypercalcemic eels (Wagner et al., 1988). In contrast, the fact that teleocalcin shows cross-reactivity with PTH antiserum (Harvey et al., 1987) suggests that teleocalcin/hypocalcin may exhibit some structural resemblances to parathyrin. The data presented here indicate that the active molecule purified from eel CS is a 32to 34-kDa protein and that its biosynthesis occurs via the synthesis of a 45-kDa precursor encoded by a major mRNA extracted from the CS. Several authors have reported that human tumors associated with humoral hypercalcemia of malignancy (HHM) synthesize and secrete a PTH-like peptide with a molecular weight of 20-30 kDa (Broadus et al., 1985; Docherty and Heath, 1986; Sat0 et al., 1986). Primary transcript mRNA of mammalian parathyroid hormone contains 850 * 50 nucleotides in human and 750 +- 50 nucleotides in bovine (Heinrich et al., 1984). These hn (heterogeneous nuclear) RNA would encode, if mRNA processing did not occur, for a 27.5-kDa protein (bovine) and a 3 1-kDa protein (human). It could be that in tumor cells mRNA processing is altered leading to the synthesis of a 30-kDa protein. Our results showing the presence of a 30-kDa PTH-like hormone in

FISH PTH-LIKE

HORMONE

the teleostean CS could mean that mRNA processing of the gene product differs from that in mammals. As previous results showed that the CS hormonal product exhibits bone-resorbing activity in vitro (Milet et al., 1979b; Lafeber et al., 1986) as well as in vivo (Milet et al., 1979b), we suggest that a relationship might exist between the bone-resorbing factor secreted by HHM-related tumors and the parathyrin-like hormone isolated from CS (PCS). This hypothesis is based on the similar molecular weight (around 30 kDa) and on biological activities shared by these molecules: antigenic site recognition by antiPTH antisera, bone-resorbing activity, and hypercalcemic activity in mammals. Work directed toward protein and gene sequencing will address the question of whether these two molecules are homologous and gene-related. ACKNOWLEDGMENTS This work was partially supported by a grant from INSERM, CRE No. 844012 and by the National Museum of Natural History (plan quadriennal).

REFERENCES Babson, A. L., Greeley, S. J., Coleman, C. M., and Phillips, G. E. (1966). The use of phenolphtalein monophosphate as a substrate for serum alkaline phosphatase. Clin. Chem. 12, 482-W. Birge, S. J., and Gilbert, H. R. (1974). Identification of an intestinal sodium and calcium-dependent phosphatase stimulated by parathyroid hormone. J. Clin.

Invest.

54, 710-717.

Broadus, A. E., Goltzman, D., Webbs, A. C., and Kronenberg, H. M. (1985). Messenger ribonucleic acid from tumors associated with humoral hypercalcemia of malignancy directs the synthesis of a secretory parathyroid hormone like peptide. Endocrinology

117, 1661-1666.

Butkus, A., Roche, P. J., Femley, R. T., Haralambis, J., Penshow, J. D., Ryan, G. B., Trahair, J. F., Tregear, G. W., and Coghlan, J. P. (1987). Purification and cloning of a corpuscles of Stannius protein from Anguilla australis. Mol. Cell. Endocrinol.

54, 123-133.

Chattier, M. M., Martelly, E., Lopez, E., and Milet, C. (1983). Effets de la calcitonine de saumon sur l’absorption intestinale du calcium chez l’anguille

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