Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 220, No. 2, February 1, pp. 645-651, 1983

COMMUNICATION Biosynthesis

of Lutropin in Ovine Pituitary [%3]Sulfate in Carbohydrate

KALYAN

RAO ANUMULA

AND

Slices: Incorporation Units’

of

OM P. BAHL’

L?epartment of Biological Seiaees, Division of Cell and Molecular Biology, State University of New York, Amherst Campus, B@iio, New York 14260 Received

June

‘7, 1982, and in revised

form

November

8, 1982

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H235S04. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the (Yand /3 subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [35S]sulfate was also incorporated into several other proteins in addition to LH. The location of ?SOi- in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-0-[35S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[%]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of 3”SOz- by chondro-lsulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.

Ovine lutropin (oLH)s is a glycoprotein hormone in which the carbohydrate constitutes almost 20% of the molecule. The carbohydrate is distributed in three “complex’‘-type asparagine-linked units, two in the

a and one in the fl subunit. A recently proposed structure of the carbohydrate units of oLH has revealed some unique structural features, particularly the presence of sulfated N-acetylgalactosamine at the nonreducing terminals of the oligosaccharide chain (1). Based on the calorimetric estimation of two sulfate groups per carbohydrate unit Parsons and Pierce (2) have reported the presence of both sulfated Nacetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) in bovine lutropin (bLH) and thyrotropin (bTSH). In order to investigate whether the sulfated GlcNAc terminals also occur in oLH, we have followed a different approach involving the metabolic incorporation of “Sein oLH in pituitary slices. This communication describes the preparation of 35SO:-labeled oLH and its characterization by immunological and physieochemieal methods. The isolation of both 35SO%--labeled N-acetylhexosaminyl-glycerols and N-acetylglucosaminitol (G1cNAcH.J indicates the presence of sulfated GlcNAc in addition to GalNAc as previously reported in oLH (1).

i This work was supported by U. S. Public Health Grant 2ROl-HD-12581. ‘To whom correspondence should be addressed. ‘Abbreviations used: oLH (bLH), ovine (bovine) lutropin or luteinizing hormone; hCG, human choriogonadotropin; bTSH, bovine thyroid-stimulating hormone; RIA, radioimmunoassay; NaDodSO,, sodium dodecyl sulfate; IPB, immunoprecipitation buffer; PBS, phosphate-buffered saline; GlcNAc, GalNAe, GlcNAcH*, and GalNAcH,, N-acetylglucosamine, N-acetylgalactosamine, N-acetylglucosaminitol, and N-acetylgalactosaminitol, respectively; Hepes, N-2-hydroxyethylpiperazine-Nðanesulfonic acid; TCA, trichloroacetic acid; CM-, carboxymethyl-. 645

0003-9861/83/020645-07$03.00/O Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved

646

ANUMULA EXPERIMENTAL

PROCEDURES

Incubation of sheep pituitary

slices with “S@.

Pituitary glands were excised from skinned lamb heads (Brennan Meat Packing Co., Buffalo, N. Y.) and finely minced (<0.5 mm) on an ice-cooled petri dish as previously described (3) except the tissue was rinsed with cold 0.01 M phosphate-buffered saline (PBS), pH 7.4. Pituitary minces, about 350 mg, were incubated in 5 ml of the medium, pH 7.4, equilibrated at 37°C in an atmosphere of COz/Oz, (5%/95%), containing 10 rtIM MgClz and KCl, 30 mM NaHCOa, 70 mM NaCl, 20 mM Hepes, 2 mM CaClz, 0.22% D-ghCOSe and MEM (Gibco), essential and nonessential amino acids with glutamine and sodium pyruvate. After a 30-min incubation at 37°C the pituitary minces were transferred to 5 ml of fresh medium containing 5 mCi of HzasSO, (0.8 Ci/rmol New England Nuclear) and the incubation was continued for 5 h with a gentle gas flow. The time course of ?SOi- incorporation into TCA-precipitable material from the medium and the tissue extract was optimum under these conditions (data not shown). The tissue was separated from the medium after cooling on ice and rinsed with 2 X 1 ml of the medium. Tissue was homogenized in 3 ml of 0.01 M PBS, pH 7.4, with a Teflon motor-driven Potter-Elvehjem homogenizer at a slow speed for 2 X 10 s with cooling. The homogenate was centrifuged at 100,OOOg for 2 h. Both the tissue extract and the medium were used for the isolation of ?SO:--labeled oLH. Andyti~~l methods. The RIA for oLH was carried out using rabbit anti-bLH and ‘%I-bLH. NIH standard bLH was used for iodination by the chloramineT procedure (4). The specific radioactivity of the labeled hormone was generally about 40 pCi/pg. For the RIA, 50,000 cpm of ‘“I-bLH and 1:6000 diluted anti-bLH were used. Of the total radioactivity 30% was immunoprecipitated in the controls (without the unlabeled hormone) and taken as zero percent activity. Hexosamines were determined with a Beckman 121 MB amino acid analyzer after hydrolysis of the samples in 4 M HCl at 100°C for 4 h. Mixtures of hexosamines and their alcohols were analyzed by 60 mM, pH 9.5 (5) borate electrophoresis (45 V/cm, 2.75 h) following N-acetylation with l[i4C]acetic anhydride. Inorganic [35S]sulfate released was determined by paper electrophoresis (45 V/cm) using pyridine:acetic acid:water, 20:20:960 (v/v), pH 4.73. Paper chromatography of sulfated sugars was performed on Whatman No. 1 paper in solvent system A [ethyl acetate:pyridine:water, 12:5:4 (v/v)] and of N-acetylhexosamines, N-acetylhexosaminitols, and glycerol in solvent system B [n-butanol:acetic acid:water, 4:l:l (v/v)]. Sugars were detected by staining with alkaline silver nitrate dip (6). Radioactivity was measured in 5 ml of Liquiscint (National Diagnostics) using a Beckman LS 7500 liquid scintillation counter. Electrophoretic strips were scanned for radioactivity on a Packard 7220/21 radiochro-

AND

BAHL

matogram scanner. Paper strips (0.5 cm) containing radioactivity were soaked in 0.4 ml of water for 2 h and counted in 5 ml of Liquiscint. N-[i4C]Acetylation of hexosamines following hydrolysis (4 N HCl, lOO”C, 4 h) of the samples was carried out by evaporating samples to dryness under vacuum with a rotary evaporator. To samples containing less than a micromole was added 0.3 ml of water, 0.2 ml saturated NaHCO,, and 25 &i of l[‘“C]acetic anhydride (10 mCi/mmol, New England Nuclear) in 50 ~1 of acetone. The reaction mixture was mixed vigorously on a Vortex. After 30 min at room temperature the procedure was repeated with 2 X 2 ~1 of nonradioactive acetic anhydride. The samples were boiled for 5 min, diluted with water, and acidified with 2 M acetic acid before passage through a AG-50 X4 H+ over AG-1 X4, acetate column. The neutral fraction was lyophilized and chromatographed in solvent system B. After scanning the paper for radioactivity the acetylated hexosamines and their alcohols migrating together with the standards were eluted with water and analyzed by borate paper electrophoresis. Periodate oxidation/Smith degradation. Purified “Se--labeled oLH (5000 cpm) together with 2 mg of unlabeled oLH was treated with 1 ml of 0.05 M sodium acetate, pH 4.5, 0.05 M NaIO, at 4°C for 48 h in the dark. Ethylene glycol(O.01 mmol) was added and after 1 h at room temperature the pH was adjusted to 9.0 with 1 M NaOH followed by the addition of NaBH, (0.5 mmol). After keeping the reaction mixture for 16 h at 4°C the pH was adjusted with 2 M acetic acid to 5.0 and the incubation mixture was dialyzed exhaustively against water and lyophilized. The oxidized and reduced oLH was dissolved in 1 ml of 0.5 M HCl and incubated at 37°C for 24 h (2). The sample was neutralized by adding an equivalent amount of solid NaHC03. Free aldehyde groups from Ci of mannoses (1) were reduced with either NaBH4 or NaB3H, in the case of nonradioactive oLH as the starting material. After passage of the reaction mixture through a column of AG-BOW X4 H+ form (200-400 mesh), boric acid was removed by evaporation as methyl borate with 5 X 2 ml of methanol at 37°C. The degradation products were analyzed for sugar composition and glycerol after fractionation by a combination of paper chromatography and high-voltage paper electrophoresis. Immunqwrecipitation

and

NaDodSO,-polyazryl-

amide gel e.!edrophoresti. ?SOf-labeled oLH was immunoprecipitated with rabbit anti-bLH serum. About 3000 cpm of the purified labeled oLH was incubated for 2 h at room temperature followed by another 14 h at 4°C with 0.5 ml of the antiserum in a final volume of 0.7 ml of the buffer (IPB), containing 1% Triton X-100, in 0.01 M PBS, pH 7.6, 5 mM EDTA, 20 mM NazSOd, and 0.02% NaNa. Appropriate amounts of Staphylococcus aureauS cells (Pansorbin, Calbi-

=SO,-LABELED

ochem) were added and incubated at room temperature for 2 h before centrifugation at 80009 for 10 min. The sedimented cells were washed twice with 2 ml of IPB and once with 0.5 ml of NaDodSo,-polyacrylamide gel electrophoresis sample buffer (7) at room temperature with 30-min intervals before each centrifugation. Finally, the cell pellet was suspended in 70 ~1 of NaDodSo,-polyacrylamide gel eleetrophoresis sample buffer and boiled for 5 min in order to extract the labeled proteins. Following centrifugation approximately 90% of the initial radioactivity was consistently present in the supernatant. Labeled proteins were analyzed on 1.5-mm-thick 12% acrylamide gels, cast in a Hoefer slab gel apparatus with a 1.5-cm-high 4% stacking gel in a buffer system described by Laemmli and Favre (7). Gels were fixed and treated with ENHANCE (New England Nuclear) according to the manufacturer’s directions, except that fluors were precipitated in the gel using 3% glycerol. Gels were dried and autoradiography was performed at -70°C with Kodak X-Omat R film. The film background was often removed with a cotton swab soaked in a fresh 1:l mixture of 5% potassium ferricyanide and 10% sodium thiosulfate, followed by thorough washing under tap water. Prme digestion of “SO,-oLH. Purified oLH (5000 cpm) was mixed with 2 mg of oLH, reduced with dithiothreitol, and S-carbamidomethylated with iodoacetamide as described for hCG (8). Pronase digestion was carried out in 0.5 ml of 0.1 M Tris-acetate, pH 8.0, 15 mM CaClz at 37°C for 96 h under toluene with a total of 1 mg of enzyme added in equal portions at 0,24, and 48 h. Digestion was terminated by boiling for 5 min and the supernatant was applied to a Sephadex G-25 fine column (1.4 X 110 cm) in 0.1 M pyridine-acetate, pH 5.0. Treatment with sulfatases. Various commercial sulfatases from Sigma Chemical Company were tested for their ability to release “SO:from peripheral hexosamines of oLH (6). Purified 35SOd-oLH (3990 cpm) was incubated with 0.8 units of chondro-4-sulfatase from Proteus vulgar-is (pH 7.5), 1.0 units of Aerobaxter aerogelzes sulfatase (pH 7.5), 148 units of HeZixpmnatia sulfatase Type H-2 (pH 5.0), or 50 units of sulfatase from limpets Type V (pH 5.0) under toluene at 37°C for 110 h. Samples were boiled for 3 min and the supernatant was subjected to high-voltage paper electrophoresis for the released “SO:determination.

RESULTS Isolation sf S5SOf-hbeled oLH. The medium and the tissue extract containing labeled oLH obtained after incubation of the sheep pituitary slices with “S@ were subjected to purification by the procedure previously used in the laboratory for the isolation of an immunologically active and electrophoretically

OVINE

647

LUTROPIN

6

FRACTION

NUMBER

FIG. 1. Sephadex G-100 chromatography of [35S]sulfated products from the incubation mixture of ovine pituitary slices. oLH activity in the medium (top) and the tissue extract (bottom) was monitored by RIA as described in the text. Column was previously calibrated with oLH and oLH-cu. Arrows represent the points of elution. homogenous preparation of oLH from sheep pituitary glands (9). The medium and the tissue extract were applied to separate columns of Sephadex G-100, superfine (2.6 X 87 cm), previously equilibrated with 0.05 M sodium phosphate, pH 6.5, at 4°C. The column was equilibrated with oLH and its subunits Fractions of 7.5 ml were collected and a 5% aliquot was counted for radioactivity. The elution profiles are shown in Fig. 1. The column fractions were monitored by RIA using ‘%I-bLH and rabbit anti-bLH serum. Fractions containing oLH and its subunits as shown by RIA were pooled (Fig. 1, pools 2 and 3, respectively). The oLH active pool 2 was exhaustively dialyzed against 0.01 M sodium phosphate, pH 6.0, and applied to a column of CM-Sephadex C-50 (0.9 X 15 cm), previously equilibrated with the same buffer. Columns were washed with the application buffer until no more radioactivity was detected. Elution was initiated with 0.05 M sodium phosphate, pH 8.0, and then changed to 0.1 M sodium phosphate, pH 8.0, containing 0.2 M NaCl. About 70% of the immunoreactive oLH from the Sephadex G-100 pool was recovered in the peak eluted with the second buffer containing NaCl in both the columns. This elution pattern is consistent with the previously reported results obtained by the same procedure used for the purification of unlabeled oLH from pituitary glands (9). In

648

ANUMULA

the medium immunoactivity of oLH did not coincide with the radioactivity and trailed beyond the radioactive peak. This was probably due to microheterogeneity at the nonreducing end caused by varying degrees of sulfation in the secreted oLH. Characterization of *SOh-oLH. The labeled oLH from the medium and the tissue extract varied in amount and specific radioactivity. Although the oLH medium had 5-fold less specific radioactivity than the tissue extract, it contained 20-fold more LH activity. Recovery of the oLH from CM-Sephadex was approximately 70% and about 50 pg of 35S01-oLH was obtained from the medium. More than 90% of the radioactivity in the purified oLH preparations from the tissue extract and the medium was immunoprecipitated by rabbit anti-bLH serum (oLH and bLH are immunologically cross-reactive), as described above. Autoradiography of the gel following NaDodSO,polyacrylamide gel electrophoresis of the purified SSSOI-oLH (data not shown) and the immunoprecipitated material showed the presence of only two labeled bands which comigrated with the (Y and /3 subunits of unlabeled oLH (Fig. 2, lane 4). It may be pointed out that relatively more label was present in the a subunit from the medium than the fl subunit (lane 2). This difference in radioactivity between the subunits could be due to the number of

AND

BAHL

oligosaccharide chains or to the fact that a subunit is secreted more into the medium. The presence of free a and fl subunits (Fig. 1, pool 3, and Fig. 2, lane 2) in the medium as well as tissue extract is rather interesting. It suggests that there may be some type of regulatory step involved in the subunit recombination. As is clear from the electrophoretic gels, ovine pituitary minces were able to incorporate %SO:- into several other proteins secreted in the medium (lanes l-3) as well as those present in the tissue (lanes 57). The relative molecular weights of these labeled proteins ranged from 12,000 to 175,000 Da. Among the labeled proteins a 27,000-Da protein was secreted into the incubation medium (Fig. 1, pool 3, and Fig. 1, lane 2) in a relatively large amount. Thus, it is obvious that the sulfation is not restricted only to LH in ovine pituitary gland. Exhaustive Pronase digestion of ?SO,-oLH resulted in glycopeptides containing almost all the initial radioactivity. No radioactivity was detected in the amino acid peak. Smith degradation of “SO,-oLH. When periodate oxidized-reduced labeled oLH was subjected to mild acid hydrolysis as described above, less than 10% of the label appeared as inorganic sulfate (Fig. 3A). The major peak containing about 35% of the label was eluted with water and chromatographed in solvent system A (Fig. 3B). The resulting major ?SO:--la-

5

6

7

8

175-

%58473527-

p”FIG. 2. Autoradiograph of NaDodSO, polyacrylamide gel electrophoresis of [?S]sulfated proteins from the incubation mixture of ovine pituitary slices. Lanes 1 to 4, asSO:--labeled proteins from the medium; lanes 5 to 7, from the tissue extract; lanes 1 and 5, Sephadex G-100, pool 1; lanes 2 and 6, Sephadex G-100, pool 3 (Fig. 1); lanes 3 and 7, proteins eluted with the first buffer from CM-Sephadex; lane 4, labeled proteins immunoprecipitated with bLH antisera from the purified oLH. Relative molecular weights (kilodaltons) were calculated from the calibration curve constructed using labeled protein standards. Lane 8, i4C-labeled protein standards, from top to bottom are myosin (2OOK), phospborylase B (93.5K) bovine serum albumin (69K), ovalbumin (46K), carbonic anhydrase (30K), and lysozyme (14.3K).

?S04-LABELED

1

I 0

OVINE

I 10 DISTANCE

I 30

20 FROM

649

LUTROPIN

ORIGIN

I 40

km)

FIG. 3. Separation of the Smith degradation products from 35SOi--labeled oLH. (A) Radioscan of the periodate-cleaved fragments following electrophoresis for 45 min. (B) Paper chromatography of the [?S]sulfated hexosamine derivatives in solvent system A. (C) Resolution of $S]sulfated hexosaminyl-glycerols and hexosaminitols by borate paper electrophoresis. The bars under the radioactive peaks indicate the portions eluted with water. (1) Sulfated hexosamine derivatives. (2) Glc-6-S4. (3) Inorganic sulfate.

beled component also migrated as a single species in the following solvent systems: (1) s-butyric acid:0.5 N NHdOH (5:3), (2) I-butanol:pyridine:water (6:4:3), and (3) ethyl acetate:pyridine:acetic acid:water (5:5:1:3). The radioactive peak was broad in solvent system 3. The hexosamine analysis of this component revealed the presence of GlcNAc, 6.2% ; GalNAc, 42.7% ; and GlcNAcHz, 52% (Fig. 4A). Therefore, it is obvious from the hexosamine composition that the major component in fact is a heterogenous mixture of compounds with identical mobility in the above solvent systems. Indeed, this component could be resolved into two fractions by borate electrophoresis (Fig. 3C). Both fractions moved slower than Glc-6SO,. The slow-migrating peak contained both GlcNAc and GalNAc whereas the fast-migrating peak contained only GlcNAeHa (Fig. 4C). In an analogous experiment using nonradioactive oLH the Smith degradation products were reduced with NaBSHA. Only the slow-moving component in borate electrophoresis was found to contain rHlglycero1 as shown by hydrolysis and chromatography in solvent system B. It is interesting to note that the oligosaccharide chain containing GlcNAc-l-sulfate undergoes further hydrolysis to yield 4-sulfo-N-acetylglucosamine which

then is reduced during the second NaBH, reduction. The ‘H-labeled GlcNAcHz was found only in the fastmigrating component from borate electrophoresis (Fig. 3C), after reduction with NaB3H4 (Fig. 4D). The slow-migrating component in fact is a mixture of 4O-sulfate N-acetylhexosaminyl (GlcN/GalN)-B-nglycerol, whereas the fast-moving component is Nacetylglucosaminitol-4-sulfate. The sulfated GlcNAc as GlcNAc-glycerol and GlcNAcHz amounted to 55% of the total hexosamines released by Smith degradation. Thus the above data indicate the presence of sulfate on both the terminal GlcNAc and GalNAc. Action of suEfata.ws on ssSO~-oLH. Chondro-4-sulfatase from Proteus vulgaris, an enzyme specific for releasing sulfate from oligoor disaccharides of chondroitin 4-sulfate (lo), was also able to release 15% of the ?SO:- from the hormone. This is probably due to some similarity of oLH carbohydrates with the enzyme substrate, N-acetylchondrosine-4-sulfate. Since the oLH oligosaccharide contains only two sulfated GlcNAc/GalNAc terminals it is expected to incorporate 35SO:- to a maximum of 56% of the total radioactivity in GalNAc-4-SOI. Therefore, it appears that 30% of the GalNAc-4-SO, terminal was in fact hydrolyzed by the P. wubaris enzyme. The limpet sulfatase on the other hand was able to release only

650

ANUMULA

OGICNAC

AND

BAHL

OGalNAc

GolNAcH20

OGlcNAcHn

B

I

I 0

1 10 DISTANCE

FIG. 4. Hexosamine analysis of borate paper electrophoresis. (A) paper chromatography (Fig. 3B). paper electrophoresis (Fig. 3C). unidentified contaminant.

a small amount of sulfate (~3%) from All other sulfatases failed to hydrolyze able ?SO$-.

20 FROM

30 ORIGIN

I 40

km)

the Smith degradation products following N-[“Clacetylation by Hexosamine composition of the sulfated material obtained by (B) Slow-moving and (C) fast-moving components from borate Radioactivity was determined as described in the text (a) An

the hormone. any detect-

DISCUSSION The present studies were undertaken to investigate the biosynthesis of oLH as well as to settle the unresolved question of the presence of SOI- on GlcNAc using 3SSOI-oLH. The above data clearly indicate that we have been able to obtain for the first time a metabolically 3SSOi--labeled homogeneous preparation of oLH. The homogeneity of the biosynthetic product was established by its immunoprecipitability with anti-bLH serum and by NaDodSOd-polyacrylamide gel electrophoresis. The latter showed only two labeled bands corresponding to the ~1 and /3 subunits on autoradiography of the gel, thus indicating ?Wincorporation into both subunits of oLH. In addition to labeled oLH both subunits were also found free in the medium as well as in the tissue extract. The ?SOiester group was shown to be located in the oligosaccharide portion of the molecule by its presence in the glycopeptides obtained by exhaustive Pronase digestion of 56S0,-oLH. The question whether the sulfate ester was present on GlcNAc in addition to GalNAc as found in bovine LH and TSH (2) was

investigated by Smith degradation of the labeled oLH. The approach using 86S0,-oLH certainly facilitated both the isolation and unequivocal characterization of Smith degradation products. Two classes of compounds, 4-0-sulfate-GalNAc/GlcNAc+D-glycerol and GlcNAcHz-4-O-sulfate, were obtained by the periodate oxidation of SSSO,-oLH followed by reduction, mild acid hydrolysis, and another reduction. The data thus indicate the presence of both sulfated GlcNAc and GalNAc in oLH. The C, location of the “SO:ester on GalNAc was further confirmed, since it was hydrolyzable by a &-specific chondroitin sulfatase. Since oLH contains 0.3 residue of terminal galactose per mole it appears that the sulfate ester group is present in two of three carbohydrate units in the hormone. Bovine LH and TSH, however, have been reported to have sulfated GlcNAc and GalNAc in all three carbohydrate units (2). In our previous studies using nonradiolabeled oLH the presence of the sulfate group on GlcNAc was not detected since only sulfated 2,5anhydrotalitol and not the sulfated 2,5anhydromannitol was found in the deamination products of the N-deacetylated oligosaccharides. Similarly a mole equivalent of terminal GlcNAc was consistently observed by gas chromatography/mass spectrometry in the methylation products of oLH glycopeptides (1). One way to explain the above data is to assume that the sulfated GlcNAc is more labile

&SO,-LABELED under the strong alkaline conditions used in both deamination and methylation studies. In a recent publication, while this work was in progress, Hortin et al. (11) were also able to show the incorporation of “SO:into bovine and rat LH but no attempt was made by the authors to purify the biosynthetic product or establish the position of the sulfate group. It may also be pointed out that we have not been able to incorporate ?SOi- in rat LH. In view of the lack of information on the structure of rat LH, it is not possible to evaluate this result. Unlike the sialyl lactosamine terminals found in “complex’‘-type carbohydrate units of plasma glycoproteins (12), the presence of sulfated N-acetylhexosamines is a unique feature of the pituitary glycoprotein hormones. It appears, however, from the autoradiographs of NaDodSO,-polyacrylamide gel electrophoresis of the medium and tissue extract that there are other proteins which are also sulfated. It is interesting to note that a 27,909-Da “SO:--labeled protein, the function of which is not known currently, was found to be secreted in relatively large amounts. Whether sulfate is in the asparagine or the Ser/Thr-linked carbohydrate units cannot be concluded from the present data: However, in a double-labeling experiment using %Ofand 2-rH]mannose, the medium and the tissue extract showed the presence of proteins with both the isotopes (data not shown), suggesting the synthesis of sulfated “complex’‘-type carbohydrate units occurring in other glycoproteins. The possibility of both sulfated and asparagineand sulfated Ser/Thrlinked carbohydrates in the same molecule, however, cannot be ruled out. Recently, the existence of sulfated oligosaccharides of the “complex” type in other tissue glycoproteins has been implicated (13). Finally, the purified metabolically labeled %O,oLH provides a suitable substrate for the detection and isolation of enzymes involved in the metabolism of sulfate ester groups in glycoproteins, such as a sulfatase or a sulfotransferase(s). The availability of a sulfatase should enable us to study the role of SO:- on the biological function of the hormone, such as the effect of SO:- on plasma half-life, receptor binding, CAMP stimulation, and steroidogenesis. It would be also interesting to know if the negatively charged sulfate ester groups in oLH have a function similar to that of sialic acid in plasma glycoproteins.

OVINE

651

LUTROPIN

Obviously, “SO,-oLH agent for a variety

should prove of studies.

to be a useful

re-

ACKNOWLEDGMENTS The authors wish to thank Dr. F. Bellino for the kind gift of “C-labeled protein standards. The authors also acknowledge James Stamos for preparing the figures and Ursula Brunn for typing the manuscript.

REFERENCES 1. BEDI, G. S., FRENCH, W. C., ANDBAHL, 0. P. (1982) J. Bid Ch.em 257,4345-4355. 2. PARSONS, T. F., AND PIERCE, J. G. (1980) Proc Nut. AC&. sci USA 77,7089-‘7093. 3. HENNER, J. A., KESSLER, M. J., AND BAHL, 0. P., (1981) J. Biol Chem 256, 5997-6003. 4. BELLISARIO, R., AND BAHL, 0. P. (1975) J. Bid Chem 250,3837-3844. 5. TAKASAKI, S., AND KOBATA, A. (1975) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 50, pp. 50-54, Academic Press, New York. 6. TREVELYAN, W. E., PROCTOR, D. P., AND HARRISON, J. W., (1950) Nature (London) 166. 444445. 7. LAEMMLI, U. K., AND FAVRE, M., (1973) J. Mel BioL 80, 575-599. 8. CARLSEN, R. B., BAHL, 0. P., AND SWAMMINATHAN, N., (1973) J. BioL Chem 253, 6810-6827. 9. SHERWOOD, 0. D., GRIMEK, H. J., AND MCSHAN, W. H., (1970) B&him Biophys. Acta 221,87106. 10. SUZUKI, S. (1972) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 28, pp. 917-921, Academic Press, New York. 11. HORTIN, G., NATOWICZ, M., PIERCE, J. G., BAENZIGER, J., PARSONS, T., AND BOIME, I. (1981) Proc Nat. Acad Sci. USA 78, 7468-7472. 12. KORNFELD, R., AND KORNFELD, S. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W., ed.), pp. l-34, Plenum, New York. 13. HEIFETZ, A., KINSEY, W. H., AND LENNARZ, W. J. (1980) J. Bid Chem 255,4528-4534.