Cyclic adenosine monophosphate and phorbol ester, like gonadotropin-releasing hormone, stimulate the biosynthesis of luteinizing hormone polypeptide chains in a nonadditive manner

We have previously reported that GnRH stimulates the synthesis of both the alpha- and beta-polypeptide chains of LH. In the present study, in order to investigate the mechanisms involved in the GnRH regulation of LH subunit synthesis, we have explored the effects of cAMP and a phorbol ester [12-O-tetradecanoyl phorbol 13-acetate (TPA)] using anterior pituitary cells in primary culture incubated in the presence of [35S]methionine. The radioactivity incorporated into alpha and LH beta immunologically related polypeptides was measured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled material immunoextracted from cells and media with specific antisera. The cAMP analog 8-Br-cAMP (at concentrations 0.25-2 mM), the cholera toxin (6-60 nM), and forskolin (10-100 microM) induced, like GnRH, an increase in the [35S]methionine incorporation into alpha- and LH beta-subunits. On the other hand, TPA (50-100 nM) also enhanced the synthesis of LH subunits. After a 5-h incubation in the presence of GnRH, 8-Br-cAMP, and TPA in different combinations, no cumulative effect was observed. These results demonstrate that intracellular cAMP and TPA are potent activators of both alpha- and LH beta-polypeptide chain synthesis, suggesting that cAMP as well as diacylglycerols may act as intracellular mediators of the GnRH effect on LH subunit synthesis.     

Gonadotropin-releasing hormone action upon luteinizing hormone bioactivity in pituitary gland: role of sulfation

The regulation of rat luteinizing hormone (rLH) bioactivity was studied in an in vitro system using isolated pituitaries from male rats. Stored and released rLH was evaluated in terms of mass (I-LH), bioactivity (B-LH), mobility in nonequilibrium pH gradient electrophoresis, and mannose and sulfate incorporation either in the presence or absence of gonadotropin-releasing hormone (GnRH). GnRH increased the biological potency of stored and released rLH. The pituitary content revealed seven I-LH species (pH 7.2, 7.8, 8.5, 9.0, 9.1, 9.3, and 9.7) and five B-LH species (pH 8.5, 9.0, 9.2, 9.4, and 9.7). The major I-LH and B-LH peaks were at pH 9.0 and 9.2, respectively. I-LH peaks at pH 7.2 and 7.8 are devoid of bioactivity; at these pH values, free rLH subunits are detectable. GnRH increases the amount of both I-LH and B-LH material secreted into the medium, and the major component migrates at pH 8.5 and is probably the alpha beta dimer. [3H]Mannose and [35S]sulfate can be incorporated into stored and released rLH (pH 7.2, 7.8, 9.0, 9.1, and 9.3 and 7.2, 7.8, 8.5, and 9.0, respectively). GnRH decreases [2-3H]mannose incorporation into secreted rLH. [35S]Sulfate was incorporated into I-LH released spontaneously into the medium; the form at pH 7.2 has no biological activity and is probably the free alpha subunit. GnRH decreases the [35S]sulfate-labeled rLH content of the pituitary concomitantly with a 500% increase in [35S]sulfate-labeled released rLH, suggesting that, soon after [35S]sulfate is incorporated, sulfated rLH is released. Sulfatase action on released rLH reveals that sulfation may be related to release of rLH but that sulfate residues are not involved in the expression of rLH bioactivity. In conclusion, GnRH stimulates carbohydrate incorporation and processing of the oligosaccharide residues giving the highest biological potent rLH molecule and also increases sulfation; this step is closely related to the step limiting the appearance of LH in the medium in the absence of GnRH.     

In vitro translation of distinct mRNAs coding for the precursors of porcine LH subunits

Conditions are described to characterize and estimate the precursors of porcine LH alpha and beta subunits and indirectly their specific mRNAs. Poly(A) RNAs extracted from castrated male pig anterior pituitaries were translated in a wheat-germ system in the presence of [35S] cysteine and [35S] methionine. The translation products were precipitated by antisera directed against reduced and carboxymethylated LH alpha and beta subunits and analyzed by high resolution electrophoresis. It is shown that the precursors of pLH alpha and beta subunits are located in two distinct congruent to 15 K proteins and represent--on the basis of the incorporation of the [35S] labeled aminoacids into proteins--congruent to 0.12% and 0.05% respectively of the total translation products. It is suggested that in the pig, as in other species, the LH alpha and beta subunits are encoded by two distinct mRNAs, and at variance with other species the leader sequence of LH alpha mRNA is longer than that of LH beta mRNA.     

The biosynthesis of sulfoquinovosyldiacylglycerol: studies with groundnut (Arachis hypogaea) leaves

The biosynthetic pathway of sulfoquinovosyldiacylglycerol (SQDG) was investigated using groundnut (Arachis hypogaea) leaf discs and 35S-labeled precursors. [35S]SO4(2-) was actively taken up by the leaf discs and rapidly incorporated into SQDG. After 2 h, 1.5% of the [35S]SO4(2-) added to the incubation medium was taken up, of which 28% was incorporated into SQDG. The methanol-water phases of the lipid extracts of the leaf discs were analyzed for the 35S-labeled intermediates. Up to 2 h of incubation, cysteic acid, 3-sulfopyruvate, 3-sulfolactate, 3-sulfolactaldehyde, and sulfoquinovose (SQ) which have been proposed as intermediates [Davies et al. (1966) Biochem. J. 98, 369-373] were not labeled. Only a negligible amount of radioactivity was observed in these compounds after incubation for 4 h and more. Addition of sodium molybdate inhibited the uptake of [35S]SO4(2-) as well as its incorporation into SQDG by the leaf discs, suggesting that 3'-phosphoadenosine-5'-phosphosulfate may be involved in the biosynthesis of SQDG. Addition of unlabeled cysteic acid to the incubation medium enhanced the uptake of [35S]SO4(2-) but did not affect its incorporation into SQDG. 35S-labeled cysteic acid was taken up by the leaf discs and metabolized to sulfoacetic acid but not incorporated into SQ or SQDG. These results show that cysteic acid is not an intermediate in SQDG biosynthesis. [35S]SQ was taken up by the leaf discs and incorporated into SQDG in a time-dependent manner. [35S]Sulfoquinovosylglycerol was also taken up by the leaf discs but not incorporated into SQDG. It is concluded that SQDG is not biosynthesized by the proposed sulfoglycolytic pathway in higher plants. Though [35S]SQ was converted to SQDG, the rates are much lower compared to [35S]SO4(2-) incorporation, which suggests that a more direct pathway involving sulfonation of a lipid precursor may exist in higher plants.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium,Chromatium vinosum: II. Biosynthesis of constituent subunits

[³⁵S]Methionine-labeled free subunits A and B of RuBP carboxylase were present in barely detectable amounts; the radioactivity in the free subunit B was approximately 1/150th of that in the subunit B contained in the holoenzyme of RuBP carboxylase. The turnover rates of subunits A and B in the holoenzyme were equal at each time during the incubation period. The ratio of subunit A to subunit B was constant throughout the incubation time both in quantity and in the level of [³H]leucine and [³⁵S]methionine incorporated. CO₂ contained in the incubation medium suppressed [³⁵S]methionine incorporation into both subunits A and B equally. These results suggest that the biosynthesis of subunits A and B is completely synchronized and may be regulated by identical mechanisms.     

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)     

LH biosynthesis and secretion in rat anterior pituitary cell cultures: stimulation of LH glycosylation and secretion by GNRH and an agonistic analogue and blockade by an antagonistic analogue.

In rat anterior pituitary cell cultures GnRH (1nM) stimulated a progressive increase in LH release into the medium from 1 to 8 h of incubation, while cellular LH showed a corresponding decrease. GnRH (1nM) neither modified the uptake nor the incorporation of [³H]-glucosamine and [³H]-proline into total protein. The incorporation of [³H]-proline into cellular LH also was unaffected by GnRH. In contrast, GnRH stimulated a 3 to 4-fold increase in [³H]-glucosamine incorporation into cellular LH. The agonistic analogue, [des GlyNH₂¹⁰]-LHRH ethylamide, mimicked the GnRH effects and was 5 to 6 times more potent than GnRH. The antagonistic analogue, [D-Phe², D-Phe⁶]-LHRH blocked the GnRH-stimulated effects. These results suggest that GnRH and agonistic analogues may preferentially regulate turnover or synthesis of the carbohydrate moiety of LH.     

Effect of cycloheximide and tunicamycin on the gonadotrophin-releasing hormone stimulated distal glycosylation of luteinizing hormone by rat pituitary cells

We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 microg/mL) or tunicamycin (0.5 and 2.0 microg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.     

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

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H₂³⁵SO₄. 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 α and β 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 ³⁵SO₄^2? 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-O-[³⁵S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[³⁵S]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of ³⁵SO₄^2? by chondro-4-sulfatase. 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.     

Intracellular assembly of human fibrinogen

Hep-G2 cells, pulse-labeled with L-[35S]methionine, incorporate radioactivity within 2 min into precursor forms of fibrinogen and into fibrinogen. Pulse-labeled intracellular fibrinogen is first composed of radioactive B beta chains, followed by nascent A alpha chains. Radioactive gamma chains accumulate in the cells and later contribute, via intermediate forms, to the assembly of fibrinogen. Following a pulse-chase incubation with L-[35S]methionine, the radioactive composition of newly secreted fibrinogen also reflects the fact that there is a large intracellular pool of gamma chains.     

Regulation of alpha and luteinizing hormone beta subunit messenger ribonucleic acids during stimulatory and downregulatory phases of gonadotropin-releasing hormone action

Decreased gonadotropin responsiveness (downregulation) to gonadotropin-releasing hormone (GnRH) following chronic in vivo and in vitro exposure to GnRH or its agonist (GnRH-A) has been previously reported. In the present studies, changes in LH subunit mRNAs in rat pituitary monolayer culture during stimulatory and down regulatory phases of GnRH action are described. Rat pituitary cells in culture, pretreated with medium alone or GnRH-A (10(-6) M) for 48 h were extensively washed and treated with graded concentrations of GnRH [10(-9) to 10(-7)] for 4 h. Medium was assayed for luteinizing hormone (LH) immunoreactivity, and total cytoplasmic RNAs were extracted by the hot phenol-guanidinium isothiocyanate method. Subunit-specific mRNAs were quantified by dot-hybridization assay using 32P-labeled subunit-specific cDNA probes. Cells pretreated with medium alone showed a dose-dependent increase in medium LH immunoreactivity, but the alpha and LH beta mRNAs showed no change over the 4-h period. Cells pretreated with GnRH-A showed no significant increase in medium LH with GnRH treatment, thus demonstrating that the cells had been desensitized by prior GnRH-A treatment. Alpha and LH beta subunit mRNAs of cells pretreated with GnRH-A did not show any significant change with further GnRH treatment. In subsequent experiments, cells were incubated with medium alone or 10(-7) M GnRH for 4, 8, or 24 h. GnRH failed to increase subunit mRNAs after 4 and 8 h incubation; after 24 h, alpha subunit mRNA showed a modest but significant increase and beta subunit mRNA showed a modest decrease compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of LH-RH on the release of sulfated lutropin: a potential in vitro bioassay for LH-RH

Sheep pituitary cells prelabelled with radioactive [35S] sulfate (35SO4(2-)) were incubated with different concentrations of LH-RH and the release of LH (lutropin) into the medium was monitored in terms of immunoprecipitable [35S] sulfated LH radioactivity and estimation of LH in the same sample by radioimmunoassay. A dose dependent response was obtained with a maximum of a 16 fold increase in immunoprecipitable 35SO4(2-) -labelled LH radioactivity in the medium which was confirmed by radioimmunoassay. Similar results were also obtained for Buserelin, a well known superactive analogue of LH-RH. However, the half maximal response for Buserelin was obtained at 3-5 nM in comparison to 80.5 nM for LH-RH. After the maximal response to LH-RH as well as Buserelin, a further increase in the concentrations caused a decrease in the release of immunoprecipitable [35S]-sulfate labelled LH into the medium. Differential labelling of stored and newly synthesized LH with radioactive [35S] sulfate and [3H]-labelled leucine revealed that there was a dose dependent increase in the [35S] sulfate labelled LH into the medium whereas the release of [3H]-leucine labelled newly synthesized LH did not show a parallel increase either at different concentrations of LH-RH or at different time intervals. The above observations strongly suggest the possibility of sulfation of LH being the potential signal indicating the storage of LH in sheep pituitary cells. Another important observation in our study was that the dose dependent response of LH-RH in the form of release of [35S]-sulfate labelled LH, which was monitored by immunoprecipitation with specific LH antiserum, can be used in an in vitro bioassay for LH-RH. We believe that a new cheap and sensitive in vitro bioassay could be developed on the basis of this observation.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Exact location of cross-linking acceptor sites

Human fibrinogen was clotted under conditions that promote latent factor XIII activity and in the presence of a radioactive substitute cross-linking donor ([14C]glycine ethyl ester). The labeled fibrin was reduced and alkylated in the presence of 6 M guanidinium chloride. After dialysis and freeze-drying, the preparation was separated into its constituent polypeptide subunits by chromatography on (carboxymethyl)cellulose in the presence of 8 M urea. Under the incorporation conditions used, the radioactivity was limited to gamma chains (one donor molecule/chain) and alpha chains (two donor molecules/chain). The labeled alpha chains were digested with cyanogen bromide and fractionated on Sephadex G-50. All the radioactivity was found in a fragment previously designated H alpha CNI, the largest of the cyanogen bromide fragments in the alpha chain. The fragment was further fragmented by digestion with plasmin, trypsin, chymotrypsin, and/or staphylococcal protease. The incorporated radioactivity was found to reside in equal amounts at two different sites located 38 residues apart. These were determined to be positions 88 and 126 in H alpha CNI, which correspond to glutamine-328 and glutamine-366 in the alpha chain.     

The frequency of gonadotropin-releasing hormone secretion regulates expression of alpha and luteinizing hormone beta-subunit messenger ribonucleic acids in male rats

The influence of GnRH pulse frequency on LH subunit mRNA concentrations was examined in castrate, testosterone-replaced male rats. GnRH pulses (25 ng/pulse) or saline to controls, were given via a carotid cannula at intervals of 7.5-240 min for 48 h. alpha and LH beta mRNA concentrations were 109 +/- 23 and 30 +/- 5 pg cDNA bound/100 micrograms pituitary DNA, respectively, in saline controls. GnRH pulse intervals of 15, 30, and 60 min resulted in elevated alpha and LH beta mRNAs (P less than 0.01) and maximum responses (4-fold, alpha; 3-fold, LH beta) were seen after the 30-min pulses. Acute LH release to the last GnRH pulse was seen after the 15-, 30-, and 60-min pulse intervals. In contrast, LH subunit mRNAs were not increased and acute LH release was markedly impaired after the rapid (7.5 min) or slower (120 and 240 min) pulse intervals. Equalization of total GnRH dose/48 h using the 7.5- and 240-min intervals did not increase LH subunit mRNAs to levels produced by the optimal 30-min interval. These data indicate that the frequency of the pulsatile GnRH stimulus regulates expression of alpha and LH beta mRNAs in male rats. Further, GnRH pulse frequencies that increase subunit mRNA concentrations are associated with continuing LH responsiveness to GnRH.     

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)     

An efficient and prolonged in vitro translational system from isolated cucumber etioplasts

Etioplasts were isolated from dark grown cucumber cotyledons pretreated with kinetin and gibberellic acid. When incubated in a cofactor enriched medium these etioplasts incorporated [35S] methionine into a hot trichloroacetic acid-insoluble fraction; this incorporation was linear for 8 h of incubation and was inhibited by chloramphenicol but not by cycloheximide. Over the same time period, the etioplasts showed continued linear synthesis of the chlorophyll precursors protochlorophyllide, Mg-protoporphyrin and protoporphyrin IX. Analysis of products of in vitro protein synthesis by etioplasts and cotyledons showed the thylakoid membrane polypeptide profiles to be identical. Continued incorporation of [35S] methionine into the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) for 8 h has been confirmed further by immunoprecipitation with anti-spinach RuBisCO. This competent in vitro translation system should be useful for future studies of chloroplast protein synthesis and gene expression.     

Effect of endothelial-cell-conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.     

Characterization of a sulfotransferase responsible for the 4-O-sulfation of terminal beta-N-acetyl-D-galactosamine on asparagine-linked oligosaccharides of glycoprotein hormones

The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2 Man alpha-. Using a chemically synthesized trisaccharide GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOCH3 (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-O-sulfation of the terminal beta-D-GalNAc. GGnM-MCO is incubated with a bovine pituitary membrane extract and [35S]3'-phosphoadenosine 5'-phosphosulfate ([35S]PAPS). The sulfated product [35S]SGGnM-MCO is separated from [35S]PAPS, PAPS degradation products and endogenous sulfated products by a two-step procedure utilizing an Ecteola cellulose column and a Sep-Pak (C18) cartridge. Characterization of the [35S]SGGnM-MCO produced in the assay indicates that sulfate is incorporated exclusively on the 4-position of GalNAc. Linear incorporation of sulfate into GGnM-MCO can be maintained for greater than 10 h. GGnM-4-sulfotransferase has a pH optimum of 7.2, requires the presence of a reducing agent, and is stimulated by, but does not require, divalent cations. Initial velocity studies indicate an apparent Km (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 microM and for GGnM-MCO of 9 microM. Incorporation of sulfate into the trisaccharide is stimulated 3-fold by the presence of basic proteins including deglycosylated LH. The stimulation by deglycosylated LH suggests that the protein component of glycoproteins that bear oligosaccharides terminating with GalNAc-GlcNAc-Man- may modulate GGnM-4-sulfotransferase.     

Identification of a tyrosine residue at a nucleotide binding site in the beta subunit of the mitochondrial ATPase with p-fluorosulfonyl[14C]-benzoyl-5'-adenosine.

The mitochondrial F1-ATPase is irreversibly inactivated by the adenine nucleotide analogue, p-fluorosulfonylbenzoyl-5'-adenosine. This inactivation is partly prevented by the presence of bound adenine nucleotides. Inactivations of the ATPase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine were most efficiently accomplished with the nucleotide-free enzyme at pH 7.0, in a buffer containing 20% glycerol. Under these conditions, 4.2 g atoms of 14C are incorporated per 350,000 g of enzyme when the ATPase is inactivated by 90% by its reaction with 2 mM p-fluorosulfonyl[14C]benzoyl-5'-adenosine. Isolation of the component polypeptide chains of the labeled ATPase showed that all of the radioactivity was associated with the two largest subunits. The isolated alpha subunit contained 0.45 g atom of 14C/mol and the isolated beta subunit contained 0.88 g atom of 14C/mol. Hence, the inactivation can be correlated with the incorporation of 14C into the beta subunit. This suggests that the hydrolytic site of the enzyme resides on this subunit. The majority of the radioactivity in a tryptic digest of labeled beta subunit is contained ina tryptic peptide that has the following amino acid sequence: Ile-Met-Asp-Pro-Asn-Ile-Val-Gly-Ser-Glu-His-Tyr-Asp-Val-Ala-Arg, where Tyr is the radioactive derivative of the tyrosine residue that was sulfonylated during the inactivation.