On the optical activity of ionized tyrosyl residues in ovine lutropin.

The effect of alkali on the circular dichroic (CD) spectra of ovine lutropin and its subunits has been studied. Mild alkaline pH induces the appearance of a new optically active band in the 250-nm region of the spectra of lutropin without any detectable alteration in the secondary structure of the protein. This change is reversible and can be correlated with ionization of 2--3 exposed tyrosyl residues in the intact hormone. In a previous report from this laboratory it was concluded that the three exposed tyrosyl residues are located in the alpha subunit, in positions 21, 92 and 93 [Burleigh, B.D., Liu, W.-K, and Ward, D.M. (1976) J. Biol. Chem. 251, 308--315]. Nitration of these residues lowers the pH at which the intensity of the 250-nm band is maximal. The importance of the tyrosyl residues of lutropin alpha (as opposed to those of lutropin beta) is also supported by the similarity of the effect of alkali on the CD spectra of lutropin and lutropin alpha. Further evidence for this involvement was also obtained by a comparison of the alkali-induced changes of refolded lutropin (alpha + beta recombinant) and the product obtained by recombination of des-(92--96)-lutropin alpha (obtained from carboxypeptidase treatment of the alpha-subunit) and lutropin beta. The results indicate that removal of tyrosines alpha 92 and alpha 93 results in a decrease of the intensity of the 235-nm band of ovine lutropin (at pH7.5) as well as that of the 250-nm band observed under alkaline conditions. It is therefore concluded that the 250-nm band observed in alkaline solutions of lutropin arises (at least partially) from the red shift produced in the short-wavelength optically active band of tyrosines alpha 21, alpha 92, and alpha 93 upon ionization.     

Studies on pituitary follitropin. V. Isolation of the subunits of the human hormone and reaction of the amino groups with acylating agents.

The alpha and beta subunits of human follitropin were isolated in a high state of purity. The tryptophan fluorescence of the native hormone and the isolated beta subunit are different. The N-terminus of the alpha and beta subunits was identified as valine and aspartic acid respectively. While recombination of the isolated alpha and beta subunits restores the electrophoretic mobility of the intact hormone, its receptor binding activity cannot be fully regenerated. Substitution of the human follitropin alpha by an ovine lutropin alpha subunit, to form a recombinant with the follitropin beta subunit, generates a complex with 2-3 receptor binding activity of the native human follitropin and the same activity as ovine follitropin. Acylation of the intact hormone does not disrupt the quaternary structure but leads to complete inactivation. Acylation studies with the subunits suggests the crucial role of the epsilon-amino groups of the alpha subunit in determining biological activity.     

Rabbit lutropin: preparation, characterization of the hormone, its subunits and radioimmunoassay.

The purification of rabbit lutropin is described. A product with a potency of 1.53 X NIH-LH-Sl was obtained as assayed by the ovarian ascorbic acid depletion assay. In a homologous radioimmunoassay, which is described, rabbit lutropin has a potency 4.83 X NIH-LH-Sl. In a radioligand assay, utilizing labeled ovine lutropin as the trace, the relative potency was 0.47 X NIH-LH-Sl measured by 50% inhibition comparison since rabbit lutropin response in this system did not parallel ovine lutropin. A counter-current distribution procedure for separation of rabbit lutropin subunits is described. Amino acid composition of the isolated subunits and intact rabbit lutropin was determined. The carbohydrate composition of the latter is presented; only amino sugar determinations are available for the subunits. The NH2-terminal amino acids are phenylalanine (alpha subunit) and alanine (beta subunit). Preliminary data on COOH-terminal amino acids are provided.     

Further characterization of the ovine lutropin alpha and beta subunits prepared by the salt precipitation method.

The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the alpha subunit. The subunits possessed low activity. The alpha and beta subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method alpha subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized alpha subunit combines with the native beta subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structur of the reduced alpha subunit. The native lutropin alpha subunit effectively combined with follitropin beta subunit generating complete follitropin activity.     

Reaction of tetranitromethane with lutropin, oxytocin, and vasopressin.

Tetranitromethane reaction with intact ovine lutropin and its isolated subunits was studied using spectrophotometric measurements, amino acid analysis, and isolation of tyrosyl peptides. Tyrosyl residues in the beta subunit (beta37, beta59) did not react with tetranitromethane in the intact hormone, but were nitrated in the isolated subunit. The sequence and extent of reaction of tetranitromethane with the tyrosyl residues in the alpha subunit was alpha21 = alpha92 = alpha93 (in intact hormone or isolated subunit) greater than alpha 41 (reacted in isolated subunit only) greater than alpha 30 (reacted in isolated subunit in 8 M urea only). Polymerization was observed as a side reaction in agreement with previous studies. The degree of polymerization appeared to be related to both primary sequence and tertiary structure, and for lutropin had the relation: alpha subunit (93% polymerized) greater than intact hormone greater than beta subunit (less than 40%). Polymerization observed with vasopressin was significantly greater than with oxytocin; for these peptides the tyrosine residues in the monomeric product were converted to 3-nitrotyrosine. Neither 3-nitrotyrosine nor tyrosine was detected in the polymerized by-products. In the tetranitromethane reaction with intact ovine lutropin, other reaction products charcterized by absorption spectra were found. Peptides isolated from these products lacked the characteristic 428 nm abosrption maxima of 3-nitrotyrosyl peptides and showed instead absorption in the 310 to 350 nm region. Similar products from tetranitromethane reactions with di- and tripeptides containing tyrosine have been observed previously (Boyd, N.D., and Smith, D.B. (1971) Can. J. Biochem, 49, 154-161), but they have not been studied in proteins. A possible relationship to the polymerization side reaction is suggested.     

Differentiation of the contribution of the two subunits of lutropin to its in vivo activity

Porcine and ovine lutropins were acetimidated at amino groups to various degrees and the effects of the modification on the induction of ovarian ornithine decarboxylase activity were examined. A drastic (84%) loss of biological activity was observed upon modification of two amino groups in porcine lutropin. The results with ovine lutropin derivatives were quite similar. Circular dichroism measurements showed no conformational changes and dissociation into subunits was not observed in the derivatives. Accordingly, the loss of biological activity was not a by-product of conformational changes. It was concluded that lutropin carries a single binding site which is constructed by surface residues or regions located on both the alpha- and beta-subunits of the hormone.     

Effects of beta subunit acylation on lutropin receptor site binding.

The effects of various modifications on the beta subunit of lutropin have been studied using the binding characteristics of the reconstituted hormone in the rat testicular radioligand assay. Conditions for iodinating lutropin and lutropin derivatives were determined which resulted in 15 per cent specific binding when tested immediately and retention of 6 to 7 per cent specific binding even after storage for 6 months. Acetimidinyl, acetyl, and carbamyl derivatives of the beta subunit were prepared and combined with unmodified alpha subunit to form reconstituted lutropin. Modification of the beta subunit was shown to have no effect on the time course of binding to testicular receptors or, with one exception, on the extent of receptor saturation. Very high concentrations of lutropin reconstituted with acetylated beta subunit showed an anomalous binding behavior. Scatchard plots of the binding data support the view that the native hormone has a unique receptor affinity which is irreversibly disrupted by separation of subunits and that derivatization of the beta subunit does not alter this parameter further. These data also suggest that there are no significant differences in the amino groups modified on the beta subunit. Competition and preincubation tests for receptor sites that reacted only with modified lutropin and not with the native hormone were negative.     

The amino acid sequence of the rabbit glycoprotein hormone alpha subunit

The amino acid sequence of the alpha subunit of rabbit (lagomorph) lutropin (lLH) has been determined. Overlapping peptides from trypsin and chymotrypsin digestions were isolated by reverse-phase high-pressure liquid chromatography (HPLC). Sequencing was by the dansyl-Edman procedure. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH alpha subunit is (asterisks denote carbohydrate attachment sites): This proposed sequence is highly homologous with the porcine, murine, ovine, and bovine glycoprotein hormone alpha subunit sequences. Two unusual proteolytic cleavages were observed: (1) a cleavage by trypsin between Asn-77 and Ala-78, and (2) a cleavage by chymotrypsin between Ala-45 and Arg-46. Similar enzymatic cleavages were previously reported for equine chorionic gonadotropin alpha subunit by Wardet al. and for these sites in the ovine LH alpha subunit by Liuet al. Chymotrypsin cleaved on the carboxyl side of methionine sulfone residues at positions 51 and 75.     

Isolation of mRNA from bovine pituitary. The cell-free synthesis of the alpha and beta subunits of luteinizing hormone

RNA derived from bovine steer pituitary was translated in wheat germ cell-free extracts containing [35S]methionine. Antisera generated against purified denatured alpha and beta subunits of lutropin were used to demonstrate the synthesis of both proteins in vitro. The immunoprecipitated products of the cell-free system were resolved on sodium dodecyl sulfate/polyacrylamide gels and it was observed that the molecular weight of the immunoprecipitated alpha subunit protein was approximately 14,000, while that of the beta protein was estimated to be 16,000. Since the molecular weights of authentic alpha and beta subunits are 10,600 and 14,000 respectively, the cell-free products presumably represented their pre-protein forms. The ratio of the immunoprecipitated subunit pre-proteins was dependent on the magnesium concentration in the translation mixtures; at 2.1 mM, translation of lutropin alpha and beta mRNAs was comparable. RNA isolated from cow pituitary tissue directed the synthesis of fivefold less of the alpha and beta immunoprecipitated proteins than did steer RNA. Since the blood levels of gonadal steroids are higher in the cow, the results supported the hypothesis that lutropin alpha and beta mRNA biosynthesis is repressed by these steroids. The data also suggest that synthesis of lutropin alpha and beta subunits is coordinately expressed in certain physiological situations.     

In vitro activation of glycoprotein hormones. Hybridization of subunits from thyrotropin, lutropin and human choriogonadotropin.

In vitro assembly of thyrotropin alpha and beta subunits led to an increase in content of alpha helix and beta sheet very similar to that found for gonadotropins. This association-dependent active folding involved the burying of three tyrosine residues tentatively assigned to Tyr alpha 41, Tyr beta 37 and Tyr beta 59 and common to all studied glycoprotein hormones. In vitro hybridizations between alpha and beta subunits of various hormones (thyrotropin, lutropin and choriogonadotropin) from different species (ovine, bovine and human) triggered the same molecular events as assembly of homologous subunits: the burying of three tyrosine residues and the increase of periodic structure of the folding. These changes are slow, time-dependent processes. Rates and yields of hybrid formation measured by sedimentation analysis and difference spectroscopy of tyrosines are identical, within experimental error, with the rates and yields measured by the recovery of the biological activity either the stimulation of chick thyroids for thyrotropin-beta hybrids or binding to porcine testis receptors for gonadotropin-beta hybrids. Whatever the origin of the alpha subunit, the thyrotropin-beta hybrids were not able to bind to testis receptors although active on chick thyroids. Rates and yields of hybrid formation essentially depended on the origin of the beta subunit. All the hybrids could be dissociated at acid pH with rates similar to those of native hormone. The extension to thyrotropin and various hybrids of the structural features of the in vitro assembly already recognized for gonadotropins strengthens the hypothesis that one deals with a basic activation process which also occurs in vivo after the synthesis of the subunits.     

Porcine follitropin. Isolation and characterization of the native hormone and its alpha and beta subunits

The properties of porcine follitropin and its subunits which have not yet been characterized are presented. The porcine follitropin obtained has a biological potency of 81 times the National Institutes of Health Porcine Follitropin P-1 preparation. Its contamination by lutropin and thyrotropin amounted to 1 and 0.5 percent by weight respectively, as measured by radioimmunoassay. The alpha and beta subunits of porcine follitropin were obtained by incubation in an acidic urea solution followed by anion exchange chromatography. The amino acid composition of porcine follitropin alpha subunit was found to be identical to that of alpha chain of porcine lutropin and thyrotropin. These porcine alpha chains differ, nevertheless, markedly in their carbohydrate composition particularly with respect to their mannose and galactose contents. The amino-terminal residue of the follitropin alpha subunit is threonyl. The carboxy-terminal end of the alpha chain is of variable length. Cysteyl residue was detected at the aminoterminal end of the follitropin beta chain with glutamic acid at its carboxy-terminal end. Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassay and amounted to 0.5 and 0.1 percent by weight respectively.     

The amino acid sequence of the rabbit lutropin beta subunit

The amino acid sequence of the beta subunit of rabbit lutropin (lLH) has been determined. The amino terminus of about 97% of the beta subunit has a two amino acid extension (pyro-Glu-Pro) compared to other lutropin beta sequences. Overlapping peptides from trypsin and chymotrypsin digestions of the performic acid-oxidized beta subunit and trypsin digestion of the S-aminoethylated cysteine beta subunit were isolated by chromatography on TSK Fractogel 40F and high-pressure liquid chromatography (HPLC). Sequencing was by a combination of the dansyl-Edman method and the direct Edman method. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH subunit is: This sequence is highly homologous to the other known lutropin beta subunits, especially rat and pig lutropin beta (91%). Partial cleavage of the peptide bond between Asp-79 and Pro-80 was observed during cyanogen bromide treatment. Rabbit thyrotropin and thyrotropin beta subunit copurified with lLH and lLH except at a final chromatography on Sephadex G-100.     

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits.

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The alpha-subunit possesses almost 3 times as much total carbohydrate as the beta-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The alpha-subunit begins with the sequence NH2-Phe-Pro (Gly or Pro) ... and terminates with isoleucine. The beta-subunit has the sequence NH2-Ser-Pro-Gly ...; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.     

Free alpha-like material from bovine pituitaries. Removal of its O-linked oligosaccharide permits combination with lutropin-beta

Further characterization of the free alpha subunit immunoreactive material, not combined with beta subunit in extracts of bovine pituitaries, shows that the only significant modifications, relative to alpha subunits themselves, are the oligosaccharide O-linked to threonine-43, and heterogeneity of the carboxyl terminus. Removal of the O-linked carbohydrate with a mixture of glycosidases from Streptococcus pneumoniae results in an alpha-like material capable of combining with lutropin beta subunit and, thus, the presence of the oligosaccharide is responsible for the inability of the free alpha-like material to combine with beta subunits. Amino acid compositions of tryptic peptides spanning the entire sequence indicate no change in amino acid sequence of the free alpha-like material as compared to lutropin alpha. Further, based on the similar behavior reverse phase high performance liquid chromatography of the tryptic peptides as compared to their lutropin alpha counterparts, it is concluded that no additional post-translational modifications are present. The N-linked oligosaccharides of the free alpha-like material most likely contain terminal O-sulfated N-acetylhexosamines (as do the asparagine-linked carbohydrates from the pituitary hormones) as indicated by the presence of 3 mol of sulfate/mol of free alpha-like material and the resistance of these oligosaccharides to enzymatic deglycosylation. The O-linked oligosaccharide does not contain sulfated residues.     

Comparison of the thermal stability characteristics of native and deglycosylated ovine pituitary lutropin

The receptor binding, immunological and biological activities of native ovine lutropin were almost completely eliminated when aqueous solutions of the hormone were kept in a boiling water bath for 30 or 60 min. Similar exposure of chemically deglycosylated lutropin revealed that this preparation was relatively more stable to heat treatment. The conformational features of deglycosylated lutropin required for receptor binding and immunological activity were significantly retained after thermal treatment. The heated deglycosylated lutropin solution still retained its ability to antagonize cyclic AMP accumulation stimulated by the native hormone in rat testicular interstitial cells. Specificity of receptor (lutropin) binding or inhibitory activity was not lost by heating of deglycosylated lutropin as revealed by lack of an effect in follitropin radio-receptor assays.     

Human pituitary lutropin. Isolation, properties, and the complete amino acid sequence of the beta-subunit.

The separation of alpha and beta subunits from human pituitary lutropin is described, and the complete amino acid sequence of the beta subunit is proposed. It consists of 115 amino acids with serine and glycine at the amino and carboxyl termini, respectively. The single carbohydrate moiety is linked to asparagine in position 30 and the single tryptophan of the lutropin molecule is located at position 8. The two methionine residues in lutropin-beta are in positions 41 and 42. In addition to COOH-terminal heterogeneity, evidence for internal peptide cleavages was observed.     

A chemical method for the deglycosylation of proteins

A simple and rapid chemical method for the deglycosylation of glycoproteins has been developed. The method involves the incubation of protein with trifluoromethanesulfonic acid at 0 degrees C from 0.5 to 2 h followed by the neutralization of the acid with aqueous pyridine at -20 degrees C. The method has been applied effectively to fetuin, ovine submaxillary mucin, ovine lutropin, and human choriogonadotropin. In 1 h almost all of N- and O-linked carbohydrates from ovine lutropin and human choriogonadotropin, with the exception of the linkage N-acetylglucosamine or N-acetylgalactosamine, were removed. Similarly, in 1 h all N-linked carbohydrates, excepting again the linkage sugar, in fetuin were degraded. Longer reaction times up to 2 h completely removed the O-linked carbohydrate chains from fetuin and ovine submaxillary mucin. The deglycosylated hormones thus prepared retained their immunological and biological activities.     

Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. Properties of the Arg beta 43 cleaved hormone

Ovine lutropin (oLH) and its beta subunit (oLH beta) were nicked by short-term incubations with endoproteinase Arg-C. Isolated oLH beta was rapidly nicked and converted from an Mr 18,000 band on sodium dodecyl sulfate-polyacrylamide gels to an Mr 13,000 band. Partial nicking of only the beta subunit in intact oLH was also observed as indicated by the appearance of small amounts of the Mr 13,000 band detected in Arg-C-treated oLH samples. The alpha subunit was protected by association with the beta subunit, but free alpha subunit was rapidly degraded. Sequence analysis of nicked oLH beta indicated that one of the peptide bonds on either side of Arg43 was cleaved by the protease, with a slight preference for the amino side of this residue. Nicked oLH beta was reassociated with oLH alpha, and the resulting dimer was separated from unrecombined subunits. The biologic activity of nicked oLH beta + oLH alpha in an LH radioligand assay was only 2% that of intact oLH.     

Identification of in vitro synthesized pituitary glycoprotein alpha subunit. Translation of a possible precursor.

Bovine pituitary RNA was translated in heterologous cell-free systems derived from wheat germ and reticulocyte lysate. Analyses of the cell-free products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed three major proteins, exhibiting apparent molecular weights of 25,000, 24,000, and 14,000. The two larger products were identified as preprolactin and pregrowth hormone by immunoprecipitation and thus demonstrated the fidelity of pituitary RNA translation. The 14,000-dalton product was shown to be immuno-precipitable with specific bovine lutropin (LH)alpha antisera. Since this protein is 3000 to 4000 daltons larger than the apoprotein form of the alpha subunits, it suggests that the subunit is synthesized in precursor form. The immunological specificity was further demonstrated by the successful competition with unlabeled alpha subunit plus the failure to immunoprecipitate this product using specific antisera to other pituitary hormones. Although specific antisera to bTSH(thyrotropin)beta and bLH(lutropin)beta failed to immunoprecipitate the 14,000-dalton product, LHbeta antisera precipitated a product with a molecular weight of approximately 18,000. Since the alpha and beta antisera specifically precipitated different products, and since a larger immunoprecipitable product was not detected, the results suggest that the two subunits are synthesized separately.