Native oLH Research Articles

Evidence for two folding domains in glycoprotein hormone alpha-subunits

We reconstituted ovine (o) LH alpha from its amino- and carboxyl-terminal fragments obtained as follows. oLH alpha was nicked at Arg46-Ser47 with Arg-C protease. Nicked oLH alpha disulfide bonds were broken by sulfitolysis, and its N-terminal peptide and C-terminal glycopeptide were separated by Sephacryl S-200 chromatography. Both fragments were mixed, reduced, and reoxidized. Reoxidation products were chromatographed on Sephacryl S-200, and an alpha-monomer fraction was recovered. The putative nicked alpha-monomer fraction was reassociated with native oLH beta, and the resulting oLH derivative was isolated by S-200 chromatography with a reduced yield of 11% (intact subunits yield, 67% oLH). This preparation was 2.6% as active as oLH in a LH receptor binding assay. Two additional oLH derivatives were prepared. Cleavage at alpha Arg46-Ser47 alone, followed by reassociation with native oLH beta, produced Arg-C-nicked oLH alpha:oLH beta (14% yield) that was 3.3% as active as native oLH. Reduction-reoxidatio...

Renotropic and gonadotropic activity in homologous and heterologous hybrids of ovine luteinizing hormone and human chorionic gonadotropin subunits.

We previously demonstrated the renotropic activity of ovine and porcine LH, i.e. the stimulation of [3H]thymidine incorporation into the renal DNA of castrated hypophysectomized rats. We conducted this study to determine which condition is required to promote renotropic activity. We prepared homologous (oLH alpha.oLH beta, hCG alpha.hCG beta) and heterologous (oLH alpha.hCG beta, hCG alpha.oLH beta) hybrids from isolated oLH- and/or hCG-derived subunits. Native oLH and all hybrids showed gonadotropic activity when examined for cyclic AMP generation in cultured Leydig tumour cells (MA-10). Native oLH and the hybrids which showed renotropic activity were all found to contain at least one of the oLH subunits. Our findings point out the important, special role of oLH subunits in promoting renotropic activity. The condition required is suggested to be presence of a common structure in the two oLH subunits. Such a structure should consequently not be present in hCG. A carbohydrate moiety of oLH containing a sulphate group is proposed as a candidate, but the assumption remains to be demonstrated.

Adenylate cyclase stimulation and luteinizing hormone-receptor interaction in plasma membranes from rat testicular interstitial cells in relation to the chemical structure of the hormone. Role of Mg2+.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.

Ovine Luteinizing Hormone. III. Relationships between the Charge Heterogeneity of Partially Purified Subunits and the Native Hormone1

Extracts of anterior pituitaries from wethers were prepared by homogenization and centrifugation at 100,000 X g. When chromatofocused on pH 10.5-7.0 gradients, eight peaks of immunoreactive ovine luteinizing hormone (oLH) were observed: six exhibited apparent pIs in the range of 9.33-8.83, one eluted unbound (apparent pI greater than 9.8), and one was bound to the column (apparent pI less than or equal to 7.0). A portion of the same extracts was subjected to gel filtration on Sephadex G-100 Superfine to resolve native oLH and its uncombined subunits. oLH, oLH alpha, and oLH beta were present at concentrations of 0.907 +/- 0.127, 0.089 +/- 0.020, and 0.010 +/- 0.023 microgram/mg tissue, respectively, which translated to oLH alpha/oLH and oLH beta/oLH molar ratios of approximately equal to 0.19 and approximately equal to 0.02. Fractions containing immunoreactive oLH or uncombined subunits (oLH alpha and oLH beta) were pooled, lyophilized, and chromatofocused. Native oLH resolved from uncombined subunits by gel filtration displayed a similar pattern of isohormones to those in crude extracts. In contrast, three purified oLH preparations exhibited distinct chromatofocusing patterns. Uncombined oLH alpha in pituitary extracts resolved from native oLH by gel filtration exhibited a higher percentage (approximately equal to 37%) of acidic components when chromatofocused, while more than 97% of purified oLH alpha focused as basic forms having pIs greater than 8.9. When uncombined oLH beta in pituitary extracts was chromatofocused, more than half of the immunoreactivity was bound to the column (apparent pI less than or equal to 7.0); purified oLH beta displayed a nearly identical pattern. These results suggest that native oLH resolved from uncombined subunits by gel filtration displays a similar chromatofocusing profile to that of oLH in crude pituitary extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovine luteinizing hormone. II. Effects of castration and steroid administration on the levels of uncombined subunits within the pituitary.

Pituitaries were removed from rams, wethers, and wethers that received Silastic implants containing 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E2) or DHT + E2. After homogenization and centrifugation (100,000 X g), aliquots of the supernatants were subjected to analytical gel filtration on Sephadex G-100 Superfine to separate native ovine luteinizing hormone (oLH) from its uncombined subunits. Immunoreactive oLH and oLH subunits were quantified in the elution profiles to examine the effects of castration and gonadal steroid administration on the intracellular levels of uncombined oLH subunits. Pituitaries from rams contained 1.41 +/- 0.26, 0.191 +/- 0.024, and 0.0246 +/- 0.0043 micrograms oLH, oLH alpha and oLH beta per mg tissue, respectively, which translated to oLH alpha/oLH and oLH beta/oLH molar ratios of approximately equal to 0.29 and approximately equal to 0.04. Castration decreased the concentrations of oLH and its subunits by approximately 50%, but did not significantly alter the oLH alpha/oLH and oLH beta/oLH molar ratios. All three steroid treatments further decreased the concentrations of oLH and oLH beta. Pituitaries from DHT-implanted wethers exhibited similar oLH alpha/oLH and oLH beta/oLH molar ratios to rams and unimplanted wethers. However, in E2- or DHT + E2-implanted wethers, there was a greater reduction in the concentration of native oLH than in the uncombined subunits. Thus, both the oLH alpha/oLH and oLH beta/oLH molar ratios were significantly higher in E2- or DHT + E2-implanted wethers than in the other groups. The apparent molecular sizes of oLH or its subunits were not significantly altered by castration or steroid administration. These results suggest that DHT and E2 decrease the concentrations of uncombined oLH beta as well as native oLH in the pituitary, but do not appear to alter the apparent molecular size of either oLH or its uncombined subunits However, because the levels of uncombined subunits were not decreased to the same degree as oLH in E2-implanted wethers, estrogens may affect the process of oLH subunit combination or may result in the production of molecular forms of oLH that are easier to dissociate.

Deglycosylation of gonadotropins with an endoglycosidase.

A commercially available endoglycosidase (N-glycanase, Genzyme, Boston, Mass.) purified from Flavobacterium meningosepticum with a specificity for cleaving asparagine-linked carbohydrate moieties in glycoproteins was tested on several pituitary and chorionic gonadotropins as substrates. All intact hormones tested were resistant to the action of the enzyme as were all beta subunits from the respective gonadotropins. All alpha subunits, however, were susceptible to the enzyme as evidenced by a decrease in molecular size when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparative experiments with ovine luteinizing hormone subunit (oLH alpha) indicated that only 35-40% of the carbohydrate was removed after N-glycanase treatment, suggesting that perhaps only one of the two carbohydrate moieties was cleavable under the conditions employed. The enzyme-modified subunit (DG-oLH alpha) was able to recombine with untreated oLH beta. An in vitro steroidogenic bioassay (rat Leydig cell) showed that the recombinant (DG-oLH alpha-oLH beta) was about 22% as potent as the native oLH, but in a testicular membrane binding assay for LH, it was equal in potency to the native hormone in competing with the radioligand.

Guanidination of Ovine Luteinizing Hormone and Effects on Activity

The free amino groups in oLH, oLHalpha and oLHbeta were guanidinated by O-methylisourea. The epsilon-NH2 groups of lysine residues reacted bo substitute these positions in the sequence with the more basic homoarginine residue. The alpha-NH2 groups did not react under the conditions used. Guanidinated oLH or the products of guanidinated oLHalpha + native oLHbeta or guanidinated oLHalpha + guanidinated oLHbeta were inactive in two bioassay systems. Native oLHalpha + guanidinated oLHbeta, however, showed potencies of 39% to 55% of that observed with the native subunit recombinant or native oLH. Possible structural implications for hormone-receptor site interactions are discussed.

The Role of the Amino Group in Subunit Association and Receptor Site Interaction for Ovine Luteinizing Hormone as Studied by Acylation

Abstract The positively charged amino groups (in physiological pH range) of ovine luteinizing hormone (oLH) and its subunits (oLHα and oLHβ) were acylated to produce negatively charged (maleylated) or neutral groups (carbamylated) in their place. In both reactions, the number of modified e-lysine amino groups in luteinizing hormone (LH), LHα, and LHβ was 10, 9, and 2, respectively. The NH2 termini were also completely acylated in both reactions. Maleylation or carbamylation did not alter the hydrodynamic volume of oLH or oLHβ significantly, although a greater Stokes radius was obtained with the oLHα derivatives compared to native oLHα as judged by Kav values from molecular exclusion chromatography. Maleylated LHβ combined with native LHα as well as the native subunits recombine, but maleylated LHα recombined with native LHβ less effectively. Efficiency of recombination was judged by the quantity of material with a hydrodynamic volume similar to LH under specified recombination conditions. Maleylated LHα and maleylated LHβ recombined very poorly. The series of recombination products with carbamylated subunits were all obtained in yields comparable to those observed with the recombination of the native subunits. Biological activities of the various recombined products were examined by both the in vivo ovarian ascorbic acid depletion assay and the in vitro radioligand receptor assay and the results agreed. Intact oLH which had been carbamylated or maleylated, as well as the recombined subunit derivatives from these reactions were completely inactive. Either the carbamylated or maleylated oLHα plus native oLHβ, and native oLHα plus maleylated oLHβ gave a low biological activity with response characteristics different from that of the native hormone. The recombined native subunits, or native oLHα plus carbamylated oLHβ, gave products that were slightly less potent than native oLH, with response characteristics that paralleled those of the native hormone. These results suggest that the charges on lysine e-amino groups in the oLHα play a role in the recombination with oLHβ, but that the charge on the oLHβ amino groups is not crucial for subunit-subunit interaction. It may also be concluded that the charge on the free amino groups of oLHβ must be positive or neutral for receptor site interaction. At least a portion of the amino groups on the oLHα must carry a positive charge for maximum hormone-receptor site interaction.

Reversible folding of human chorionic gonadotropin at acid pH or upon recombination of the α and β subunits

Gonadotropic hormones share the property of being made of two non-covalently bound subunits, the CY and /I subunits. Isolated subunits have been found to be inactive either by biological or by radioligand receptor assays [ 1,2]. Circular dichroism (CD) measurements performed by several authors 13-51 on native ovine luteinizing hormone (oLH) and its isolated subunits strongly suggest at least a partial unfolding of the polypeptide chains upon dissociation and refolding upon reassociation and subsequent recovery of the biological activity. Recently it has been found by Aloj et al. [6] that native human chorionic gonadotropin (hCG) and hLH, but not their subunits, enhance ANS fluorescence, which seems to be a sensitive probe of the associated state. However, this property is so far limited to these luteinizing hormones since, for example, native oLH exhibits only a weak binding affinity for ANS (7 X lo3 M-’ at 37°C) and a very low enhancement (ten times less than hCG) of the quantum yield of fluorescence as found by Aloj et al. [6] and by Garnier (unpublished). It was suggested by studies of the chemical reactivity towards tetranitromethane or iodine that some tyrosine residues were exposed by dissociation of the subunits of ovine, bovine and porcine LH [7-lo]. The same conclusion was reached by difference spectroscopy [4] and CD measurements of oLH 13-51. Evidence IS presented here [fiat acid dissociation of hCG also resulted in a reversible unfolding of the subunits during which tyrosine residues were pcrturb-