Wan-Kyng Liu

A reevaluation of the amino acid sequence of human follitropin β-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin β-subunit sequence (hFSHβ), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSHβ with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.

Differential response of growing and regressing chicken ovaries to gonadotropic hormones

Abstract Steroid hormone secretion was investigated by growing left and regressing right ovaries from chick embryos under the stimulation of purified equine LH (eLH), equine FSH (eFSH), ovine LH (oLH), and ovine FSH (oFSH) in organ culture. Radioimmunoassay was used to measure the amount of steroid hormone released into the culture medium during 6 hr of incubation at 41°. Stimulation of steroid hormone production by mammalian gonadotropin was dose dependent. Studies on 17β-estradiol (E2) production from the growing left ovary of the 15-day-old embryo showed that steroid biosynthesis was stimulated fourfold over the basal rate of the control ovary by mammalian LH, whereas mammalian FSH could stimulate production by only 2.5-fold. Gonadotropin stimulation of testosterone (T) production by the growing ovary of the 15-day-old embryo showed a pattern similar to E2 production. The E2 production of the regressing right ovary was stimulated 3.7-fold by mammalian LH and 3.2-fold by mammalian FSH. Testosterone production by the regressing ovary was stimulated sevenfold above basal level by gonadotropin. By comparing the concentrations of hormone required for the half-maximum effect (ED50) for oLH, eLH, and eFSH, we found that the regressing ovary required 5 times more hormone for the same stimulating effect observed in the growing ovary. The regressing ovary required 10 times more oFSH than the growing ovary to achieve the same degree of stimulation. The results indicated that the regressing ovary has lower response to gonadotropic hormone stimulation than the growing ovary and embryonic chicken ovary receptors are more sensitive to LH-like activity than FSH-like activity. Furthermore, the right ovary, although regressing, still maintains this ability to discriminate between LH or FSH structure.

Hybrids from equine LH: alpha enhances, beta diminishes activity.

LH hybrids were prepared by combining eLH alpha and eLH beta with the corresponding subunits of oLH, pLH and hCG. Recombinants were isolated by gel filtration and assessed by SDS-polyacrylamide gel electrophoresis under both dissociating and non-dissociating conditions. All combinations of subunits produced hybrid LH molecules. Hybrids prepared by combining eLH beta with oLH alpha, pLH alpha or hCG alpha were very inactive in rat radioligand and Leydig cell in vitro bioassays. Hybrids prepared with eLH alpha were very active in both assays. The greatest potentiating activity was observed when eLH alpha was combined with pLH beta. The resulting hybrid was 49 times as active as pLH in stimulating steroidogenesis by Leydig cells.

Studies of disulfide bond location in ovine lutropin β subunit

By combinations of selective chemical cleavage (cyanogen bromide), selective enzymatic cleavage (trypsin, thermolysin), and random cleavage (partial acid hydrolysis) a series of disulfide-containing peptides have been isolated from ovine lutropin beta subunit. These peptides suggest six disulfide linkages between half-cystine residues in positions 23-72, 26-110, 93-100, 34-88, 9-90, and 38-57. The latter pair was placed by elimination of other possibilities. The first three pairs are in agreement with a report by Chung, D., Sairam, M. R. and Li, C. H. (1975) Int. J. Peptide Protein Res. 7, 487-493; the pair 93-100 has also been detected by Reeve, J. R., Cheng, K. W. and Pierce, J. G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155, using partial reduction and alkylation. In an attempt to improve the efficiency of enzymatic attack, a preliminary partial reduction as per Reeve et al. [16] was done. In this instance a peptide suggesting an additional disulfide linkage between half-cystines 23-26 was obtained as well as peptides consistent with the 23-72 and 26-110 placements. This was interpreted as an artifactual opening and recombining during partial reduction-reoxidation to produce the 23-26 linkage. The placement of three disulfide bonds (34-88, 9-90, and 38-57) is in disagreement with the pairings Chung et al. [15] suggest for these six half-cystine residues. Six reasons for uncertainty in the placement of disulfide bonds are discussed. It is concluded the definitive placement of the disputed three disulfide bonds in ovine lutropin beta subunit remains an open question.

Preparation of lutropin with acetyl or acetimidinyl substituents on the amino groups of the β-subunit

The free amino groups in ovine lutropin beta subunit were acylated with acetic anhydride and methyl acetimidinate-HCl to produce the corresponding acetyl and acetimidinyl ovine lutropin beta derivative. These two derivatives recombined with ovine lutropin alpha as well as native ovine lutropin beta, but produced lutropin derivatives which were 33-50% less active than the ovine lutropin alpha + ovine lutropin beta in biological assays.

Functional Groups in Ovine Luteinizing Hormone and Their Receptor Site Interactions

Selective chemical reactions have long been used in the field of hormone research for the study of functional group relationships to biological activity. An early example of such a study applied to ovine LH is that of Li, Simpson and Evans (1) who employed ketene to acetylate LH (ICSH) and found that acetylation under very mild conditions inactivated the hormone. Geschwind and Li (2) found periodate oxidation inactivated ovine LH. Although in that study periodate was selected for its attack on vicinal hydroxyl groups in the carbohydrate moiety, these authors appreciated that other reactions might be taking place. In a later study from that same laboratory Gan et al. (3) demonstrated that periodate oxidation of ovine LH not only attacked the carbohydrate moiety but also the disulfide bonds in the molecule. Thus, although periodate oxidation inactivates ovine LH it is not known whether this inactivation results from destruction of the carbohydrate moieties, destruction of the disulfide bonds, or both. Our own laboratory made an extensive study of the effect of several reagents and various conditions upon the activity of ovine LH (4). Included in those studies were some on the effects of oxidation by performic acid which attacks the disulfide bonds and the thioether group of methionine. This reagent completely inactivated ovine LH. We also studied the effect of reduction with sodium and liquid ammonia on the biological activity of the hormone. Although the hormone was virtually inactivated by this treatment, it is difficult to be certain in such an experiment that there was not some reoxidation to the native hormone to account for the small residual activity observed. There are also other technical difficulties in dealing with a hormone in the reduced form at the very dilute concentrations which must be used for bioassay.

LH-Binding Inhibitors from the Corpus Luteum

The initial observations of LH receptor binding inhibitor activity were quite serendipitous. A graduate student (Dr. Kuo-Pao Paul Yang) attempted to use some ovaries from psuedopregnant rats as a source of receptors for luteinizing hormone. These ovaries had been stored for quite some time at -20°. When Paul attempted to measure LH-binding in a crude homogenate of these ovaries no significant binding was observed. However, in the residuum after 1,2 or 3 washings there was significant specific binding for LH. Our first studies (Yang et al., 1976a) demonstrated that the binding inhibition resided in both a low molecular weight (dialyzable) and a high molecular weight (non-dializable) fraction. We were aware of the earlier studies of Bhalla and Reichert (1974) concerning an aqueous ethanol solubilized fraction from testicular tissue which interacted directly with gonadotropins as shown by physical properties. Reichert and Bhalla had suggested the material behaved as though it were a solubilized portion of the hormone receptor. We demonstrated, at least for the low molecular weight form of the LH.RBI, that such a direct interaction of our material with LH was not obtained.

Carbohydrate components of the glycopeptides of ovine lutropin

The three tryptic glycopeptides from ovine lutropin, in which two were from the alpha-subunit (alpha-56 and alpha-82 glycopeptides) and one from the beta-subunits (beta-13 glycopeptide), have been isolated and their carohydrate compositions analyzed. The results indicate that the alpha-56 glycopeptide has the highest amount of carbohydrate, whereas the beta-13 glycopeptide has the least. In general, each of the glycopeptides has similar distribution of various sugars, i.e. high in mannose and glucosamine and low in fucose, sialic acid, galactose and galactosamine. Within the limit of experimental error, the sum of their carbohydrate composition is in agreement with the published data on the intact hormone or separated subunits.