Leo E. Reichert

Chemical Studies of Luteinizing Hormone from Human and Ovine Pituitaries

Publisher Summary This chapter discusses chemical studies of luteinizing hormone (LH) from human and ovine pituitaries. These studies have found that ovine LH can be dissociated into subunits. The chapter also discusses the studies of disulfide bonds in the ovine LH. Disulfide cross linkages have been established for numerous proteins. The approach is straightforward. One simply breaks the protein into smaller peptides using the several specific cleavage procedures available to protein chemists, isolates the disulfide-containing peptides, and identifies which parts of the known sequence provided the peptide, and thus, which particular half-cysteine residues must be cross linked by disulfide bonds. The chapter also discusses various procedures in which many variants of Papkoff-Samy procedure have been taken into account to induce a separation of the human LH subunits. However, the procedures encountered several difficulties and the conclusions were somewhat subjective. The conclusions revealed that (1) the human LH appears either to dissociate less readily or reassociate more readily, (2) the human LH-α is less soluble in the lower phase of the Papkoff-Samy system than other species of LH, and (3) the insolubility of human LH-α favors co-precipitation of the human LH-β as a form of denatured LH that may have an indefinite composition ratio of the two subunits.

The effects of desialylation on the biologic and immunologic activity of human pituitary luteinizing hormone.

Abstract A highly purified preparation of human pituitary luteinizing hormone was treated with neuraminidase resulting in 94% removal of the sialic acid moiety. This led to a marked loss of biologic activity as measured by the ventral prostate weight and ovarian ascorbic acid depletion assays, and enhancement of the radioimmunologic activity.

Studies on Luteinizing Hormone and Its Subunits: Development and Application of a Radioligand Receptor Assay and Properties of the Hormone-Receptor Interaction,

Publisher Summary This chapter presents studies on luteinizing hormone (LH) and its subunits. It also describes the development and application of a radioligand receptor assay and properties of the hormone-receptor interaction. The use of biological assays for gonadotropins has progressed from the earlier qualitative tests to the more modern quantitative approaches. Biological assays suffer from ubiquitous and unavoidable problems related to the use of whole animals, which are reflected by the lack of adequate sensitivity and relatively poor precision. The high affinity of target tissues for tropic hormones offers the possibility for the development of sensitive, highly specific, and precise in vitro bioassays, utilizing the principles of competitive protein binding-saturation analysis. Such systems would have the advantages of relative independence from factors related to plasma half-life and in vivo metabolism and would allow structure-function studies that might not be feasible if whole animal assays were the only ones available. The chapter also discusses the application of this system to quantitation of LH activity in human and animal pituitary fractions, to the fundamental aspects of hormone-receptor binding, and to structure-function relationships of LH.

Evidence for the presence of a low molecular weight follitropin binding inhibitor in bovine follicular fluid.

The interaction of FSH with granulosa cell receptors has previously been studied by Nimrod et al. (1), Louvet and Vaitukaitis (2) and Lee and Takahashi (3) in the rat and by Zeleznik et al. (4) in the porcine. In this report, we describe the binding of FSH to bovine granulosa cells and summarize studies suggesting the presence in bovine follicular fluid of a low molecular weight inhibitor of FSH binding (FSH-BI) to bovine granulosa cell receptors.

A comparison of the properties of FSH from several species as determined by a rat testis tubule receptor assay.

The activity of purified ovine, bovine, porcine, rabbit, rat, and equine pituitary FSH and PMSG were compared with that of human FSH in the hCG-augmented ovarian growth assay of Steelman and Pohley (S-P) and in a rat testes tubule tissue receptor assay (TRA). Each of the pituitary FSH fractions studied showed greater activity in the TRA than in the S-P assay. In the TRA, all response curves were parallel with the exception of that for equine pituitary FSH. Highly purified LH preparations of the same species as the FSHs tested, were also examined in the TRA. Ovine and bovine LH showed no detectable FSH activity, while LH from the other species examined showed low activity, which presumably was due to residual FSH contamination. PMSG was an exception in that it showed greater activity in the in vivo S-P assay than in the in vitro TRA. This unique relationship is attributed to the very long plasma half-life of PMSG compared to that of the other FSH fractions tested. The more general phenomenon of greater in vitro vs in vivo activity of the FSHs can be explained by the insensitivity of the TRA to factors of metabolism that influence complete expression of in vivo activity.

Properties of particulate and detergent-solubilized adenylate cyclase of rat testis

Basal, fluoride and follitropin stimulated activities of adenylate cyclase have been studied in the testes of immature rats. The enzyme was maximally activated (about twice the basal activity) by low concentrations of follitropin, with an apparent Km of about 9 . 10(-10) M. Both Mg2+ and Mn2+ activate the enzyme but the apparent Ka for Mg2+ is about 10 times that for Mn2+. However, the apparent Km values for MgATP2- and MnATP2- are nearly the same (1.4 . 10(-4) M) and the cation activation of the enzyme is mainly through an increase in V. Ca2+ inhibited all expressions of testicular adenylate cyclase activity. Follitropin and fluoride stimulated adenylate cyclase activity at all Mg2+ concentrations but did not significantly affect the apparent Ka for Mg2+ or the Km for the substrate (MgATP2-). The stimulatory effect of the hormone or fluoride is mainly through an increase in V. Testicular adenylate cyclase could be solubilized with Triton X-100 or Lubrol-PX. The detergent-solubilized enzyme exhibited Km for substrate and Ka values for divalent cations similar to those of the membrane-bound enzyme and remained responsive to stimulation by fluoride. The stimulatory effect of follitropin, however, was lost. Responsiveness to follitropin was also lost by membrane-bound adenylate cyclase after treatment with phospholipase, although the fluoride effect was unchanged. These results reflect the essential role of lipids in the regulation of the follitropin-specific response.

Bovine luteinizing hormone. Circular dichroism and thermal difference spectra.

The circular dichroism of bovine luteinizing hormone in the far ultraviolet (200-240 nm) and near ultraviolet (240-320 nm) is reported as a function of pH, reduction and denaturation. The significance of side-chain ellipticity in the native and fully randomised hormone is critically examined, using denatured and reduced ribonuclease and insulin as bases for comparison. Disulphide groups make no measurable contribution to the side-chain ellipticity. Judged by the spectra of the subunits both isolated and recombined, those treatments which promote subunit disociation without causing chain unfolding, reversibly decrease the ellipticity in the near ultra-violet with minimal effect in the far ultraviolet. Thermal perturbation difference spectroscopy of bovine luteinizing hormone and its subunits shows that, in common with the ovine and porcine hormones, there are two tyrosine residues located at the interface between the subunits and inaccessible to water. Only two or three of the five water-accessible tyrosine residues are reactive to N-acetylimidazole.

Proteinases of the pituitaries of several fish and of a whale

Abstract Pituitaries from the salt water fish pollock, cod and hake have been shown to possess proteinase activity optimal at pH 4, but with no activity in the alkaline pH range. Pituitaries from the fresh water fish, carp, possessed markedly less proteinase activity at pH 4 than the salt water fish. Pituitary tissue from a marine mammal, the humpback whale, showed some proteinase activity in the acid range, and relatively marked activity in the alkaline range as compared with that present in fish pituitaries. Enzymes of pollock pituitaries active at pH 4 have been partially characterized with respect to inhibitors.

FSH Receptors in Rat Testes: Chemical Properties and Solubilization Studiies

The interaction of protein hormones with specific receptors is believed to initiate a series of events at the molecular level leading ultimately to a physiologically significant response. We have previously demonstrated the presence luteinizing hormone (LH) specific receptors in Leydig cell homogenates (1, 2) and follicle stimulating hormone (FSH) specific receptors in seminiferous tubule homogenate (3) of mature rat testes. These studies allowed development of tissue receptor assays for hLH (2, 4) and hFSH (5) suitable for measurement of gonadotropin activities in pituitary fractions as well as for various types of structure-function studies. We have extended our earlier binding studies with FSH (3) and in this report describe our recent observations on the interaction of 125I-hFSH with testicular receptors as well as preliminary studies on the chemical properties of the receptor and attempts directed towards its solubilization.

Purification and Properties of Pituitary and Urinary Luteinizing Hormone

We have for some time been interested in the development of methods for purification of luteinizing hormone (LH, ICSH) from a variety of sources, and have recently initiated studies on the comparative chemical and biological properties of this hormone. In the following sections, we will outline procedures which have proved effective for the purification of LH from human, ovine, bovine, porcine and equine pituitary glands and from human urine, and will summarize current information relative to its comparative physical and chemical properties.