Immunoreactive Parathyroid Hormone in Circulation of Man

WE have reported that parathyroid hormone (PTH) is secreted from the parathyroid in vivo as a polypeptide of eighty-four amino-acids, identical to the hormone stored in the glands (molecular weight of 9,500), but that the hormonal polypeptide is cleaved after it enters the general circulation¹. A large hormonal fragment from this cleavage, with a molecular weight of approximately 7,500, has been identified in the circulation. The fragment differs immunologically from the hormone secreted and extracted from the glands¹. To analyse the biological significance of the metabolism of the hormone and the chemical nature and hormonal activity of the large circulating fragment, we have developed radioimmunoassays that specifically measure the amino-terminal (N-assay) and carboxyl-terminal (C-assay) regions of the hormonal molecule. We now report that much higher concentrations of immunoreactive hormone are found in the general circulation by the C-assay than by the N-assay. The studies with the N-assay indicate that the large fragment has lost a portion of the amino-terminal sequence required for biological activity⁹. Since the fragment is present in much higher concentration than native uncleaved hormone, we must conclude that much of the immunoreactive PTH detected in the circulation is biologically inactive.     

Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.     

Synthesis of Mono-PEGylated Growth Hormone Releasing Peptide-2 and Investigation of its Biological Activity

The purpose of this study was to investigate an efficient synthetic route to the mono-PEGylated growth hormone releasing peptide-2 (GHRP-2) and its biological activity in vivo. The commercially available key PEGylating reagent, mPEG-NHS ester, was successfully utilized to the synthesis of mono-PEGylated GHRP-2, during which the PEGylation profiles of GHRP-2 were monitored by high-performance liquid chromatography (HPLC). The product was purified by cation exchange chromatography, and its biological activity was conducted in rats. The desired mono-PEGylated GHRP-2 as the major product was readily obtained in anhydrous aprotic solvent, such as dimethyl formamide (DMF) and dimethylsulfoxide (DMSO), when the molar ratio of mPEG-NHS ester to GHRP-2 was fixed to be 0.8:1. The products were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The evaluation of the biological activity for the products showed that the mono-PEGylated GHRP-2 gave a more stable activity than GHRP-2, suggesting that PEGylation led to the increase in the half-life of GHRP-2 in plasma without greatly impairing the biological activity. PEGylation of the GHRP-2 is a good choice for the development of the GHRP-2 applications.KEY WORDS: growth hormone, growth hormone releasing peptide-2, mono-PEGylated GHRP-2, mPEG-NHS, PEGylation     

New data on the biological activity of pituitary somatotropin fragment 77-107

Some new evidence on the biological activity of somatotropin fragment 77-107 is given. This fragment was prepared from whale somatotropin by tryptic hydrolysis. Beside the previously established ability of the hormone to increase the width of the tibial epiphyseal cartilage in hypophysectomized rats ("tibia" test) two other properties of the fragment indicative of its growth-promoting activity were established. The fragment enhances DNA biosynthesis in cultured human fibroblasts and increases the somatomedin content in blood serum of hypophysectomized rats. However, the fragment unlike the native hormone does not exert any metabolic action on adipose tissue "in vitro", i. e. does not stimulate the nonesterified fatty acid release into the medium. A comparison of the biological activity spectrum of native somatotropin and of its fragment 77-107 suggests that the biochemical information required for the realization of a prolonged growth-promoting effect and a relatively rapid action of the hormone on lipid and carbohydrate metabolism is contained in different parts of the polypeptide chain.     

Characterization of a novel Xenopus tropicalis cell line as a model for in vitro studies

Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.     

cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3 - cotransporter pNBC1 in human embryonic kidney (HEK293) cells

Background

The fission yeast Schizosaccharomyces pombe is widely-used as a model organism for the study of a broad range of eukaryotic cellular processes such as cell cycle, genome stability and cell morphology. Despite the availability of extensive set of genetic, molecular biological, biochemical and cell biological tools for analysis of protein function in fission yeast, studies are often hampered by the lack of an effective method allowing for the rapid regulation of protein level or protein activity.

Results

In order to be able to regulate protein function, we have made use of a previous finding that the hormone binding domain of steroid receptors can be used as a regulatory cassette to subject the activity of heterologous proteins to hormonal regulation. The approach is based on fusing the protein of interest to the hormone binding domain (HBD) of the estrogen receptor (ER). The HBD tag will attract the Hsp90 complex, which can render the fusion protein inactive. Upon addition of estradiol the protein is quickly released from the Hsp90 complex and thereby activated. We have tagged and characterised the induction of activity of four different HBD-tagged proteins. Here we show that the tag provided the means to effectively regulate the activity of two of these proteins.

Conclusion

The estradiol-regulatable hormone binding domain provides a means to regulate the function of some, though not all, fission yeast proteins. This system may result in very quick and reversible activation of the protein of interest. Therefore it will be a powerful tool and it will open experimental approaches in fission yeast that have previously not been possible. Since fission yeast is a widely-used model organism, this will be valuable in many areas of research.     

Hormone als Schadstoffe?

Environmental estrogens Endocrine disrupters are environmental substances which interfere with the hormone system of organisms and thereby induce adverse effects. They exert their biological activity either by disrupting hormone metabolism or by imitating the biological action of the endogenous hormones. In the aquatic environment, an important group of endocrine disrupters is represented by the estrogen‐active compounds, which mimic the female sex hormone, 17β‐estradiol. Both laboratory experiments and field studies on fishes have demonstrated that already very low concentrations of environmental estrogens are able to induce disturbances in the hormone system and hormone‐regulated processes of fishes.     

Binding of parathyroid hormone to bovine kidney-cortex plasma membranes

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with (125)I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. (75)Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca(2+) inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.     

A rapid, high-yield method of producing mono-[125I]A14 iodoinsulin

We have developed a rapid method for producing homogeneous mono-[125I]A14 iodoinsulin with high specific activity and yield. After iodination by the lactoperoxidase method, the labeled peptides were applied to a C18 Porasil pre-column, washed with aqueous buffer to eliminate the free [125I]-iodide and placed "in-line" with a C-18 HPLC column; mono-[125I]A14 iodoinsulin was then eluted isocratically with 29% acetonitrile in 16 minutes. The labeled hormone was extremely stable, and proved suitable for various biological studies.     

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both2,3 and2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.     

Cleavage of growth hormone by rabbit liver plasmalemma enhances binding

Several studies have shown that proteolytic cleavage can enhance the biological activity of the growth hormone (GH) molecule. It seemed possible, therefore, that proteolytic modification of GH might be a normal function of GH-target tissues. Plasmalemma-enriched fractions isolated from rabbit liver were found to contain a proteinase(s) which cleaves the large disulfide loop of human and rat GH. The proteolytic activity was specific to plasmalemma-enriched fractions in that much lower activities were observed in microsomal-enriched fractions prepared from the same livers. The plasmalemmal proteinase(s) may be a trypsin-like enzyme because proteolytic activity was decreased by two serine proteinase inhibitors. Inhibition by unlabeled human GH of 125I-GH binding to receptors did not prevent cleavage of the tracer; therefore, hormone-receptor interaction was not required for cleavage of the GH molecule. In binding studies, cleaved GH associated more readily than did intact hormone with rabbit liver receptors. These studies suggest that plasmalemma-enriched fractions prepared from rabbit liver contain a proteinase which cleaves the GH molecule in a highly specific manner. Moreover, it is unlikely that inactivation of GH is the function of this limited proteolysis because cleaved hormone is bound preferentially by at least a subset of receptors in rabbit liver.     

Preparation and characteristics of porcine growth hormone.

A recently published method for preparation of porcine growth hormone resulted in a highly purified protein with good biological activity. However, after storing for some months the originally homogeneous hormone separated again into several fractions when rechromatographed on ion exchange columns. The biological activity, found in one of these fractions, was clearly diminished compared with the activity of a freshly prepared hormone. In the present paper a modified procedure is described for the isolation of a more stable porcine growth hormone. The influence of ions, involved in buffers of the same molarity and the same pH, upon ion exchange chromatography of porcine growth hormone is discussed. The purified hormone shows high biological activity in the tibia test and is free of activities of other pituitary hormones. The molecular weight is about 20 000; only phenylalanine is found as N-terminal as well as C-terminal amino acid; the amino acid composition resembles neither that of porcine growth hormone described in literature nor that of human growth hormone.     

Conformational and dynamic considerations in the design of peptide hormone analogs

Efforts to understand the chemical-physical basis for peptide hormone and neurotransmitter action requires integration of conformational parameters and biological properties. Since most peptide hormones are conformationally flexible, the question arises as to which of the manifold of conformations is of biological significance. In molecular terms, it is necessary to carefully distinguish chemical-physical features important to binding (the binding message) from those involved in transduction (the biological activity message). One approach to this involves the design, synthesis, and conformational analysis of semirigid hormone analogs. The distinction between binding and transduction can best be examined by evaluation of full biological profiles of partial agonists, antagonists, and analogs with prolonged biological activity. Using this multidisciplinary approach, we have prepared several semirigid [Pen¹]-oxytocin antagonist analogs and evaluated their conformational properties and biological activities. Specific conformational features can be related to inhibitory activities in several cases. On the basis of structure–activity relationships and conformational considerations, we have designed a series of conformationally restricted cyclic and acyclic analogs of the linear peptide α-melanotropin. Some of these peptides have exceptionally prolonged in vivo activity (weeks), and others exhibit superagonist potency (10,000 times the native hormone). We have evidence that potency and prolonged activity have different structural and conformational requirements. It is suggested that potency is primarily a function of receptor recognition (the binding message), whereas prolonged activity is related to transduction (the biological activity message).     

Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding.

Recent studies on the glucagon antagonist des-His1-[Glu9]glucagon amide have resulted in pure inhibitors of the hormone, suggesting that the inhibitory properties may be centered around position 9. The present study was designed to investigate the chemical characteristics of substitutions in position 9 of glucagon that determine binding affinity and biological activity. Twenty replacement analogs of position 9 of glucagon were synthesized and assessed for their ability to bind to the glucagon receptor in rat hepatocyte membranes and to activate adenylate cyclase. Any substitution of aspartic acid 9 was accompanied by a severely diminished capacity to transmit the biological signal, while retaining receptor binding affinity. These results are an indication of an uncoupling of receptor binding and biological activity at this locus and define a central role of aspartic acid 9 in glucagon activity. Single replacement or deletion of either His1 or Asp9 in glucagon caused a 20- to 50-fold decrease in cyclase activity, whereas these same changes made in tandem caused virtually complete loss of activity, with decreases of 10(4)-to 10(6)-fold. These observations have led us to speculate that, at the molecular level, the region of glucagon required for transduction of the biological response may be distinct from the binding region and is mediated by a coupled interaction between His1 and Asp9 of the hormone and a complementary functional site of the glucagon receptor.     

Somatostatin analogs which define the role of the lysine-9 amino group

Structure-activity studies of the lysine residue in the highly active cyclic hexapeptide somatostatin analog cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) confirm the importance of the lysine amino group for biological activity through the loss of activity seen on replacement of lysine by ornithine, arginine, histidine and p-amino phenylalanine. Three analogs containing thialysine, gamma- and delta-fluorolysine were equipotent to the parent as inhibitors of insulin, glucagon, and growth hormone release. The pKa's of the amino groups in these equiactive peptides ranged from 8.23-9.4. The lack of a correlation between the basicity of the amino groups and the biological activities suggests that deprotonation is not required for biological activity.     

Effects of estrogen and growth hormone on steroid sulfatase activity and estrogen binding in rat liver

It is now well established that the activity of certain liver enzymes displays sex differences and that administration of human growth hormone to male rats alters the liver metabolism in a "female" direction. In this work we studied steroid sulfatase activity and binding of estradiol-17 beta in livers from intact rats and found a sex difference, with considerably higher enzyme activity in male as compared to female liver tissue. Continuous infusion of native and recombinant human growth hormone and estradiol-17 beta to male rats reduced sulfatase activity to "female" levels. A specific binding of estradiol-17 beta with receptor properties was found in the rat livers, but the concentration of binding sites did not change after administration of growth hormone or estradiol in this group of intact animals. Our data confirm previous reports that continuous administration of human growth hormone "feminize" liver metabolism, and since estradiol was found to have an identical effect on sulfatase activity it is suggested that the effect of estradiol-17 beta in this respect may be indirect, mediated via an altered secretory pattern of rat growth hormone.     

Heterogeneity of chicken growth hormone (cGH). Identification of lipolytic and non-lipolytic variants

The identification and biological activity of chicken growth hormone (cGH) charge variants is described. On the basis of electrophoresis and immunoreactivity chicken pituitary glands contain at least two "charge" variants (Rf = 0.22 and 0.3) which have different net charge but similar molecular weight (26,300 d). Both are immunoreactive but show different bioactivity with adipose explants, band 0.22 being lipolytic whereas band 0.3 appears to be inactive. The abundance of these cGH bands vary with age, both being higher in young birds and lower in adults. These results suggest that cGH variants may have different biological actions.     

The mobile receptor hypothesis and "cooperativity" of hormone binding. Application to insulin.

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone. A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from "negative cooperativity." (2) Receptor occupancy and biological response may be coupled in a non-linear fashion. By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of "cooperativity." These considerations may also be pertinent to interpretations of other hormone-receptor systems and of various ligand-macromolecule interactions.     

The origin of glucocorticoid hormone oscillations

Oscillating levels of adrenal glucocorticoid hormones are essential for optimal gene expression, and for maintaining physiological and behavioural responsiveness to stress. The biological basis for these oscillations is not known, but a neuronal "pulse generator" within the hypothalamus has remained a popular hypothesis. We demonstrate that pulsatile hypothalamic activity is not required for generating ultradian glucocorticoid oscillations. We show that a constant level of corticotrophin-releasing hormone (CRH) can activate a dynamic pituitary-adrenal peripheral network to produce ultradian adrenocorticotrophic hormone and glucocorticoid oscillations with a physiological frequency. This oscillatory response to CRH is dose dependent and becomes disrupted for higher levels of CRH. These data suggest that glucocorticoid oscillations result from a sub-hypothalamic pituitary-adrenal system, which functions as a deterministic peripheral hormone oscillator with a characteristic ultradian frequency. This constitutes a novel mechanism by which the level, rather than the pattern, of CRH determines the dynamics of glucocorticoid hormone secretion.