Characteristics of a testosterone-estradiol binding globulin (TEBG) in goldfish serum

Neuroendocrine tissues in teleost fish aromatize androgen to estrogen at extraordinarily high rates. As part of a project in which we are studying the dynamics of sex steroid uptake, metabolism, and receptor binding in goldfish (Carassius auratus) brain and pituitary, we have identified and characterized a sex-steroid-binding component of serum. This protein has been designated a testosterone-estradiol-binding globulin (TEBG) since it bound testosterone (T) and estradiol-17 beta (E2) with high affinity (Kd = 1.9 and 2.1 nM, respectively) whereas other steroids were less effective ligands (5 alpha-dihydrotestosterone greater than progesterone = 11-ketotestosterone greater than estrone = estriol = diethylstilbestrol greater than cortisol). Scatchard analysis, disc gel electrophoresis and sucrose gradient centrifugation all indicate that T and E2 are bound by the same protein. The number of available serum binding sites (Bmax = 10(-7) M) greatly exceeded reported maximal levels of T and E2 in the same species and showed no obvious sex or seasonal differences. However, the steroid-TEBG interaction was unstable, exhibiting very short half-times of association (less than 15 min) and dissociation (less than 10 min). On the basis of comparison of the physicochemical characteristics of TEBG with other intracellular androgen-binding proteins in goldfish brain (androgen receptor, aromatase), we predict that the serum-binding protein would not limit but rather enhance exchange of T and E2 in central tissues.     

Genetic variations in human testosterone-estradiol binding globulin

The human testosterone-estradiol-binding globulin (hTeBG) is a plasma heterogeneous glycoprotein with high affinity for a number of circulating steroid hormones. The heterogeneity originates from differential glycosylation of a common protein precursor. Analysis of desialylated hTeBG by isoelectric focusing (IEF) has revealed that microheterogeneity could be partly attributed to variability in sialic acid content or rearrangement of amino acid composition. We have studied this possibility by the analysis of desialylated serum hTeBG by Western blotting of proteins previously separated on IEF-gels. Two distinct well-defined IEF patterns were identified. The most frequent consisted of two major IEF-bands of equal color intensity. The other pattern consisting of four IEF-bands was present in only 5.55% of the total serum samples analyzed. Family studies showed that these phenotypes were autosomally inherited with a simple Mendelian transmission and allele frequencies had an excellent agreement between the observed and expected phenotypes. Androgen affinity constants and serum concentrations of hTeBG variant were similar to those of normal hTeBG. Molecular analyses of each of the exons of hTeBG gene by denaturing gradient gel electrophoresis revealed the presence of a point mutation in exon 8. The studies presented herein confirm and extend previous reports on the existence of structural variants of hTeBG. In addition, the mutation reported in this study is probably the same as that recently identified within numerous ethnic groups throughout the world, thus further supporting the concept of a two allele gene worldwide concoding hTeBG.     

Solid phase method for measurement of the binding capacity of testosterone-estradiol binding globulin in human serum

A solid phase method for measuring the binding capacity of serum testosterone-estradiol binding globulin (TeBG) is described and compared with other methods. TeBG, a glycoprotein, is adsorbed from serum or plasma onto a solid phase matrix of concanavalin-A, a carbohydrate-specific adsorbent. The TeBG binding capacity is determined by Scatchard analysis of the binding of radioactive testosterone at physiologic pH, in standard test tubes, and without the addition of albumin. Transcortin binding of testosterone is inhibited by the addition of cortisol.The levels of TeBG binding capacity determined with this solid phase method showed an excellent correlation with levels determined by procedures using equilibrium dialysis (with added cortisol) or ammonium sulphate precipitation. The serum TeBG binding capacity was 0.798±0.064 (mean±SE) μg/100 mL in men (n=32), 1.06±0.13 in women (n=10), 2.18±0.19 in women taking oral contraceptives (n=4), 6.2±2.9 in hyperthyroid women (n=2), and 11.6±3.1 in pregnant women (n=5). The serum TeBG binding capacity determined in heparinized plasma did not differ from that determined in serum. The within-assay variation is 9.6% and the between-assay variation is 11.2%.This solid phase method for measurement of serum TeBG binding capacity is simple, precise, and reproducible, and gives values which correlate well with those determined by other methods.     

Corticosteroid binding globulin, testosterone-estradiol binding globulin, and androgen binding protein belong to protein families distinct from steroid receptors

The cDNA nucleotide sequences and the deduced amino acid sequences of human corticosteroid binding globulin (hCBG), human testosterone-estradiol binding globulin (hTeBG), and rat androgen binding protein (rABP) were determined. Studies of the steroid binding sites suggest they are toward the carboxy-terminus in hTeBG and rABP and more central in hCBG. hCBG has remarkable sequence homology with members of a superfamily whose functions have diverged; these include thyroxine-binding protein, serine protease inhibitors, egg white proteins, and angiotensinogen. hTeBG and rABP have a 68% amino acid sequence identity. Hybridization studies suggest that hTeBG is probably even more closely related, if not identical, to hABP. The carboxy-terminal sequences of hTeBG and rABP are also similar to that of protein S, a vitamin-K-dependent clotting factor. There were no nucleotide or amino acid sequence homologies between hCBG, hTeBG, or rABP and other steroid binding proteins such as steroid receptors, albumin, alpha-fetoprotein, and vitamin D binding protein. We conclude that the "extracellular steroid binding proteins" and steroid receptors do not appear to have descended from a common ancestor.     

Measurement of the testosterone binding parameters for both testosterone-estradiol binding globulin and albumin in individual serum samples

This report describes a solid phase method for the characterization of testosterone binding to both albumin and testosterone-estradiol binding globulin (TeBG). TeBG is adsorbed from serum samples onto a solid phase matrix of concanavalin A covalently linked to 4B Sepharose. The binding of testosterone is then examined both in the presence and absence of the endogenous serum albumin. Analysis of the resulting Scatchard plots permits determination of the TeBG binding capacity, TeBG association constant and a parameter of albumin binding equivalent to the product of its affinity and capacity for binding testosterone. Results showed that the TeBG capacity was lower in men than in women (18.4 +/- 5.8 vs. 33.1 +/- 19.2 nM, p less than 0.01). The association constant was greater in men (1.59 +/- 0.35 vs. 1.19 +/- 0.32 x 10(9)M-1, 10(9)M-1, p less than 0.01). There was no difference in the albumin binding parameter (43.8 +/- 18.3 vs. 46.6 +/- 15.5, NS). These parameters can then be used to calculate the distribution of the circulating testosterone into albumin bound, TeBG bound and unbound fractions.     

There are two forms of androgen binding protein in human testes. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin

To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.     

Distribution patterns of neurotensin-like immunoreactive cells in the gastro-intestinal tract of higher vertebrates

Summary The endocrine system of the gastro-intestinal tract of selected species representing the five higher vertebrate classes was investigated with reference to occurrence and distribution of neurotensin-like immunoreactive cells. Using antibodies against C-terminal and N-terminal fragments of neurotensin and against the C-terminal sequence of xenopsin it was demonstrated that the intestine of all species studied contains endocrine, neurotensin-like immunoreactive cells. However, large differences in localization and frequency of these neurotensin-like immunoreactive cells were found. Except for a teleostean fish, neurotensin-like immunoreactive cells in the gastro-intestinal tract were more frequent in non-mammalian vertebrates than in mammals. In contrast to mammals, where the highest density of neurotensin-like immunoreactive cells was present in the ileal mucosa, in the non-mammalian vertebrates studied the corresponding cells were most abundant in the pyloric-duodenal junction. The exact mapping of neurotensin-like immunoreactive cells is presented throughout the entire gastro-intestinal tract of six species (Rattus, Coturnix, Lacerta, Rana, Xenopus, Carassius) including a quantitative evaluation of sequential serial sections.     

Nuclear glycoproteins in higher vertebrates

Nuclear glycoproteins recognized by Concanavalin A have been isolated from pig, rabbit and chicken tissues. Mono and bidimensional electrophoresis patterns of proteins loosely and tightly bound to DNA have been examined. The tissue specificity rather than species-specificity appears to be a quite general property of these proteins, suggesting for them a role in the mechanism of regulation of chromatin functions.     

Levels of testosterone-estradiol-binding globulin TeBG and of testosterone in pregnancy with relation to the sex of the foetus (author's transl)]

A practical and economic method for the quantification of testosterone-estradiol-binding globulin TeBG is described. The procedure premits the differentiation without overlap of the TeBP levels in males, non-pregnant females and during pregnancy. Mean titles were 1/5, 1/93 and 1/360 respectively. During pregnancy, we found high levels of TeBG and increased plasma testosterone, with mean values of 143.4 nanograms/100 ml. We have found no significant differences in TeBG levels, or in maternal blood testosterone levels in relation to fetal sex; however, plasma testosterone levels were significantly different among new born of different sex, with mean values of 96.25 nanograms per cent for males and 78.21 nanograms per cent for females.     

Isoelectric focusing of Gc (vitamin D binding globulin) in parentage testing

Summary Gc subtyping with isoelectric focusing (IEF) in paternity cases allowed us to increase the exclusion probability (P) of Gc in Whites to 31% from a (P) of 16% using agarose electrophoresis. Both IEF and agarose are necessary to identify certain rare variants, therefore it is recommended that both techniques be performed in cases of disputed parentage and population studies.     

Thyroxine binding globulin (TBG) assay by radial immunodiffusion.

Thyroxine-binding globulin was measure by radial immuno-diffusion using ten microliters of serum. The mean value in a normal group was 1.95 +/- 0.38 mg/dl. The values obtained by this method were in good agreement with the thyroxine-binding capacity determined by paper electrophoresis (r = 0.956).     

Concentration decrease of corticosteroid binding globulin (CBG) in plasma of the mare throughout pregnancy

A significant decrease of CBG binding capacity in plasma of the mare throughout pregnancy was demonstrated using equilibrium dialysis and gel equilibration methods. As indicated with immunoelectrophoresis experiments, the pregnancy related fall of CBG binding capacity was linked to an actual decrease in blood CBG concentration. This result contrasts sharply with data on most other mammalian species, with the exception of the gestating rhesus monkey.     

Progesterone measurement using "stripped" corticosteroid binding globulin (CBG)

Modifications in an existing competitive protein binding assay for progesterone have been made which provide a readily available and rich source of binding sites on the corticosteroid binding globulin (CBG). The primary and most important modification is the rapid removal of endogenous steroids from plasma by gel filtration at an elevated temperature. The ‘stripped’ protein retains full CBG activity, but is cleared of 95% of its endogenous steroids. This stripping procedure provides not only increased number of binding sites, but in conjunction with the other modifications also eliminates some of the variability in the assay.     

Circadian basis of seasonal timing in higher vertebrates

Abstract

The two dominant environmental oscillations shape biology and survival of species: the day–night cycle and the succession of the seasons in the year. Organisms have adapted to anticipate these variations by evolving internal circadian (ca.- about, diem- day) and circannual clocks. The former enables the organisms to regulate physiological functions on a daily basis, and the latter on the annual basis. In mammals, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus contain master pacemaker and orchestrate peripheral clocks in synchrony with the daily 24 h light-dark cycle, while in birds circadian pacemake is an interacting system principally located in the retina, pineal and the hypothalamus. In this mini review, we discuss the role of circadian clocks in regulation of seasonal timing in higher vertebrates, with reference to birds and mammals.     

Distribution of phenylalanine hydroxylase (EC 1.14.3.1) in liver and kidney of vertebrates.

The range of phenylalanine hydroxylase activity was determined by measuring the conversion of radioactive phenylalanine to tyrosine in liver and kidney of various vertebrates. Rodents (rats, mouse, gerbil, hamster and guinea pig) were found to have the highest liver phenylalanine hydroxylase activity among all animals studied. They are also the only species that possessed a significant kidney phenylalanine hydroxylase activity which was about 25% of that found in the liver of the same animal. The synthetic dimethyl-tetrahydro-pteridine, used as a cofactor for the enzyme assay in most studies, catalyzed non-enzymatic hydroxylation of phenylalanine to tyrosine. Inclusion of boiled-blank and strict control of timing between incubation and product measurement were essential precautions to minimize erroneous results from substrate contamination and non-enzymatic hydroxylation.