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G L Gunsalus

Publications and source records attributed to G L Gunsalus.

At least 37 records · Page 2Linked to original sources
PubMed1986

Regulation of pituitary gonadotropin-releasing hormone receptors by androgens in the male rabbit.

The regulation of pituitary GnRH receptors was studied in adult male rabbits after castration and androgen replacement with testosterone (T) or 7 alpha-methyl-19-nortestosterone acetate (U-15,614; T analog) supplied by Silastic capsules implanted sc. Castration increased pituitary GnRH receptors significantly, from 99.3 to 329.5 fmol/mg protein within 4 weeks, without a change in the equilibrium association constant. Serum LH concentrations increased from 0.45 to maximum levels of 2.6 ng/ml by day 8 after orchiectomy; these levels persisted throughout the 4 weeks of study. Serum FSH reached maximum levels of 33.6 ng/ml 5 days after castration. T replacement with 250, 500, and 1000 micrograms/kg X day, prevented a postcastration rise in both pituitary GnRH receptor concentrations and gonadotropin secretion, while 100 micrograms/kg X day prevented an increase in GnRH receptors, but did not completely inhibit hypersecretion of gonadotropins. Administration of T analog at doses of 6.25 and 12.5 micrograms/kg X day partially suppressed the castration-induced increase in pituitary GnRH receptor concentrations, while 25, 50, and 100 micrograms/kg X day suppressed GnRH-binding sites to the levels found in intact controls in 15 of 16 rabbits. By contrast, none of the T analog doses was able to prevent completely LH and FSH hypersecretion. The fact that both T and T analog induced dose-dependent stimulation of prostate and seminal vesicle weights indicates that there are tissue-specific differences in the sensitivity to androgens. We conclude that in the male rabbit 1) pituitary GnRH receptors significantly increase after castration; 2) this increase may partially mediate the postcastration hypersecretion of LH and FSH; 3) castration-induced effects can be prevented by androgen replacement. These results are similar to those obtained in rats, where castration increases LHRH receptors, but contrast with results in mice and hamsters, where castration either reduces or does not change receptor levels. This indicates significant species differences in the response of pituitary GnRH receptor concentrations to elimination of the negative feedback effects of androgens.

Androgens↗
PubMed1986

Differential regulation of testicular transferrin and androgen-binding protein secretion in primary cultures of rat Sertoli cells.

Specific RIAs for rat transferrin (rTF) and androgen-binding protein (rABP) were used to determine whether the secretion of these proteins was coordinately regulated in the Sertoli cell under a variety of conditions. Sertoli cell-enriched primary cultures were prepared from the testes of 20-day-old rats, and rTF and rABP were assayed in medium from the same culture. There was a strong effect of cell density on both rABP and rTF secretion per cell, with increased secretion per cell at high densities. Human TF (hTF), FeSO4, and desferrioxamine had little or no effect on rTF secretion. The age of the animal at the time of preparation of cells for culture had a strong effect on the pattern of rTF and rABP secretion in vitro; however, the effects of animal age, time in culture, and medium supplementation differed for the two proteins. In cultures prepared from 20-day-old animals, insulin, epidermal growth factor, and testosterone stimulated both rTF and rABP secretion, although to different extents. Retinoic acid was required for the stimulation and maintenance of rTF secretion, but had no effect on rABP secretion in the presence of insulin, hTF, and epidermal growth factor. Conversely, FSH and isoproterenol stimulated rABP, but not rTF, secretion. These data suggest that the secretion of rABP and rTF by Sertoli cells is under differential control.

Aging↗
PubMed1986

The effects of opioid receptor antagonists suggest that testicular opiates regulate Sertoli and Leydig cell function in the neonatal rat.

beta-Endorphin and other peptides derived from proopiomelanocortin are synthesized in testicular Leydig cells. To better understand the possible function of these and other endogenous opioid peptides in the testis, the opioid antagonists naloxone and nalmefene were administered intratesticularly to hemicastrated 5-day-old rats. Both naloxone and nalmefene potentiated testicular hypertrophy induced by unilateral orchidectomy at 11 days of age. Unexpectedly, at least a 100-fold lower dose of nalmefene was required to produce maximal hypertrophy than that previously reported for naloxone. Leydig and Sertoli cell functions were evaluated, respectively, by measurement of basal testosterone production in vitro and rat androgen-binding protein (rABP) in serum. The optimal dose of naloxone for hypertrophy (1 microgram/testis) suppressed testosterone production and had a nonuniform effect on rABP secretion (either had no effect or produced a slight increase). By contrast, the optimal dose of nalmefene for hypertrophy (0.01 microgram/testis) not only suppressed basal testosterone secretion, but also uniformly increased rABP levels in serum. Larger doses of this opioid antagonist, up to 1 microgram/testis, were not as effective on the three parameters measured (hypertrophy, testosterone secretion, and rABP levels). These results suggest that this agent has both antagonistic and agonistic activities in the testis. At the doses that produced optimal effects on hypertrophy, systemic administration of these antagonists produced no effects. The results of these studies suggest that intratesticular opiates exert a suppressive effect on Sertoli cell growth and rABP secretion. In addition, these peptides may modulate testosterone secretion by Leydig cells.

Androgen-Binding Protein↗
PubMed1986

Measurement of a follicle-stimulating hormone-responsive protein of Sertoli cell origin using an enzyme-linked immunoblot assay.

Many proteins secreted by Sertoli cell-enriched cultures are maximally stimulated by a combination of FSH and testosterone. Since very few are stimulated primarily by FSH, we thought it pertinent to identify such proteins. Sertoli cell-enriched cultures were prepared from testes of 20-day-old rats and grown in serum-free medium containing insulin, transferrin, and epidermal growth factor and in such medium supplemented with FSH, testosterone, or FSH plus testosterone. Media were fractionated using HPLC, and proteins were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A protein designated CMB-2, with an apparent mol wt of 22,000, was shown to increase in response to FSH. Antiserum was raised using denatured protein eluted from SDS-polyacrylamide gels as the antigen, and a specific immunoassay using a combination of SDS-polyacrylamide gel electrophoresis and Western blotting was developed. The production of CMB-2 by primary Sertoli cell-enriched cultures was found to increase in a dose-dependent manner in response to FSH (30-1000 ng/ml); secretion was not significantly affected by testosterone (2 X 10(-13) M). An investigation of the tissue distribution of CMB-2 showed that the puberty, CMB-2 is secreted into the rete testis and accumulates in the epididymis in high concentration. We conclude that CMB-2 will be a useful marker to study the action of FSH on the rat testis.

Androgen-Binding Protein↗
PubMed1985

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)

Androgen-Binding Protein↗
PubMed1985

The role of the carbohydrate moiety on the size heterogeneity and immunologic determinants of human testosterone-estradiol-binding globulin.

Human testosterone-estradiol-binding globulin (hTeBG) has been purified to apparent homogeneity by several laboratories using procedures which, in most instances, were labor intensive. In this report, hTeBG was purified from pregnancy serum by a newly developed two step procedure involving sequential affinity chromatography and ion-exchange high performance liquid chromatography (ion-exchange HPLC). The purity of the final product was confirmed by silver stained SDS-polyacrylamide gel and reverse phase HPLC monitored at 206 nm. hTeBG purified by ion-exchange-HPLC maintained binding activity by Dextran coated charcoal (DCC) assay and size heterogeneity on SDS-polyacrylamide gels which were indistinguishable from those of the proteins purified by conventional chromatography. Removal of the carbohydrate moiety from the molecule by both enzymatic and chemical treatment reduced the apparent molecular size and eliminated lectin binding of hTeBG subunits. Deglycosylation did not, however, abolish or alter the distribution of the protomeric forms of this subunit. We conclude that hTeBG is a dimer whose monomer exhibits two protomeric forms which is not a result of carbohydrate heterogeneity. In addition, disialylated and deglycosylated hTeBG exhibited antigenic determinants identical to the native protein.

Carbohydrates↗
PubMed1985

Rats homozygous for the grc complex have defective transport of androgen-binding protein to the epididymis, but normal secretion into the blood.

Male rats homozygous for the growth and reproduction complex (grc), which is closely linked to the major histocompatibility complex, are sterile due to a uniform arrest of spermatogenesis after formation of primary spermatocytes. The present study was conducted to determine whether grc influenced the secretion of the Sertoli cell product androgen-binding protein (rABP) in two F2 hybrid crosses. One was an F2 hybrid of the R10 and ACP strains and the other was an F2 hybrid population derived from the R16 and BI strains. These crosses were chosen because they allowed examination of the effects of grc on rABP secretion when this gene complex was associated with two different major histocompatibility complex backgrounds. In both crosses the rABP content of testes and epididymides from rats homozygous for grc were markedly reduced whereas heterozygotes were normal. By contrast, the concentration of rABP in plasma was normal or near normal at all ages. These observations are compatible with the hypothesis that grc influences the secretion of rABP from the apical portion of Sertoli cells into the tubular lumen and its subsequent transport to the epididymis, but has little or no effect on rABP secretion from the base of Sertoli cells into the blood. In other studies we have shown that infertile animals that have received prenatal x-ray or that are heterozygous for the Hre gene have patterns of rABP secretion similar to that of grc/grc homozygotes. It is therefore possible that the abnormal rABP secretion into tubular lumen in these animals is secondary to germ cell depletion, which is a factor common to grc/grc, Hre/+, and x-ray-treated rats.

Androgen-Binding Protein↗
PubMed1985

Effect of vitamin A deficiency upon gonadotropin response to gonadotropin-releasing hormone.

There is a monotypic change in basal serum gonadotropin levels following retinol treatment of chronically vitamin A-deficient (VAD) male rats. The present study was undertaken to investigate the hypothesis that the specific increase in serum follicle-stimulating hormone (FSH) represents a change in gonadotrope responsiveness to gonadotropin-releasing hormone (GnRH). To this end, a test dose of GnRH was given to VAD rats pre-, 5 days post-, and 10 days postreplacement of vitamin A (PVA). In VAD rats, basal serum FSH and luteinizing hormone (LH) levels were higher than those of controls. Increased LH/testosterone ratios, both in basal levels and in the secretory response to GnRH, suggested Leydig cell hyporesponsiveness in VAD animals. Both the FSH and LH responses to GnRH were maximal at 1 h, declining thereafter. Although the absolute increments in FSH and LH 1 h after GnRH in VAD rats were greater than in controls, the percent increase in FSH tended to be lower in VAD rats and to increase after vitamin A replacement. The specific enhancement of FSH release PVA became evident only when assessing total secretion of FSH and LH after GnRH. Luteinizing hormone response to GnRH increased PVA, but not significantly, while FSH secretion after GnRH increased both 5 and 10 days PVA, times during which basal FSH levels were also increasing. These changes in FSH secretion could not be attributed either to increases in endogenous GnRH or to changes in testosterone or estradiol levels. Basal serum androgen binding protein levels, elevated in VAD animals, did not respond to the acute increases in FSH after GnRH and remained high PVA, suggesting no acute change in Sertoli cell function. Thus, the PVA increase in FSH secretion unmasks a partial inhibition of the gonadotrope present in the retinol-deficient, retinoic acid-fed male rat.

Androgen-Binding Protein↗
PubMed1985

Purification of testosterone-oestradiol-binding globulins from mammalian sera by anion-exchange high performance liquid chromatography.

Testosterone-oestradiol-binding globulin (TeBG) has been isolated from serum or plasma of several species using procedures that yielded highly purified protein, but which required multiple and tedious chromatographic steps. In this report we describe a procedure for the isolation of TeBG which involves two chromatographic steps: androgen affinity chromatography followed by anion-exchange high performance liquid chromatography (anion-exchange HPLC). The purity of the final product was confirmed by silver staining following fractionation on sodium dodecyl sulphate-containing polyacrylamide gels. The size heterogeneity and specific binding activity of TeBGs purified from human, rabbit, or bull serum (or plasma) by this technique was indistinguishable from preparations obtained by conventional chromatography. The present technique shortened the entire purification procedure to about 5 working days and yielded milligram quantities of highly purified protein. Bases on our experience with serum or plasma from the human, rabbit, and bull, this approach should be suitable for isolation of TeBG from a wide range of species.

Animals↗
PubMed1985

Glucocorticoid receptors and responsiveness of normal and neoplastic human adrenal cortex.

Glucocorticoids have been postulated to directly inhibit adrenocortical steroid production in laboratory animals. To investigate this in the human, we measured specific [3H]dexamethasone-binding sites in cytosol samples from normal and neoplastic human adrenal tissues. All nine normal adrenocortical samples, six adenomas (four cortisol-producing and two aldosterone-producing), and two hyperplastic adrenocortical samples studied were devoid of measurable specific glucocorticoid-binding activity. In contrast, steroid binding with characteristics of the glucocorticoid receptor (concentration of binding sites, 32-146 fmol/mg cytosol protein; Kd, 1.7-3.1 X 10(-9) M) was readily detectable in cytosol of all three adrenocortical carcinomas and all three pheochromocytomas examined. To elucidate the in vivo role of glucocorticoids as direct regulators of adrenocortical function, five patients with hypopituitarism receiving varying oral maintenance doses of dexamethasone were given ACTH iv. Increasing the orally administered dexamethasone dose from 1 to 8 mg/day did not alter the plasma cortisol response to a 4-h infusion of 250 micrograms synthetic ACTH in these patients. Collectively, these data cast doubt on the proposal that synthetic glucocorticoids directly suppress adrenocortical function in the human. Whether glucocorticoid receptors in tumor tissue could mediate the dexamethasone-induced suppression of hypercortisolism occasionally reported in patients with adrenocortical neoplasia remains to be investigated.

Adolescent↗
PubMed1984

Antibodies against the beta-subunit of ovine luteinizing hormone can decrease the clearance of human chorionic gonadotropin in rhesus monkeys.

Contraceptive vaccines based on active immunization against gonadotropic hormones are being investigated in humans and other primates. Immunization against the beta-subunit of ovine luteinizing hormone (oLH beta) reduces fertility in rhesus monkeys by inducing inadequate luteal phases and preventing corpus luteum rescue by rhesus chorionic gonadotropin (rhCG). These effects result from the cross-reactions of the oLH beta-antibodies with rhCG and rhLH. We used human CG (hCG), which also cross-reacts strongly with anti-oLH beta to examine how the circulating oLH beta-antibodies affect the metabolic clearance rates (MCR) of hCG in rhesus monkeys. 125I-hCG was injected into four nonimmunized and seven immunized monkeys and blood was collected at frequent intervals over 7 days. Total and immunoprecipitable radioactivity did not differ significantly, suggesting that the radioactivity in the plasma consisted almost entirely of 125I-hCG. This was confirmed by column chromatography. The MCR (mean +/- SE) was significantly lower (p less than 0.001) in six immunized monkeys (0.35 +/- 0.06 liters/day) as compared to controls (1.19 +/- 0.09 liters/day). The hCG disappearance curve in control monkeys was best described by a two-compartmental system (slow and fast) while an additional third (intermediate) compartment of distribution was typical for immunized animals. The half-lives of hCG for the two exponentials corresponding to the slow and fast components of distribution were not significantly different between the two groups. One immunized monkey had a MCR (1.44 liters/day) that was much greater than the MCR of the other six.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The heterogeneity of rat androgen-binding protein in serum differs from that in testis and epididymis.

Fractionation of testicular, epididymal, and serum extracts containing rat androgen-binding protein (rABP) on a Concanavalin A-Sepharose (Con A) column resolved two peaks of immunoreactive protein. The first peak was present in the void volume, and the other was bound by the column and specifically eluted by alpha-methyl-D-glucoside. These two peaks of immunoreactive rABP have been designated form I and form II for the portions of rABP that do not and do bind, respectively, to Concanavalin A. In the course of studying this heterogeneity, we observed that the distribution of the two forms of rABP was the same in the blood and cytosols prepared from testis and epididymis of young rats before the formation of the blood-testis barrier; that is, the ratio of form I to form II ranged from 1:1 to 1:2. Similar heterogeneity was observed in extracts of the reproductive tract from mature animals. However, the blood of adult rats contained reduced amounts of form I relative to form II, so that their ratio was about 1:5. Subsequent studies of infertile rats heterozygous for the Hre gene (Hre/ +), in which total rABP secretion was decreased, and of their normal littermates, indicated that the reduced amount of form I ABP in the sera of mature rats is typical of adult animals regardless of strain or genetic abnormality. The reduced amount of form I relative to form II observed in the blood of adult rats could result from either reduced secretion or increased metabolic clearance of form I in the blood compartment. To distinguish between these possibilities, the blood clearance of the two forms was estimated after orchiectomy. The disappearance rate of form I was not significantly different from that of either form II or unfractionated serum. These results are consistent with reduced release into blood of form I relative to form II rABP rather than increased clearance of form I in adult animals.

Aging↗
PubMed1984

Human testicular androgen-binding protein shares immunodeterminants with serum testosterone-estradiol-binding globulin.

In the human, there are two glycoproteins, testosterone-estradiol-binding globulin (hTeBG) and androgen-binding protein (hABP), which bind testosterone. Although these two proteins have similar physicochemical properties, they can be distinguished on the basis of origin and lectin binding. hTeBG is made in the liver and exhibits high affinity for Concanavalin A (Con A), while hABP from the testes is only partially bound to this lectin. That is, when testicular extracts were applied to Con A-Sepharose columns, a portion of the testosterone-binding material showed no interaction with the lectin and eluted in the void volume (peak I), while the remainder interacted strongly and could be eluted with alpha-methyl-D-glucoside (peak II). These observations are consistent with the proposal that peak I contains only hABP, whereas peak II contains hTeBG and/or hABP with carbohydrate units that permit binding to Con A. To further study the properties of these binding proteins, a hTeBG RIA using a monospecific antiserum was employed to compare the proteins in testes and serum. The results indicated that the testosterone-binding activities in peaks I and II of testicular extracts could not be distinguished immunologically from hTeBG in sera of normal women. These findings suggested that hTeBG and hABP share common epitopes. We next determined whether hABP was secreted into the blood or amniotic fluid by fractionating these fluids in Con A-Sepharose columns. Unlike testicular extracts, male serum and amniotic fluid contained single immunoreactive and steroid-binding species which bound specifically to Con A. We conclude from these observations that hABP (peak I), peak II activity, and hTeBG have common immunodeterminants and that if hABP is secreted into the blood of men, then its carbohydrate chains bind to Con A, making it indistinguishable from hTeBG under these conditions.

Adult↗
PubMed1984

The epididymis contributes minimally to serum androgen-binding protein in the rat: a whole body kinetic study.

The half-times, MCRs, and secretion rates of androgen-binding protein (rABP) were determined in male rats under a variety of conditions. After orchiectomy, the disappearance of endogenous immunoassayable rABP from serum was described by a single exponential term with half-lives of 21 +/- 0.2 and 20 +/- 0.8 h at 25 and 90 days, respectively. The MCR (milliliters per g/day) was not affected by age or hormonal status of the animals. The secretion rate of rABP into the blood was higher in the immature animals than in adults. The decrease in serum rABP concentrations after 20-25 days of age was due to a decrease in the rate of secretion into blood rather than an increase in MCR, a finding consistent with the observation that after formation of the blood-testis barrier, most of the rABP is secreted into the seminiferous tubular lumen. The disappearance curve after injection of purified epididymal rABP was best described by two exponential terms. The first component disappeared very rapidly and the second more slowly, with a half-time corresponding to that of endogenous rABP. The MCR calculated from the latter component was the same as that for endogenous rABP. Having established the kinetic parameters for rABP in serum, a series of experiments was conducted to determine whether it was possible for the epididymis to release this protein into the blood. The apparent half-time of rABP measured in rats in which the testes had been removed and the epididymides left intact was found to be 65 +/- 3 to 70 +/- 5 h in three separate experiments. This increase over the actual half-life of rABP (20 h) was due to the release of rABP from the epididymis into the blood. A similar experiment was performed in an identical group of animals (testes removed, epididymides intact) that had been treated with testosterone via Silastic implants. In these animals the apparent half-time (24 +/- 4 to 28 +/- 2 h; three experiments) closely approximated the actual half-life (20 h). These findings indicate that androgens retarded degeneration of the epididymides, thus minimizing their release of rABP into blood. Our experimental findings suggest the following conclusions. The dramatic rise and subsequent decline of serum rABP concentrations that occur before puberty are due to changes in the secretion rate rather than in the MCR, which is unaffected by age or hormonal states.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1984

On the androgen microenvironment of maturing spermatozoa.

Adult anesthetized male rats were submitted to in vivo micropuncture of the seminiferous and epididymal tubules and reproductive tract vasculature to obtain fluids for analysis of testosterone, 5 alpha-dihydrotestosterone, and androgen-binding protein (ABP). Androgen and ABP concentrations were determined by RIA. The highest concentrations of testosterone (73.14 +/- 5.12 ng/ml) were in testicular interstitial fluid. A significant downhill concentration gradient exists between testosterone concentrations in testicular interstitial fluid and seminiferous tubule fluid (50.24 +/- 2.26 ng/ml); another significant decrease occurs between seminiferous tubule fluid and rete testis fluid (17.85 +/- 2.11 ng/ml). 5 alpha-Dihydrotestosterone concentrations were highest in intraluminal caput epididymidal fluids (58.73 +/- 6.48 ng/ml) as were ABP concentrations (33.30 +/- 2.40 mu leq/microliter). Intraluminal sperm concentrations were also determined, and from these data, fluid reabsorption by the efferent ducts and epididymal tubules were calculated. Eighty-nine percent of the fluid leaving the testis is reabsorbed between the rete testis and caput epididymidis, and 96% is reabsorbed between rete and cauda. It was calculated that large losses of androgen and ABP also occur from the lumen of the excurrent duct system. These losses may be due to metabolism, diffusion from the lumen, or uptake by cells.

Androgen-Binding Protein↗
PubMed1983

Demonstration of heavy and light protomers of human testosterone-estradiol-binding globulin.

Human testosterone-estradiol-binding globulin (hTeBG) was purified from pregnancy serum by sequential ammonium sulfate precipitation, affinity chromatography, and hydroxylapatite chromatography. An overall purification of 2800-fold was achieved with a 27% total yield. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS). The equilibrium dissociation constant (Kd) at 4 degrees C for 5 alpha-dihydrotestosterone (DHT) was estimated to be 1.94 +/- 0.95 X 10(-9) M. Analysis of the purified protein revealed microheterogeneity with regard to size on polyacrylamide gel in the presence of SDS and to charge on isoelectric focusing gels. The apparent molecular weight of native hTeBG determined by gradient gel electrophoresis was 115,000. SDS-polyacrylamide gel electrophoresis indicated that hTeBG is comprised of two molecular weight components of 53,000 and 46,000, which are designated as heavy (hTeBGH) and light (hTeBGL) protomers, respectively. Photolysis of purified hTeBG with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one [( 3H]delta 6-testosterone) resulted in specific labeling of its binding sites. Analysis of photolabeled products by SDS-polyacrylamide gel electrophoresis revealed two radioactive products with electrophoretic mobilities identical to those of the hTeBGH and hTeBGL. The ratio of hTeBGH to hTeBGL was about 10:1. The H and the L protomers were separated and examined by peptide mapping using protease V8 and chymotrypsin. Comparison of the fragmentation patterns produced by these proteases revealed that hTeBGH and hTeBGL components were nearly identical. Removal of sialic acid or carbohydrate residues from hTeBG did not affect the presence of two molecular components. Isoelectric focusing of native hTeBG demonstrated three isoelectric variants with pIs at 4.75, 4.85 and 4.90. After treatment with neuraminidase and other glycosidases, only two isoelectric species were observed with more alkaline pIs. Although purified hTeBG appeared heterogeneous with regard to size and charge, it was remarkably homogeneous in its ability to absorb to Concanavalin A-Sepharose. We conclude that hTeBg, like the androgen binding proteins of the rabbit and rat, is a dimer whose monomer exhibits two protomeric forms.

Chemical Phenomena↗