Localización inmunocitoquímica de la serpina CBG-símil en el sistema reproductor de mamíferos hembra / Immunocytochemical localization of the CBG-like in the mammalian female reproductive system

La participación de la globulina que une corticoides (CBG) en el proceso de la inducción de la reacción acrosomal, ha sido claramente demostrada in vitro en espermatozoides humanos. Esta molécula fue aislada primariamente desde fluido folicular y su presencia, detectada inmunológicamente, ha sido demostrada por nosotros en folículos ováricos, epitelio tubular uterino y en endometrio de humanos y bovinos. Estos resultados nos llevaron a ampliar el estudio de la probable presencia de esta molécula en otras especies mamíferas, con la hipótesis de que si está involucrada en mecanismos previos a la fecundación en humanos y bovinos, su presencia podría estar también jugando un rol molulatorio en especies con procesos de interacción gamética básicamente comparables. El objetivo del presente trabajo fue determinar inmunocitoquímicamente la presencia y distribución de la CBG-símil en el sistema reproductor de cerdos, perros, gatos, conejos y ratas, Las muestras fueron obtenidas, en los distintos estadios del ciclo reproductivo, desde piezas quirúrgicas, a excepción de las de cerdo que se obtuvieron desde su matadera local. Muestras de ovario, tuba uterina y útero fueron procesadas para inmunocitoquímica (ICQ) con anticuerpos (Ac) poli y monoclonales anti hCBG que reconocieron antígenos comunes dentro de las distintas especies animales. En todas ellas la ICQ reveló una intensa reacción positiva a nivel de los folículos ováricos, en las células secretoras del epitelio de la mucosa tubárica y en las células epiteliales de las glándulas y de la mucosa endometrial. En general, la inmuno tinción se manifestó muy intensa hacia el periodo ovulatorio y muy escasa en los periodos de niveles esteroidales bajos. Sin embargo, existieron diferencias particulares entre los distintos animales debido, probablemente, a la variación antigénica por la lejanía de la especie con la molécula generadora del Ac. Muy interesante resultó el estudio del animal en el cual se produjo el Ac policlonal, éste confirmó la presencia de la CBG endógena, determinada por una intensa inmunorreactividad en los preparados controles (sin Ac específico), y que por tanto, la única fuente de ellos fue la proveniente de la generación de sus propios Ac. Nuestros resultados nos permiten sugerir que en las especies estudiadas existe CBG-símil, la cual se distribuye en áreas morfológicas del sistema reproductor, similares a las observadas en humanos y en bovinos

Evidencias inmunocitoquímicas sobre la presencia de la serpina CBG-símil en el sistema reproductor de la hembra en bovinos / Inmunocytochemistry evidence on the presence of the serpina CBG-like in the reproductive system of the female bovine

La reacción acrosomal es un proceso esencial para la fecundación en mamíferos. Recientemente, ha sido demostrado que la globulina que une corticoides (CBG), asociada a progesterona, induce la reacción acrosómica en niveles fisiológicos, in vitro. En la búsqueda de un componente morfológico in situ, utilizando rastreo inmunocitoquímico, nuestro grupo ha demostrado su presencia en células secretoras de ciertas regiones del sistema reproductor femenino humano. Si esta molécula, a travéz de la reacción acrosomal, esta involucrada en los mecanismos que llevan finalmente a la fecundación en humanos, parece razonable suponer que tiene una participación similar en otras especies mamíferas con procesos reproductivos básicamente comparables. El objetivo del presente trabajo fue determinar, a travéz de la inmunocitoquímica (ICQ), la presencia y distribución de la CBG-símil en el sistema reproductor de bovinos hembras, en los distintos estadios del ciclo reproductivo. Muestras de ovarios, útero, tuba uterinay células epiteliales tubáricas en cultivo fueron procesadas para la ICQ. Para ello, se utilizaron anticuerpos policionales contra CBG humana (comercial y otro producto de laboratorio) capaz de reconocer determinantes antigénicos comunes. La ICQ demostró, a nivel ovárico una reacción positiva preferentemente en células de folículos en estadios superiores de desarrollo. A nivel de la tuba uterina la inmunotinción se presentó claramente en células no ciliadas del epitelio tubárico. En el endometrio la reacción se concentró en las células epiteliales de las glándulas endometriales, sin observarse inmunorreacción a nivel estromal. La inmunotinción fue muy intensa en los períodos de mayor actividad estrogénica para, prácticamente, desaparecer en vacas preñadas. Las células de cultivo oviductales revelaron una población con reacción intensamente positiva y otra claramente negativa. Nuestros resultados nos permiten sugerir que el bovino existe la presencia de CBG-símil, la cual se distribuye en áreas morfológicas similares a las observadas en humano, y su concentración depende del ciclo ovárico. Tales elementos permitirían sugerir que esta particular molécula juega un rol similar en la especie bovina

Studies on the role of glycosylation in the origin of the electrophoretic variants for rat corticosteroid-binding globulin.

The glycoprotein corticosteroid-binding globulin (CBG) migrates as doublet bands in PAGE and SDS-PAGE, and as numerous bands in isoelectric focusing (IEF). This study deals with the origin of this heterogeneity. Desialation of rat CBG with neuraminidase does not abolish the doublet in either PAGE or SDS-PAGE, indicating that the doublet does not arise as a result of differences in sialic acid residues. Treatment of the separated upper and lower variants of native CBG with N-glycosidase F (PNGase-F) shows a differential pattern of deglycosylation over time indicating either differences in the number, type, or location of sugars attached to each of the variants. Rate of deglycosylation is quicker and more extensive for the upper variant when compared to the lower variant. PNGase-F treatment of 1% SDS-denatured CBG does not abolish the CBG doublet seen in SDS-PAGE, indicating that there is variation in the protein moiety. Sugars could not be detected on PNGase-F treated CBG using either wheat germ aglutinin horse radish peroxidase conjugate, concavilin-A HRP conjugate, or the digoxigenin glycan detection system. While the results clearly show differences in glycosylation between the CBG variants, differences in the protein moiety may also occur to give rise to the heterogeneity seen in CBG. The latter is supported by the fact that desialated CBG migrates as two bands in IEF. Migration in IEF is based solely on charge, and since only sialic acid residues are charged in N-linked glycosylation, any heterogeneity seen for the desialated glycoprotein must reside within the protein moiety itself. The presence of O-glycosylation containing an N-acetylgalactosamine with a beta 1-3 linkage to galactose could not be demonstrated using O-glycosidase. N-terminal blockage could not account for the variation, as both the upper and lower variants were able to be sequenced resulting in identical sequences for the first 13 amino acids. The data presented supports the hypothesis that the differences in the sugar as well as the protein moiety are responsible for the heterogeneity seen for CBG.

Identification of Trp-371 as the main site of specific photoaffinity labeling of corticosteroid binding globulin using delta 6 derivatives of cortisol, corticosterone, and progesterone as unsubstituted photoreagents.

Immunopurified human corticosteroid binding globulin (CBG) was photolabeled with delta 6-[3H]cortisol, delta 6-[4-14C]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone. The maximal levels of specific incorporation, as estimated with tritiated photoreagents, were 0.21, 0.14, and 0.08 mol of label/mol of CBG, respectively. Tryptic cleavage of photolabeled CBG gave in all cases a major radioactive peptide that was no longer detectable when a 100-fold molar excess of cortisol was added to the photoreagents. Edman sequencing of tryptic peptides photolabeled with delta 6-[3H]cortisol or delta 6-[3H]corticosterone showed that these peptides correspond to residues 357-378 of the human CBG sequence. The major peak of radioactivity of these peptides was eluted at the 15th cycle (Trp-371). The radioactive tryptic peptides photolabeled with the four steroid photoreagents were subcleaved with alpha-chymotrypsin. The major part of radioactivity was recovered in the T-[*X]-S-S-L-F hexapeptide 370-375 (major peptide) and in the D-H-F-T-[*X]-S-S-L-F nonapeptide 367-375, at the second and fifth Edman cycles, respectively, whereas no PTH derivative could be identified at these cycles, thus suggesting Trp-371 as the main site of photolabeling for all tested photoreagents. Mass spectrometry of tryptic peptides photolabeled with delta 6-[3H]cortisol and delta 6-[3H]corticosterone and of chymotryptic peptides photolabeled with delta 6-[3H]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone showed molecular masses corresponding to the addition of delta 6-steroid photoreagents to the peptide.

[Isolation and purification of transcortin (cortisol binding globulin--CBG) using two-step chromatography]. / Izolacja i oczyszczanie transcortyny globuliny wiazacej kortyzol--(CBG)--za pomoca dwustopniowej chromatografii.

The isolation of cortisol and progesterone binding globulin (CBG) from pregnant women serum was performed using affinity and hydrophobic chromatography. The purity and specificity of isolated transcortin was tested by agarose gel electrophoresis using racket and cross immunoelectrophoresis and specific CBG antibodies. High purity and immunoreactivity of the isolated globulin destitute of other proteins contamination, were obtained.

Expression of biologically active human corticosteroid binding globulin by insect cells: acquisition of function requires glycosylation and transport.

Human corticosteroid binding globulin (hCBG) is a 50- to 55-kDa serum glycoprotein that binds cortisol and progesterone with high affinity. To map the steroid-binding domain and to investigate the folding pathways of hCBG, we have established an expression system based on infection of insect cells with a recombinant baculovirus encoding hCBG. Infected Spodoptera frugiperda (Sf9) cells secrete immunoreactive hCBG at high levels (16-24 pmol per 10(6) cells per 40 h), and the recombinant protein binds cortisol with an affinity and specificity equivalent to that of human serum-derived hCBG. Thus, this system has the potential to provide large amounts of wild-type and mutant hCBGs for physical-chemical analysis. Cotranslational asparagine-linked glycosylation is essential for acquisition of steroid-binding capability, as shown by the lack of cortisol-binding activity of unglycosylated hCBG secreted in the presence of tunicamycin. Golgi-associated oligosaccharide processing, however, is not required for activity, as demonstrated by the endoglycosidase H susceptibility of the fully active, secreted glycoprotein. Comparison of the steroid-binding properties of intracellular and secreted hCBG with that synthesized in vitro in the rabbit reticulocyte lysate system suggests that this protein undergoes a maturation process during transport through the secretory pathway. This system will be useful for identifying the molecular determinants of biological function in hCBG.

Interaction between corticosteroid binding globulin and activated leukocytes in vitro.

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.

Identical properties of aldosterone and corticosterone binders and their presence in rat brain and kidney.

The [3H]corticosterone binders from rat brain and kidney were characterized by binding affinity and chromatographies, and compared with the binders for [3H]aldosterone and [3H]triamicinolone acetonide. Corticosterone-binding globulin-like molecules at very high concentrations in crude extracts were completely eliminated by a DEAE-gel adsorption procedure. [3H]Aldosterone binder in the renal, DEAE-treated fraction was recovered in a single peak by gel-filtration chromatography and by ultracentrifugation in linear sucrose gradients, independent of hormone-binding and tungstate, a stabilizer of the binder. The Stokes' radius and sedimentation coefficient of the renal aldosterone binder were 6.6 nm and 9.3S, respectively, indicating an apparent molecular weight of 263,000. Corticosterone-preferring binder also existed in the DEAE-treated fraction. Both aldosterone and corticosterone binders were found in the brain and kidney preparations. Comparison among the binders showed identical values of Stokes' radius and elution pattern from DEAE-Toyopearl in a linear salt gradient regardless of the organ and the hormones. Scatchard analyses of [3H]aldosterone and [3H]corticosterone binding showed for each ligand only one group of high-affinity sites with the equivalent dissociation constants, 4-7 nM. The orders of steroids in competing for the two high-affinity sites were equivalent: corticosterone greater than or equal to aldosterone much greater than triamcinolone acetonide, and that for the triamcinolone acetonide binding was triamcinolone acetonide much greater than aldosterone greater than or equal to corticosterone. Hydroxyapatite column chromatography separated the aldosterone and corticosterone binders from the triamcinolone acetonide binder, but not the aldosterone binder from the corticosterone binder. It is concluded that aldosterone and corticosterone binders distinct from triamcinolone acetonide binder exist in rat brain and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the role of glycosylation for human corticosteroid-binding globulin: comparison with that for thyroxine-binding globulin.

The role of glycosylation on the secretion and the stability of human corticosteroid binding globulin (CBG) was studied. Cells of the human hepatoma line were labeled by [35S]methionine in presence of or absence of tunicamycin (TM). Media or cells were harvested at 0, 3, 6, and 20 h after the addition of excess unlabeled methionine. Media and cell lysates were incubated with anti-CBG serum and immune complexes were precipitated with Staphylococcus aureus protein A (Pansorbin). Immunoprecipitates were analyzed by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation of T4-binding globulin (TBG) was also carried out with anti-TBG serum. Fluorographic analysis revealed three forms of CBG: CBG1, a glycosylated, mature, and secretory form with apparent mol wt of 70 K; CBG2, a glycosylated precursor which due to incomplete carbohydrate processing has an apparent mol wt of 54 K; and CBG3, a nonglycosylated form consisting of the 40 K core protein. In absence of TM, CBG1 was observed in media and CBG2 was detected in cell lysates. The proportion of CBG1 increased during the chase, whereas that of CBG2 decreased, indicating that CBG was secreted after processing of the oligosaccharides on CBG2. In presence of TM, CBG3 was found both in media and cell lysates. The sum of CBG3 in the medium and the cell lysate decreased during the chase, whereas that of CBG1 and CBG2 remained unchanged. Similar to CBG, TBG1 (mature form, 60 K) and TBG2 (partially processed glycosylated form, 54 K) were observed in media and cell lysates, respectively, in absence of TM. However, TBG3 (nonglycosylated, 44 K) was not detected in medium. These results indicate that glycosylation is not a key factor for the secretion of CBG but is important for its stability. On the other hand the glycosylation is indispensable for the secretion of TBG.

Biological half-life and transfer of maternal corticosteroid-binding globulin to amniotic fluid in the rabbit.

Rabbit corticosteroid-binding globulin (CBG) from the serum of pregnant and nonpregnant females differs in terms of charge microheterogeneity, and both forms were, therefore, radiolabeled and injected iv into 23- to 27-day pregnant rabbits (n = 6) to assess their biological half-lives and possible transfer to the fetal compartment. After an initially rapid distribution phase, the serum half-lives of both forms of [125I]CBG were essentially identical (approximately 13 h) and did not vary at different gestational ages. There was also no difference in the transfer of either form of [125I]CBG from maternal to fetal compartments in any of the animals studied. Moreover, [125I]CBG showed no sign of degradation and retained its steroid-binding activity in fetal urine and amniotic fluid. Twenty-two hours after administration of [125I]CBG to rabbits (n = 2) at 23 days gestation, its mean level in fetal urine (7 cpm/microliter) and amniotic fluid (2.5 cpm/microliter) was much higher than that in fetal blood (0.6 cpm/microliter). More importantly, the specific activities of [125I]CBG in fetal urine and amniotic fluid were comparable to that in maternal serum, and approximately 2 orders of magnitude higher than that in fetal serum. Taken together, these results suggest that CBG in fetal urine and amniotic fluid is largely of maternal origin, and that maternal CBG crosses the fetal kidney preferentially.

Transcortin biosynthesis and intracellular distribution of rat liver polyribosomes synthesizing transcortin.

Transcortin biosynthesis in rat has been examined using liver slices technique. The incorporation of 14C-labeled amino acids into the anti-transcortin-precipitable material of liver slices has been measured and compared with that of serum transcortin. It was shown that liver synthesized transcortin with an apparent mol. wt of 66 kDa on SDS-electrophoresis which co-migrated with authentic rat serum transcortin. In order to determine an intracellular distribution of transcortin synthesizing polyribosomes, the binding character of [125J]anti-transcortin-IgG to free and membrane-bound rat liver polyribosomes has been studied. It was shown that after incubation of [125J]anti-transcortin-IgG with liver membrane-bound polyribosomes, the radioactivity was associated with the discrete polyribosome fraction in heavy polyribosome region. In similar experiments the radioactivity of [125J]anti-transcortin-IgG bound to the free polyribosomes was distributed throughout the polyribosome region. The results of our experiments obviously demonstrated that serum transcortin was synthesized exclusively on membrane-bound polyribosomes of rat liver; free polyribosomes were devoid of detectable antigenic material able to bind antibodies to transcortin.

[Isolation and comparative characteristics of rat transcortin in normal conditions and during experimental circulatory insufficiency]. / Vydelenie i sravnitel'naia kharakteristika transkortina krysy v norme i pri éksperimental'noi nedostatochnosti krovoobrashcheniia.

Corticosteroid binding protein, transcortin, was isolated using biospecific and hydroxyapatite chromatographic procedures. Mr-60,000 of transcortin was evaluated by means of electrophoresis; isoelectric points of the protein and of its complexes were detected. The association constants of transcortin with cortisol at 4 degrees constituted 2.8.10(8) M-1/mol of protein from intact animals containing one binding site and 9.8.10(8) M-1/mol of protein from impaired rates containing 0.39 binding site per a molecule. The maximal cortisol-binding activity of transcortin was shifted towards more alkaline pH value under the pathological conditions. Circular dichroism spectra of the transcortin-cortisol equimolar complexes were studied. The proteins studied were dissimilar in their main physicochemical properties and patterns of the steroid binding.

Selective changes in binding and immunological properties of human corticosteroid binding globulin by free fatty acids.

The steroid hormones, progesterone (P4) and cortisol (F), have different biological activities but are both bound to human corticosteroid binding globulin (CBG) with similar affinity. This study examines the effect of physiological concentrations of FFA on the binding of these steroids to purified CBG and to the serum of pregnant women. It also analyzes the influence of the FFA environment on the immunological behavior of CBG. Unsaturated fatty acids (UFA) had a dose-dependent inhibitory effect (P less than 0.001) on steroid binding to CBG which was offset by saturated fatty acid-induced potentiation of binding (P less than 0.01) when both were present with CBG. UFAs inhibited P4 binding more than F binding. Comparable results were obtained with pregnant serum or with pure CBG. UFAs seemed able, depending on their concentration, to promote different molecular states of CBG, some with enhanced F binding and significantly reduced P4 binding, and others in which both P4 and F binding was markedly reduced. Scatchard analysis of steroid binding to purified CBG indicated that the UFAs influenced the association constant (Ka) and the number of binding sites (n) for F and P4 binding differently. Low concentrations (less than 16 microM) of arachidonic acid (C20:4) slightly potentiated F binding, with no change in Ka and a 1.6-fold increase in n; this concentration of C20:4 reduced n for P4 binding by 40% and did not affect Ka. Higher C20:4 concentrations (greater than 32 microM), reduced the Ka for F binding but did not apparently change n; for P4 binding, Ka was sharply reduced and n increased. The apparent equilibrium dissociation constant (Kd) for both F and P4 binding varied nonlinearly and differently with increasing C20:4 concentration. Immunoelectrophoresis and immunoautoradiography showed a reduction, or loss, of CBG immunoreactivity in the presence of UFA. The extent of these changes varied with the concentration and class of the UFA. These results indicate that FFA induce conformational changes in CBG which may modulate its activity and so influence the role of this protein in both the endocrine and immune systems.

Purification and properties of squirrel monkey (Saimiri sciureus) corticosteroid binding globulin.

Corticosteroid binding globulin (CBG), a serum glycoprotein which binds glucocorticoids and progestins with high affinity, is widely distributed throughout the animal world. Although its charge and size characteristics have largely been conserved across species, we found the behavior of CBG in squirrel monkey (Saimiri sciureus) serum during fractionation by polyacrylamide gel electrophoresis or Sephadex chromatography was consistent with a molecule about twice the size of that found in most species. To more fully understand the basis for this difference, we purified the protein by sequential affinity and DEAE-Sepharose chromatographies. The final product was obtained in greater than 60% yield and was found to migrate as a single homogeneous band when examined by electrophoresis at pH 8.3 in polyacrylamide gels varying total acrylamide concentration or under conditions of severe protein overload. The steroid binding specificity of the purified protein was identical with that of the protein in the starting serum. The ultraviolet absorption spectrum of the isolated CBG-steroid complexes revealed that the protein had no pyridine nucleotide cofactor or nucleic acid. Amino acid analyses showed that the composition of the squirrel monkey protein is quite similar to that of CBG molecules from other species but distinct from albumins, hemoglobin, or rabbit progesterone receptor. In contrast to the single protein band observed following electrophoresis under normal conditions, separations in the presence of sodium dodecyl sulfate (SDS) resolved the pure protein into two bands: one at 54,000 daltons and one at 57,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple method for the purification of murine corticosteroid binding globulin: characterization of a monomer.

A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH2-terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (Mr) of murine CBG was determined to be 52,000 and the sedimentation constant S20 degrees, w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15-3.25 and two between pH 3.40-3.50.

Comparative structural analyses of corticosteroid binding globulin.

Some physicochemical characteristics of corticosteroid binding globulin (CBG) in several species have been determined. Molecular radii were determined from Ferguson plots and were used in conjunction with sedimentation coefficients determined by sucrose density gradient centrifugation to calculate the molecular weights of the CBG. These were found to range from 44,200 (dog) to 60,000 (turtle) for most species. The squirrel monkey was found to have a molecular weight twice that of other species (119,800). Purified CBG was prepared from human, rat, and guinea pig sera. The molecular weights of the purified material, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate, were in excellent agreement with those determined by Ferguson analysis. Careful examination of the purified proteins by electrophoresis at pH 8.3 revealed that each consisted of two closely related electrophoretic variants. Tryptic peptides were prepared from the purified proteins and separated by reversed phase HPLC chromatography. The peptide patterns were identical for the three proteins with the exception of three hydrophilic peptides. Amino terminal sequence analysis of the rat and human proteins revealed no apparent homology, however. The immunologic relatedness of the three purified proteins was also examined, but no crossreactivity was observed. The results obtained suggest that while the molecular size and hydrophobicity of peptides have been conserved across species considerable surface differences must exist.

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum.

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.

Application of liquid-liquid partition chromatography in the simultaneous purification of sex-hormone-binding globulin and corticosteroid-binding globulin.

Two human serum proteins, corticosteroid-binding globulin (CBG) and sex-hormone-binding globulin (SHBG), were purified to homogeneity by the application of a combination of three different modes of chromatography. Human pregnancy serum was fractionated with ammonium sulphate. SHBG (50% pellet) and CBG (80% pellet) were then purified by affinity chromatography on tresyl-activated Sepharose with 15-aminopentadecanoic acid (for SHBG) and 1,12-diaminododecane (for CBG) as spacers and 17 zeta-aminoethyl-5 alpha-androstan-3 beta,17-diol (for SHBG) and 17 alpha-hydroxy-4-androsten-3-one-17 beta-carboxylic acid (for CBG) as specific ligands for these two proteins. The eluate was injected into a Mono Q anion-exchange column. Fractions containing SHBG or CBG were finally purified by liquid-liquid chromatography on Lipar-Gel 750. This chromatographic sequence clearly separated SHBG and CBG from other proteins, mainly serum albumin, without a loss of protein or binding activity.

Transport and intracellular localization of cortisol-asialotranscortin complexes in rat liver.

The influence of desialylation of human transcortin on transport of the transcortin-cortisol complex into the liver cells and its intracellular distribution was investigated in perfused rat liver. Under experimental conditions used the half-time of cortisol in perfusion medium was decreased more than 200 times in presence of asialotranscortin compared to that of native transcortin. Experiments with [3H]cortisol and [131I]asialotranscortin demonstrated a simultaneous uptake of cortisol and asialotranscortin by the hepatocytes. Distribution of [3H]cortisol and [131I]asialotranscortin in subcellular fractions showed the following pathway of cortisol-asialotranscortin complex: cell membrane, lysosomes, cytoplasm. The complex dissociates in lysosomes.

Mineralcorticoid receptor from rat kidney. Interaction with heparin and purification to a CBG-free stage.

Purification of the mineralcorticoid receptor is a particularly challenging problem. This receptor is present in target tissues at concentrations lower and is less stable than any other steroid receptor. Addition of molybdate ions (20 mM) to rat kidney cytosol enhances stability of mineralcorticoid-specific binding sites: the inactivation rate at 0 degrees C decreases from 7.2 to 1.7% per hour in the absence of aldosterone, and from 1.8 to 0.3% per hour in the presence of hormone. Rates of inactivation in the presence of molybdate are thus compatible with purification procedures. Also, the corticosteroid-binding globulin (CBG) is an important contaminating component of kidney cytosol because it cannot be specifically blocked preliminarily to affinity chromatography. We show that when kidney cytosol is incubated with heparin covalently linked to Sepharose (Sepharose-heparin), after 30 min at 0 degrees C more than 80% of the mineralcorticoid-specific binding sites interact strongly with Sepharose-heparin while CBG is not bound at all. The mineralcorticoid-specific binding sites can be recovered from Sepharose-heparin by washing with heparin (2 mg/ml; recovery up to 90%), KCl (0.3 M; recovery up to 90%); and, less efficiently, with total liver RNA (2 mg/ml; recovery up to 55%) and dextran sulfate (2 mg/ml; recovery up to 40%); little or no recovery is achieved with chondroitin sulfate, sonicated DNA, pyridoxal-5-phosphate, dextran, d-glucosamine and d-glucuronic acid. With demonstration that also the mineral-corticoid receptor binds to heparin, this property has become a general hallmark of steroid receptors. If the "heparin" binding site of steroid receptors is of physiological significance it remains to be established. By application of the newly found property of the mineralcorticoid receptor, an overall 10-fold purified, CBG-free preparation of this receptor can be obtained from kidney cytosol with a single chromatography on Sepharose-heparin.