Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin

Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.     

Increase of transferrin receptors in regenerating rat liver cells after partial hepatectomy

The change of transferrin receptors in regenerating rat liver cells after partial hepatectomy was demonstrated. The binding of 125I-labeled transferrin to the cells began to increase after 24 h of partial hepatectomy and reached about double that of the non-operated normal rat liver cells. A Scatchard analysis of binding parameters showed 1.62 X 10(5) (Ka: 1.04 X 10(7) M-1) in normal cells and 3.36 X 10(5) (Ka: 1.23 X 10(7) M-1) in regenerating cells. This reflected on increase of transferrin receptor number and little change occurred in the binding affinity between transferrin and its surface receptor.     

Binding constants of NZB myeloma antidextrans for dextrans and isomaltose oligosaccharides determined by affinity electrophoresis.

Association constants of dextrans (Ka) and oligosaccharides (Kia) from NZB myeloma antidextrans (PC3858 and PC3936) were studied by affinity electrophoresis. With linear dextrans or with those with a low degree of branching, Ka ranged from 2.7 X 10(3) to 5.4 X 10(4) ml/g for PC3858 and from 1.3 X 10(4) to 2.6 X 10(5) ml/g for PC3936. Completely linear alpha-(1 leads to 6)-linked dextrans, LD7 and D3, showed relatively high affinities for the two NZB antidextrans. With oligosaccharides, the Kia value increased as the number oa alpha-(1 leads to 6)-linked glycosyl residues increased. Isomaltoheptaose (IM7) showed the highest Kia (1.9 X 10(4) M-1 for PC3858 and 1.63 X 10(4) M-1 for PC3936), whereas isomaltose (IM2) had the lowest Kia (2.36 X 10(2)M-1 for PC3858 and 1.32 X 10(2)M-1 for PC3936). Pullulan and glycogen showed very weak affinity for PC3936, but they did not react at all with PC3858. These findings indicate that NZB myeloma antidextrans, PC3858 and PC3936, are specific for internal chains of alpha-(1 leads to 6)-linked dextrans. Data on the precision with which Ka and Kia can be determined are presented.     

High-affinity binding of thyroid hormones to neuroblastoma plasma membranes

The binding of thyroid hormones to isolated plasma membranes was studied in NB41A3 neuroblasts. Saturable binding of L-T3, D-T3 and L-T4 was observed. Binding was time-dependent, with equilibrium reached in less than 60 min and maximal binding occurring between pH 7.4 and 7. Saturation experiments demonstrated two classes of sites for L-T3: a high-affinity site with Ka 8.4 X 10(9) M-1 and a low-affinity site with Ka 7.3 X 10(6) M-1.L-T3 and D-T3 inhibited each other's binding, L-T3 being several-times more potent. Affinity labeling of isolated membranes with bromoacetylated thyroid hormones disclosed stereospecific binding to SDS-PAGE bands with approximate molecular masses of 27 kDa (preferentially labeled by BrAc-L-T3), 32 kDa (preferentially labeled by BrAc-D-T3), and 48 and 87 kDa (preferentially labeled by BrAc-L-T4). Binding of BrAc-L-T3 to the 27 kDa band accounted for 3.4% of total binding, was selectively inhibited by excess L-T3, and may be involved in intracellular transport of L-T3.     

Production of anti-human thyroglobulin and anti-thyroid hormone antibodies in rabbits immunized with human thyroglobulin

Two rabbits (TG-1, TG-2) were immunized with human thyroglobulin (HTg) and bled serially. Antisera were obtained at different times after the first immunization and kept separately and studied. In both rabbits production of anti-HTg, and anti-thyroid hormone antibodies such as anti-thyroxine (T4) and anti-triiodothyronine (T3) antibodies was observed. Binding parameters of anti-HTg antibodies with HTg, T4, and T3 were calculated in two selected antisera (70-day and 249-day). The Scatchard's plots of these antibodies were all curve-linear and were analyzed in two components: one, higher binding constant (Ka1) and smaller binding capacity (Cap1) and the other, lower binding constant (Ka2) and larger binding capacity (Cap2). Ka1 values of anti-HTg, anti-T4, and anti-T3 antibodies in sera from TG-1 obtained from 70-day and 249-day bleeding were 1.1 X 10(10) M-1, 6.0 X 10(9) M-1. 7.9 X 10(8) M-1 and 1.7 X 10(10) M-1, 6.5 X 10(9) M-1, 1.0 X 10(9) M-1, respectively. Those from TG-2 were 1.7 X 10(10) M-1, 1.8 X 10(9) M-1, 6.4 X 10(8) M-1 and 2.0 X 10(10) M-1, 3.1 X 10(9) M-1, 1.6 X 10(9) M-1, respectively. The significance of the production of anti-HTg and anti-thyroid hormone antibodies in rabbits immunized with HTg in relation to the antigenic structure of HTg molecule was discussed.     

Ligand properties of diethylstilbestrol: studies with purified native and fatty acid-free rat alpha 1-fetoprotein and albumin.

We report the equilibrium binding parameters for the interactions of the estrogen analogue diethylstilbestrol (DES) with highly purified rat alpha 1-fetoprotein (AFP) and serum albumin preparations. At 25 degrees C and pH 7.4, an association constant (Ka) of about 1.5 X 10(6)M-1 and 2 sites/mole are measured with the DES-AFP system, whereas for the DES-albumin interaction, we find a Ka of approximately 2 X 10(5)M-1 and about 11 sites/mole of protein. The removal of fatty acids from pure AFP causes a reversible 3 fold increase of the number of DES binding sites; the same delipidation procedure applied to albumin slightly diminishes its DES binding parameters. We also demonstrate the capability of DES to displace competitively estradiol-17 beta (E2) from its high affinity sites on the estrophilic rat AFP. Finally, the binding behaviour of the two serum proteins towards the synthetic estrogen is compared to their interaction with the natural hormones. The physiological and pharmacological relevance of these data is discussed.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.     

Characterisation of monoclonal antibodies raised against testosterone

Using the antigens testosterone-17 beta-hemisuccinate and testosterone-3-(o-carboxymethyl) oxime, each coupled to bovine serum albumin, we have produced 44 monoclonal antibodies to testosterone. Of the 17 monoclonal antibodies raised against the 17 beta-linked antigen 8 showed extremely low affinity for testosterone (Ka less than or equal to 8 X 10(7) M-1) and none had an affinity greater than 5 X 10(9) M-1. Of the 27 monoclonal antibodies raised against the 3-linked antigen 2 had affinities less than 8 X 10(7) M, 7 had affinities greater than 5 X 10(9) M-1 and one had an affinity (Ka = 9 X 10(10) M-1) greater than that of a high affinity rabbit antiserum (Ka = 6 X 10(10) M-1). The affinity constant (Ka = 5 X 10(9) M-1) measured in the serum of the mouse whose spleen gave rise to the greatest number of high affinity antibodies, was significantly higher than those measured in the sera of the remaining mice (Ka = 0.7 - 3 X 10(8) M-1). The cross-reactions of the monoclonal antibodies varied widely but none showed an overall improvement in specificity when compared with the corresponding rabbit antisera. Results suggest that as well as the structure of the steroid antigen careful selection of the spleen donor facilitates the development of monoclonal antibodies with good binding characteristics.     

Carbohydrate-binding proteins of human serum: isolation of two mannose/fucose-specific lectins

Two lectins with specificities for mannose and fucose have been isolated from human serum by affinity chromatography. One mannose-binding protein (MBP 1) has a native Mr of 700,000 with subunits of Mr 32,000 and has specificities for N-acetylglucosamine, N-acetylmannosamine and glucose as well as for mannose and fucose. The other mannose-binding protein (MBP 2) has a native Mr of 200,000 with subunits of Mr 28,000 and is specific only for mannose and fucose. MBP 2 appears to recognize the core sugars of asparagine-linked oligosaccharides as well as the terminal sugars. Both lectins are calcium-dependent, requiring approx. 0.095 mM calcium for half-maximal binding. MBP 1 binds maximally between pH 7-9, whereas MBP 2 has a pH optimum of 6-7. The binding activity of both proteins decreases rapidly below pH 5. The apparent association constants (Ka) for binding to mannon are 2.1 X 10(8) M-1 for MBP 1 and 1.3 X 10(8) M-1 for MBP 2. These data provide further evidence of the complex nature of mammalian carbohydrate recognition systems.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Binding of triiodothyronine to the nuclear matrix of the rat liver. The effect of thyroid hormones on the phosphorylation of nuclear matrix proteins

The interaction of thyroid hormones with rat liver nuclear matrix proteins was studied. It was shown that the nuclear matrix contains the sites which bind triiodothyronine with a high affinity (Ka = 1.07 X 10(9) M-1) and limited capacity (maximal binding capacity--28.5 fmol triiodothyronine/100 micrograms protein). Electrophoretic analysis of triiodothyronine-binding matrix proteins revealed that the molecular mass of the major triiodothyronine-binding fraction is 50 000-52 000 Da. Injections of triiodothyronine to thyroidectomized animals stimulated the phosphorylation of all protein fractions of the nuclear matrix.     

Androgen and estrogen binding in rat skeletal and perineal muscles.

Specific in vitro binding of [3H]testosterone (T), 5ALPHA[3H]dihydrotestosterone (DHT), and [3H[estradiol (E2) was demonstrated in the 30 000 X g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 X 10(9) M-1, and 2.3 X 10(9) M-1 respectively) whereas DHT binding is of lower affinity (Ka = 5.0 X 10(7) M-1).] In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 X 10(9) M-1, 0.3 X 10(9) M-1, and 0.5 X 10(9) M-1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.     

Isolation of two forms of rat alpha-fetoprotein and comparison of their binding parameters with estradiol-17beta.

In polyacrylamide gels, highly purified rat alpha1-fetoprotein shows a molecular heterogeneity, i.e. a "slow" and a "fast" moving fraction. We have isolated by electrophoretic fractionation and subsequent elution these two forms of alpha1-fetoprotein, and we have studied comparatively the binding parameters for estradiol-17beta of whole alpha1-fetoprotein preparations and of the isolated forms. We have shown that the number of binding sites per molecule of whole alpha1-fetoprotein is always, in our experimental conditions, a fractional number, inferior to unity (0.3). Furthermore, the analysis of the binding parameters of the "two forms" of alpha1-fetoprotein allows discrimination between different classes of binding sites. For the "slow" fraction, the number of predominant binding sites per molecule of protein is close to unity (0.7-0.9), whereas for the "fast" fraction, a very low fractional value is found (0.1). The corresponding association constants are reproducibly different for the two fractions: Ka = 0.1.10(8) M-1 for the "slow" alpha1-fetoprotein, and Ka = 0.7.10(8) M-1 for the "fast" alpha1-fetoprotein. Traces of a very high affinity (10(9) M-1) minor class of binding sites are demonstrated in the "slow" fraction. These results point to the existence of a molecular population of alpha1-fetoprotein, some forms of which have a strong or very strong affinity, and some a negligible affinity, for estrogens.     

Role of glycans in the binding of human serotransferrin and lactotransferrin to human alveolar macrophages

The optimal conditions of the binding of human lactotransferrin to human alveolar macrophages have been determined and the necessity to measure the binding in absence of bovine serum albumin was demonstrated. In these conditions, diferric lactotransferrin and iron-free lactotransferrin are reversibly bound with the following parameters: association constant Ka = 2 and 5 X 10(6) M-1, respectively, and the number of binding sites N = 1.2 and 1 X 10(7), respectively. The binding of the two forms of lactotransferrins was inhibited by various neoglycoproteins, the highest inhibition being obtained with L-fucosyl, then, in the following decreasing order: D-mannosyl greater than N-acetyl-D-glucosaminyl greater than D-galactosyl. In the same conditions, the binding of serotransferrin (Ka = 2 X 10(7) M-1 and 1.6 X 10(7) M-1; N = 5 X 10(4) and 8 X 10(4) for diferric and iron-free protein, respectively) was not inhibited. These results suggest that the recognition of lactotransferrin is mediated by one or several membrane lectins, the fucose being one of the sugar playing an important role in the association. On the contrary, the binding of serotransferrin does not depend on a membrane lectin system.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence

A putative growth hormone receptor from detergent-solubilized rabbit liver membranes and the growth hormone binding protein from rabbit serum have been purified 59,000- and 400,000-fold, respectively, primarily by affinity chromatography. Both purified proteins exhibit high affinity binding for human growth hormone; K alpha = 9-30 x 10(9) M-1 for the liver receptor and K alpha = 6 x 10(9) M-1 for the binding protein. The apparent molecular weight of the liver receptor is 130,000 by reduced sodium dodecyl sulfate gel electrophoresis, while that of the binding protein is 51,000. Both contain N-linked carbohydrate. The amino-terminal sequences of the liver growth hormone receptor and the serum binding protein were found to be the same, indicating that the binding protein corresponds to the extracellular domain of the liver receptor. Ubiquitin was found covalently linked to the liver receptor but not to the serum binding protein. The amino acid sequences of several peptides from the liver receptor were also determined after tryptic and V8 protease digestion.     

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

Identification of receptors in a system of functionally heterogeneous proteins specifically bound to androgens in rat liver cytosol

The methods of androgen receptor (RA) isolation and identification in rat liver cytosol were studied. It was shown that male rat liver contains a system of specific androgen (A)-binding proteins consisting of at least three main components: RA, delta 4-androstendione (delta 4-A)-binding component and an unusual estrogen-binding protein interacting also with A and the first two components in females. The identity of one of A-binding components to RA was proved by cumulative properties of this component which are similar to those of RA from other tissues. These properties are as follows: 1) high values of apparent association constant, Ka, for 3H-R1881 (2.8 +/- 0.3 X 10(8) M-1) and 3H-5 alpha-dihydrotestosterone (3H-DHT) (5.0 +/- 0.4 X 10(8) M-1); 2) low binding capacity--approximately 10 fmol/mg of protein of nonfractionated cytosol; 3) pronounced specificity of affinity for active A (DHT, R1881, testosterone); 4) large size of the protein molecule (6.5 +/- 0.25 nm); 5) ability to decrease this size to 3.2 +/- 0.08 nm in a high ionic strength buffer; 6) precipitation at low concentrations of ammonium sulfate: 7) strong interaction with heparin-Sepharose. The properties of the delta 4-A-binding component do not coincide with those of RA: it has a low Ka for 3H-delta 4-A (1.15 +/- 0.5 X 10(6) M-1), a high binding capacity (1.22 +/- 0,12 pmol/mg of protein of nonfractionated cytosol) and can bind various delta 4-3-ketosteroids irrespective of the degree and nature of their biological activity. It was concluded that preliminary isolation of rat liver RA on heparin-Sepharose can be used for differential identification and characterization of this protein.