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1.
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.  相似文献   

2.
Dihydrotestosterone (DHT) does not seem to be the active specific metabolite of testosterone in hypertrophic sebaceous glands of subjects affected by male pattern baldness (MPB) and several results indicate that probably 3-beta-androstanediol (beta DIOL) could be an active form of testosterone in those glands. Cytosol and serum from several patients affected by MPB and subjected to hair autotransplantation, was incubated with both beta DIOL and 3-alpha-androstanediol (alpha DIOL). Binding patterns indicate that alpha DIOL binds to cytosolic proteins probably due to the contaminating sex hormone binding globulin (SHBG), whereas beta DIOL exhibits an atypical binding process in cytosol in the presence of high concentrations of non radioactive beta DIOL. This binding increases progressively up to 2 pmol/mg protein at the limit solubility conditions for the non radioactive steroid. This pattern is not observed in serum from the same patients, where the binding of beta DIOL is typically restricted to the SHBG. These results strongly suggest the existence of a specific beta DIOL-binding protein in the hypertrophic sebaceous glands and explain the lack of specific receptor for DHT in these tissues.  相似文献   

3.
The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [3H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the Kd value was 3.3 X 10(-8) M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the Kd value was 2.5 X 10(-8) M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to 3/4 of that in the untreated cytosol. The profile of glycerol gradient centrifugation indicated that [3H]methyltrienolone-bound receptor migrated in the 8-9 S region in both untreated and triamcinolone-blocked cytosols, but the 8-9 S peak in triamcinolone-blocked cytosol was reduced to about 3/4 of that of untreated cytosol.  相似文献   

4.
The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.  相似文献   

5.
The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.  相似文献   

6.
The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.  相似文献   

7.
The binding of methyltrienolone (R 1881) in crude human hyperplastic prostate cytosol was determined by a charcoal assay. Maximum binding was observed after 2-3 h of incubation at 0 degrees C. This binding decreased steadily thereafter and reached 41% of the 2-hour values after 96 h of incubation. In human hyperplastic prostate, the binding of 3H-R 1881 was competed by low concentrations of R 1881, R 5020 and progesterone and by high concentrations of dihydrotestosterone (DHT) and 17 alpha-methyl-DHT. In the rat prostate, on the other hand, this binding was competed by low concentrations of DHT and 17 alpha-methyl-DHT and only by high concentrations of progesterone and R 5020. The apparent association constant (Ka) of R 1881 was determined in three human prostates and found to be 0.2-0.4 X 10(9) liters/mol; the number of binding sites ranged from 101 to 158 fmol per mg of protein. These findings constitute further evidence for the existence of relatively large amounts of a progesterone-binding component in human hyperplastic prostate.  相似文献   

8.
When 0.5 M sodium thiocyanate is added to uterine cytosol previously labeled with excess [3H]-17 beta-estradiol, no change can be detected in the steady-state cytosol concentration of [3H]estradiol-receptor complex for at least 20 h at 4 degrees C. However, the rate of exchange of bound estradiol in the presence of NaSCN was found to be substantially higher than that in the absence of the chaotropic salt. In the presence of NaSCN, the dissociation rate of the complex increases about 10-fold (K-1 SCN = 1.10 x 10(-2) min-1 vs. K-1 = 1.07 X 10 (-3)min-1) while the rate of association increases about 2-fold (K1 SCN = 1.2 X 10(7) min-1M-1 vs.K1= 7.4 X 10(6) min-1 M-1). The Kd changes 6.4-fold (Kd SCN = 9 X 10(-10) M vs. Kd = 1.4 x 10(-10 M) with no decrease in the number of binding sites as shown by Scatchard plots of saturation experiments. This effect of NaSCN can be exploited to assay preformed estrogen-receptor complex by exchange with [3H]estradiol at low temperature. When the sample containing preformed complex is incubated overnight (16 h) at 4 degrees C with excess [3H]estradiol in the presence of 0.5 M NaSCN, there is a quantitative exchange of nonlabeled for estradiol without loss of binding sites. Hormonal steroids other than estrogens do not interfere, and the exchange estradiol is bound with high affinity. Precision, accuracy, and linearity of the method are highly satisfactory.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
S M Ho  P Ofner 《Steroids》1986,47(1):21-34
The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.  相似文献   

12.
Many liver processes are sexually dimorphic. In particular, the microsomal content of specific enzymes and the synthesis of specific proteins are under sex steroid hormone control. Because the liver of male rats is strikingly androgen responsive, we sought evidence for an androgen receptor in this tissue. We detected and characterized both cytosolic and nuclear androgen-binding proteins. Both forms bind [3H]R1881 (methyltrienolone, 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratriene-3-one) with the high affinity, low capacity, and specificity for androgens and antiandrogens characteristic of androgen receptors. No high-affinity binding of [3H]DHT could be detected in unfractionated cytosol because of the rapid metabolism of this ligand; however, binding of a DHT metabolite to the high-capacity male-specific estrogen binder (MEB) of cytosol was observed. Both gel filtration and heparin-Sepharose affinity chromatography separate the cytosolic androgen receptor from MEB. Incubation of cytosol in the absence of sodium molybdate resulted in androgen-binding activity which was retained by DNA-cellulose. Castration of male rats results in a time-dependent loss of both cytosolic and nuclear androgen binding, as well as a loss in MEB activity. Androgen-binding activity is low in livers from female rats, but can be induced by testosterone treatment. An intact pituitary is necessary for maintenance of androgen-binding activity, as hypophysectomy results in complete loss of activity.  相似文献   

13.
The binding of triton X-100 to bovine serum albumin has been shown to exhibit positive cooperativity. Subsequent equilibrium dialysis studies indicate that the binding of Triton X-100 to sheep serum albumin likewise shows positive cooperativity, the first two stepwise equilibrium constants being K1 = 1.24 X 10(4) M-1 and K2 = 1.62 X 10(4) M-1. However, the mechanism for Triton X-100 binding to human serum albumin differs in that the binding isotherm indicates the binding sites are independent and identical. In the latter case the binding is described by the Scatchard model with an equilibrium constant of K = 7.2 X 10(3) M-1. The studies were conducted at 16 degrees C in pH 7.0, I = 0.05 phosphate buffer.  相似文献   

14.
A new theory on the complex growth hormone (GH) mediated promotion of tissue growth has been developed on the basis of in vitro studies of growth hormone receptors on human peripheral mononuclear cells (PMC). It is hypothesized that GH acts through its tissue receptors and regulates its own receptors through two different pathways: first GH directly downregulates GH binding, second a partially GH-dependent serum factor (the so-called SM-B) enhances GH binding. Some experimental evidence for this hypothesis is presented using a new method to investigate GH receptors in circulating human blood cells: The effect of trypsin, antitrypsin, growth hormone, somatomedin-B (SM-B) and anti-SM-B-antiserum on GH binding to PMC was studied. Trypsinization of cells leads to a decrease both of specific binding and of binding affinity (affinity constant after 60 minutes of trypsinization 0.5 X 10(6) M-1 versus 1.5 X 10(6) M-1 in untreated control cells). Exposure of PMC to antitrypsin activities was followed by an increase of binding affinity and specific binding (affinity constants with 10 KIU 1.9 X 10(6) M-1, with 100 KIU 2.4 X 10(6) M-1, with 1000 KIU 3.6 X 10(6) M-1). This antitrypsin effect exceeds the binding values expected after blocking trypsin activities possibly being present in the incubation medium. In a subset of experiments the partially GH-dependent serum factor SM-B was used as the antitrypsin moiety and was shown to increase specific GH binding to PMC in a similar manner as did antitrypsin (with 1000 ng SM-B affinity constant 12.0 X 10(6) M-1, specific binding 9.7%).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
A method is described for the determination of binding capacity (Ro) and dissociation constant (Kd) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [3H]dihydrotestosterone3 (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (Ro = 12,105 ± 4305 (S.D.) sites/cell; Kd = 1.0 ± 0.7 (S.D.) × 10?9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.  相似文献   

16.
The present study was undertaken to determine cytosol binding properties of [3H]methyltrienolone, a synthetic androgen, in comparison with [3H]dexamethasone, a synthetic glucocorticoid, under conditions of glucocorticoid excess in skeletal muscle. Male hypophysectomized rats received either seven daily subcutaneous injections of cortisone acetate (CA) (100 mg X kg-1 body wt) or the vehicle, 1% carboxymethyl cellulose. Following treatment, both [3H]dexamethasone and [3H]methyltrienolone-receptor concentrations were decreased from those in vehicle-treated rats by more than 90 and 80%, respectively, in CA-treated animals. Scatchard analysis of [3H]methyltrienolone binding in muscles of vehicle-treated animals became nonlinear at high concentrations of labeled ligand and were reanalyzed by a two-component binding model. The lower affinity, higher capacity component, which was attributed to binding of methyltrienolone to a "dexamethasone" component, disappeared in muscles of CA-treated rats and Scatchard plots were linear. Receptor concentrations of the higher affinity lower capacity "methyltrienolone" component were similar in muscles of vehicle-treated and CA-treated groups. From competition studies, the high relative specificities of glucocorticoids for [3H]methyltrienolone binding in muscles of vehicle-treated animals were markedly reduced by CA treatment. In addition, the binding specificity data also showed strong competition by progesterone and methyltrienolone for [3H]dexamethasone binding and estradiol-17 beta for [3H]methyltrienolone binding. These results demonstrate that most of the [3H]methyltrienolone binding is eliminated under in vivo conditions of glucocorticoid excess. Furthermore, the competitiveness of various steroids for receptor binding suggests that rat muscle may not contain classic (ligand-specific) glucocorticoid and androgen receptors.  相似文献   

17.
In the present study, “in vitro” evidences are shown for the existence of a highly active 3α-hydroxysteroid dehydrogenase in the crude cytosol of rat muscle homogenates; the use of 5α-dihydrotestosterone (DHT) is therefore compromised in receptor binding measurements because of its extensive metabolism. The synthetic anabolic androgen, methyltrienolone (MT) palliates this disadvantage of DHT. Both steroids, as well as testosterone, appear to be bound to an 8–8.5 S androgen receptor on sucrose density gradient. The androgen receptor in the vastus and the levator ani bulbocavernosus complex (LA/BC) shows similar association constants, but the number of binding sites in LA/BC is about 5 times higher than in vastus. Otherwise, the total number of muscle androgen receptors seems to be invariant in adult and aged rats. The binding to these macromolecules can thus be measured “in vitro” provided specific and sensitive methods are utilized.  相似文献   

18.
A variant of human transferrin with abnormal properties.   总被引:5,自引:0,他引:5       下载免费PDF全文
Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.  相似文献   

19.
The binding equilibrium of deuteroporphyrin IX to human serum albumin and to bovine serum albumin was studied, by monitoring protein-induced changes in the porphyrin fluorescence and taking into consideration the self-aggregation of the porphyrin. To have control over the latter, the range of porphyrin concentrations was chosen to maker dimers (non-covalent) the dominant aggregate. Each protein was found to have one high-affinity site for deuteroporphyrin IX monomers, the magnitudes of the equilibrium binding constants (25 degrees C, neutral pH, phosphate-buffered saline) being 4.5 (+/- 1.5) X 10(7) M-1 and 1.7 (+/- 0.2) X 10(6) M-1 for human serum albumin and for bovine serum albumin respectively. Deuteroporphyrin IX dimers were found to bind directly to the protein, each protein binding one dimer, with high affinity. Two models are proposed for the protein-binding of porphyrin monomers and dimers in a porphyrin system having both species: a competitive model, where each protein molecule has only one binding site, which can be occupied by either a monomer or a dimer; a non-competitive model, where each protein molecule has two binding sites, one for monomers and one for dimers. On testing the fit of the data to the models, an argument can be made to favour the non-competitive model, the equilibrium binding constants of the dimers, for the non-competitive model (25 degrees C, neutral pH, phosphate-buffered saline), being: 8.0 (+/- 1.8) X 10(8) M-1 and 1.2 (+/- 0.6) X 10(7) M-1 for human serum albumin and bovine serum albumin respectively.  相似文献   

20.
K Mujoo  M Ali    M K Sahib 《The Biochemical journal》1983,216(1):227-231
Monospecific anti-[rat alpha-foetoprotein(alpha-FP)] immunoglobulin G was coupled to CNBr-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an adsorbent. The immunoaffinity column was used to isolate alpha-FP from neonatal-rat skin. Purified skin alpha-FP was found to be immunologically and electrophoretically similar to serum alpha-FP. It yielded a single band with mol.wt. 68000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, on polyacrylamide-gel electrophoresis under non-denaturing conditions, the alpha-FP displayed slow- and fast-moving variants similar to those observed in serum alpha-FP. A Scatchard plot of oestradiol binding to the alpha-FP yielded an association constant of 2.5 X 10(9)M-1 by dextran-coated-charcoal and 0.75 X 10(8)M-1 by Sephadex-gel-filtration procedures respectively. Skin explants from newborn rats were found to incorporate [14C]leucine into immunoprecipitable intracellular alpha-FP. Cycloheximide inhibited the synthesis of alpha-FP in skin explant culture. Our results indicate that newborn-rat skin contains alpha-FP that is similar to serum alpha-FP and which may arise in neonatal-rat skin as a result of synthesis in situ.  相似文献   

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