Photoaffinity labelling with [3H]R1881 of androgen receptors from human cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities. Partially purified receptors were covalently labelled with [3H]R1881 (triated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from normal individuals showed a single radioactive peak, Mr approximately 97 K. Cytosols from patients with decreased nuclear transfer showed a similar peak, Mr approximately 97 K; cytosols from patients with partial androgen insensitivity (PAIS) or receptor positive complete androgen insensitivity (CAIS, R+) showed a peak Mr approximately 30 K-43 K.     

Genital fibroblasts from normal individuals and patients with androgen insensitivity: demonstration of reduced or absent levels of a specific protein (Mr approximately 41K, pI approximately 6) in receptor negative cells

Genital fibroblasts were obtained from normal individuals and from patients with a variety of syndromes of defective androgenization (complete androgen insensitivity, partial androgen insensitivity, microgenitalia, hypospadias, infertility). Cells were labelled with [35S]methionine, and patterns of protein synthesis compared by two-dimensional gel electrophoresis, with isoelectrofocusing electrophoresis gels or non-equilibrated pH gradient electrophoresis (NEPHGE) gels as the first dimension. A protein (mol. wt approximately 41K, pI approximately 6) was found on NEPHGE gels to be reduced or absent in fibroblasts in which androgen receptor levels were abnormal. The protein was unaltered by prior incubation with 1-100 nM dihydrotestosterone for 48 h, and was present in cells both from normal controls, and from patients with abnormal sexual differentiation showing normal androgen receptor levels. The coincidence of low or absent 41K with low or absent androgen receptors suggested the possibility that it may constitute a steroid-binding moiety of the androgen receptor. To test this possibility cytosols from normal foreskins or normal cultured fibroblasts were adsorbed with testosterone-sepharose affinity resin to remove androgen receptors. Cytosols so treated showed levels of 41K on NEPHGE indistinguishable from those in untreated cytosols, or in cytosols treated with underivatized sepharose. We therefore conclude that the 41K protein, while an accurate marker of the presence or absence of androgen receptors over a range of clinical disorders, is neither an androgen-induced protein nor an androgen-binding protein.     

Analysis of renal and hippocampal type I and type II receptors by fast protein liquid chromatography

Type I and Type II adrenal steroid receptors from rat renal and hippocampal cytosols were studied by the technique of Fast Protein Liquid Chromatography. Type I receptors were labelled with [3H]aldosterone plus excess RU26988, and Type II receptors with [3H]dexamethasone. On a Mono Q anion exchange column the molybdate-stabilized renal and hippocampal Type I receptors both eluted as single symmetrical peaks at 0.27 M NaCl, with a recovery of approximately 90% and 60-fold purification (renal) and 10-15-fold (hippocampal). Molybdate-stabilized Type II binding sites from both hippocampal and renal cytosols co-eluted with the Type I sites. On Superose gel filtration renal Type I receptor-steroid complexes consistently eluted two fractions later than hippocampal Type I complexes, suggesting that the renal complexes are smaller; Type II receptor-steroid complexes from both cytosols co-eluted, consistently one fraction behind hippocampal Type I sites. Sequential gel filtration and anion exchange chromatography achieved a 1000-fold purification of renal Type I binding sites, with an overall recovery of 10%.     

Effect of endogenous corticosterone on the determination of dexamethasone receptor levels in rat liver cytosol

The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developing rats has been studied. Liver cytosols from adrenalectomized rats were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of unbound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in under-estimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors.     

Steroid hormone receptors in MCCLX, a transplantable lactogen-dependent mammary tumor of the rat

MCCLX is a transplantable rat mammary tumor which, for sustained growth, requires the elevated levels of circulating lactogen provided by pregnancy or the implantation of an estrogen pellet. High affinity receptors for estradiol, as well as for the glucocorticoids, dexamethasone and triamcinolone acetonide and the progestin R5020 were measured in the cytosols of these tumors. Estrogen binding capacities were significantly lower in the cytosols of tumors from estrogen pellet treated animals compared with tumors from pregnant animals. Ligand exchange assays demonstrated that nuclei of tumors from estrogen-treated rats contained 3-4 times the estrogen receptors but that there was a definite decrease in total estrogen binding capacity compared with tumors from pregnant rats. It was concluded that this lactogen-dependent tumor contains steroid receptors with molecular properties similar to those of normal target tissues, including estrogen receptors capable of nuclear translocation, the levels of which are modulated by the specific growth conditions.     

Hepatic Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial stabilization by molybdate

In structure and general mode of action, the Ah receptor is very similar to the receptors for steroid hormones. Molybdate previously has been shown to be highly effective at preserving ligand-binding function in steroid receptors during their exposure to elevated temperature or high ionic strength and at stabilizing steroid receptors as high molecular weight oligomeric complexes. Since such stabilization by molybdate can be very useful during characterization and purification of receptors, we tested the effects of molybdate on the Ah receptor to determine if the Ah receptor, like the receptors for steroid hormones, might be stabilized. In hepatic cytosols from C57BL/6N mice and Sprague-Dawley rats, molybdate concentrations up to 30 mM in homogenizing and analysis buffers did not alter the concentration of specific Ah receptor sites detected by binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. However, inclusion of 20 mM molybdate in the homogenizing buffer did significantly protect unliganded Ah receptor from thermal inactivation at 20 degrees C and from KCl-induced loss of ligand-binding ability. In accord with previous reports, 20 mM molybdate in homogenizing and analysis buffers greatly increased the concentration of detectable glucocorticoid receptor in rat hepatic cytosol and estrogen receptor in rat uterine cytosol. Exposure to 0.4 M KC1 caused the glucocorticoid receptor from rat liver to shift sedimentation from approximately equal to 8 S to approximately equal to 4 S and caused a severe loss of specific glucocorticoid binding. Presence of 20 mM molybdate stabilized the glucocorticoid receptor as a single discrete peak sedimenting at approximately equal to 8 S. In contrast, the Ah receptor from rat liver exposed to 0.4 M KC1 in the presence of molybdate sedimented as biphasic peaks; one peak (approximately equal to 9.5 S) corresponded to the form of Ah receptor observed at low ionic strength, while the other peak (approximately equal to 5.5 S) corresponded to the form of Ah receptor seen in cytosol treated with 0.4 M KC1 in the absence of molybdate. Addition of heparin to hepatic cytosols from mice or rats shifted sedimentation of Ah receptor from approximately equal to 9.5 S to approximately equal to 5.5 S. Molybdate, again, provided stabilization in the approximately equal to 9.5 S form, but only for about one-half the total Ah receptor content in both rat and mouse hepatic cytosols. In sum, molybdate is far less effective at stabilizing rodent Ah receptors than it is at stabilizing steroid receptors in the same species.     

A monoclonal antibody to a 48 K antigen in oestrogen-dependent human breast cancer cells

A mouse was immunised with an antigen(s) purified by oestradiol-Sepharose affinity chromatography of pooled oestrogen-receptor positive cytosols from human breast cancer tissue. One antibody secreting clone was identified which precipitated labelled antigen and which also stained MCF-7 cells. Culture supernatant and ascites fluid were used for immunofluorescence, SDS-PAGE-Western blotting, photoaffinity labelling and binding studies. The antibody staining of MCF-7 cells was inhibited by preincubation in oestrogen-receptor positive cytosol but was unaffected by oestrogen-receptor negative cytosol. MCF-7 cells stained whether cultured in the presence or absence of oestradiol. The oestrogen-receptor negative cell lines MDA-MB-231 and MDA-MB-330 did not stain. Binding studies with 16-alpha-iodooestradiol using breast cancer tissue cytosols followed by immunoprecipitation showed activity only with oestrogen-receptor positive cytosols with optimal binding activity at 4 degrees C, unaffected by molybdate, but reduced at 25 degrees C or in the presence of 0.4 M KCl. Binding studies with MCF-7, MDA-MB-231 and MDA-MB-330 cytosols and nuclear fractions only showed activity with the MCF-7 cytosol and MCF-7 particulate fractions. The antibody recognised a 48 K species in both MCF-7 cytosol and nuclear fractions but not in the cytosol and nuclear extracts of oestrogen-receptor negative cell lines. Photoaffinity labelling using 16 alpha-iodooestradiol suggests the 48 K antigen does not bind oestradiol directly. The relationship of this antigen to the classical oestrogen-receptor and receptor complex awaits further clarification.     

A protein in crude cytosol regulates glucose-6-phosphatase activity in crude microsomes to regulate group size in Dictyostelium

Dictyostelium discoideum form groups of approximately 2 x 10(4) cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). The CF signal transduction pathway involves CF-repressing internal glucose levels by increasing the K(m) of glucose-6-phosphatase. Little is known about how this enzyme is regulated. Glucose-6-phosphatase is associated with microsomes in both Dictyostelium and mammals. We find that the activity of glucose-6-phosphatase in crude microsomes from cells with high, normal, or low CF activity had a negative correlation with the amount of CF present in these cell lines. In crude cytosols (supernatants from ultracentrifugation of cell lysates), the glucose-6-phosphatase activity had a positive correlation with CF accumulation. The crude cytosols were further fractionated into a fraction containing molecules greater than 10 kDa (S>10K) and molecules less than 10 KDa (S<10K). S>10K from wild-type cells strongly repressed the activity of glucose-6-phosphatase in wild-type microsomes, whereas S>10K from countin(-) cells (cells with low CF activity) significantly increased the activity of glucose-6-phosphatase in wild-type microsomes by decreasing K(m). The regulatory activities in the wild-type and countin(-) S>10Ks are heat-labile and protease-sensitive, suggesting that they are proteins. S<10K from both wild-type and countin(-) cells did not significantly change glucose-6-phosphatase activity. Together, the data suggest that, as a part of a pathway modulating multicellular group size, CF regulates one or more proteins greater than 10 KDa in crude cytosol that affect microsome-associated glucose-6-phosphatase activity.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

Effects of ATP and pyrophosphate on properties of glucocorticoid-receptor complexes from rat thymus cells

Cytosols from rat thymus cells incubated with glucocorticoid contain nonactivated and activated receptors and mero-receptor complexes, in relative amounts that depend on the incubation conditions. These forms can be separated by a rapid minicolumn chromatographic technique based on their differential affinities for DNA, DEAE, and hydroxylapatite. We have used this method to examine the effects of ATP, pyrophosphate (PPi), and related compounds on cytosolic complexes. In addition to ATP, already known to promote activation at 0 degrees C, PPi, ADP, and other triphosphates at millimolar concentrations promoted activation of nonactivated complexes. AMP and Pi had little effect. ATP and PPi at millimolar concentrations also reduced binding of activated complexes to DNA. Characterization of the ATP- and PPi-activated complexes by gel filtration and ion exchange chromatography revealed two DNA-binding forms. One was essentially identical (Stokes radius of approximately 5.4 nm, elution from DEAE at approximately 50 mM KCl) to the normal activated complex obtained directly from cells incubated at 37 degrees C. The other had a Stokes radius of approximately 3.1 nm and had no affinity for DEAE. Analysis by minicolumns and gel filtration showed that ATP and PPi prevented formation of mero-receptor complexes, a process which occurs relatively rapidly in untreated thymus cytosols. These compounds did not alter properties of preformed mero-receptor. The accumulation of 3.1-nm complexes in thymus cytosols in which formation of mero-receptor is prevented suggests that this form is an intermediate, normally short-lived, in the conversion of 5.4 nm complexes to mero-receptor.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Corticosteroid receptors in the avian kidney

The binding $\text{in vitro}$ of tritiated aldosterone to domestic duck ($\text{Anas platyrhynchos}$) kidney tissue has been investigated. Using tissue from animals on a normal diet, tritiated aldosterone was specifically bound to kidney cytosol with an apparent equilibrium dissociation constant of about 9 nM and number of binding sites in the 20 fmol/mg protein range. These values did not show statistically significant changes when the cytosol originated from animals with salt activated nasal glands. Kidney cytosols labeled with tritiated aldosterone sedimented with a single peak at 8S in a linear sucrose gradient (10–30%) and this peak was quenched by excess, radioinert aldosterone. Following incubation of labeled cytosols with crude nuclei, the cytosols became depleted of the label and aldosterone was translocated to the Tris-soluble and Tris-insoluble, 0,4 M KCl soluble nuclear fractions. Kidney cytosols metabolized aldosterone extensively to a compound presumed to be 3α,5β-tetrahydroaldosterone. However, only unchanged aldosterone became receptor-bound. It was concluded that the duck kidney possesses aldosterone receptors, though competition studies indicated that the specificity of these receptors might be different from those described in the mammalian kidney.     

Protein C in bovine plasma after warfarin treatment. Purification, partial characterization, and beta-hydroxyaspartic acid content

Protein C was purified from the plasma of a cow treated with the vitamin K antagonist warfarin. The purified protein appeared not to bind Ca2+ ions in contrast to protein C from an untreated animal. The gamma-carboxyglutamic acid content of the abnormal protein C was reduced to approximately 10% of normal, whereas the beta-hydroxyaspartic acid content was only slightly decreased, suggesting that vitamin K is not involved in the postribosomal hydroxylation of the aspartic acid residue in position 71 of the light chain of protein C. The abnormal and normal proteins were activated at the same rates by thrombin, but normal protein C was more rapidly activated by the thrombin-thrombomodulin complex. Compared to normal protein C, the abnormal one had virtually no anticoagulant activity.     

Estrogen receptors in the adrenal cortex of the possum (Trichosurus vulpecula)

1. Specific [3H]estradiol binding activity with characteristics of estrogen receptors was found in the cytosols and nuclear extracts of the adrenal cortex proper and special zone of the brushtail possum (Trichosurus vulpecula). 2. The specific estradiol receptor had a sedimentation coefficient on sucrose gradients of approximately 9S and a molecular weight on gel filtration of more than 200,000. The adrenal cortex cytosol binds [3H]estradiol with high affinity (Ka 5.5 X 10(9) M-1), and limited capacity (Bmax 62.7 fmol/mg cytosol prot). In competition experiments with different steroids the receptor showed a high affinity for four estrogens and a very low affinity to androgens, progesterone and cortisol. 3. There was no difference in the affinity and maximum binding capacity of the cytosols from cortex proper in male and female animals, but the binding capacity of the special zone of females was half that of cortex proper. Estradiol receptors were found in the kidney, liver, lung, testis and muscle but only in the adrenal and prostate was the binding capacity relatively high compared with the uterus. 4. The specific binding capacity of [3H]estradiol to cytosols of adrenal cortex at different stages of the estrus cycle and pregnancy was unrelated to that of the uterus. In the adrenal the receptor concentration was lowest at estrus, when uterine concentration was high, while in late pregnancy the binding of adrenal cortex and uterus cytosols was almost the same. 5. The possible physiological significance of the presence of a specific estrogen receptor in male and female possums is discussed.     

Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies

In studies from several laboratories evidence has been adduced that renal Type I (mineralocorticoid) receptors and hippocampal "corticosterone-preferring" high affinity glucocorticoid receptors have similar high affinity for both aldosterone and corticosterone. In all these studies the evidence for renal mineralocorticoid receptors is indirect, inasmuch as the high concentrations of transcortin (CBG) in renal cytosol make studies with [3H]corticosterone as a probe difficult to interpret, given its high affinity for CBG. We here report direct binding studies, with [3H]aldosterone and [3H]corticosterone as probes, on hippocampal and renal cytosols from adrenalectomized rats, in which tracer was excluded from Type II dexamethasone binding glucocorticoid receptors with excess RU26988, and from CBG by excess cortisol 17 beta acid. In addition, we have compared the binding of [3H]aldosterone and [3H]corticosterone in renal cytosols from 10-day old rats, in which CBG levels in plasma and kidney are extremely low. Under conditions where neither tracer binds to type II sites or CBG, they label an equal number of sites (kidney 30-50 fmol/mg protein, hippocampus approximately 200 fmol/mg protein) with equal, high affinity (Kd 4 degrees C 0.3-0.5 nM). Thus direct tracer binding studies support the identity of renal Type I mineralocorticoid receptors and hippocampal Type I (high affinity, corticosterone preferring) glucocorticoid receptors.     

Identification of proteins involved in intracellular copper metabolism. Low levels of a approximately 48-kDa copper-binding protein in the brindled mouse model of Menkes disease

Little is known about copper metabolism at the cellular level. The brindled mouse is an animal model of Menkes disease which is an inborn error of copper metabolism. Control and brindled mice were used to identify copper-binding proteins with possible roles in normal copper metabolism that are affected by the defect in the brindled mice. When 64Cu-labeled hepatic or renal cytosols from control mice were applied to Mono Q or Superose columns, a approximately 48-kDa protein coeluted with the protein fractions which contained the radiolabeled copper. Large decreases in copper binding were detected in these fractions from the brindled mice. The same column fractions which showed decreased copper binding showed large decreases in the levels of the approximately 48-kDa protein. Decreased copper binding and approximately 48-kDa protein were not simply secondary to the abnormal hepatic and renal copper levels that are found in the brindled mice since although their liver copper levels are low, their kidney copper levels are high. Elevated levels of an approximately 80-kDa heat shock protein were also detected in the hepatic and renal cytosols from the brindled mice. Consistent with expression of the primary defect in both the liver and kidney, the levels of the approximately 48- and approximately 80-kDa proteins were affected similarly in both organs. Irrespective of how the low levels of the approximately 48-kDa protein may be related to the basic defect in the brindled mice, the data are consistent with an important role for the approximately 48-kDa protein in intracellular copper metabolism.     

Increased autophosphorylation of insulin-like growth factor-I and insulin receptors in placentas of diabetic women

Autophosphorylation of insulin and insulin-like growth factor (IGF)-I receptors were measured in lectin purified receptor preparations from placentas of normal and diabetic patients. The basal and insulin or IGF-I stimulated phosphorylation of the approximately 94 kD protein, corresponding to beta-subunit of the insulin and IGF-I receptors, were approximately 2 times greater (p less than 0.05) in placentas from diabetic patients with poor glycemic control (as judged by their serum HbA1c level) compared to the normals. The magnitude of IGF-I or insulin stimulation of the phosphorylation of the 94 kD protein was comparable in placentas from both diabetic and normal patients. Immunoprecipitation and immunodepletion of IGF-I receptor by alpha-IR3, a monoclonal antibody to IGF-I receptor, revealed the increased basal phosphorylation of the approximately 94 kD protein in placentas of diabetic patients to be associated with IGF-I and insulin receptors. The magnitude of IGF-I and insulin stimulated phosphorylation of the immunoprecipitated and immunodepleted IGF-I receptor, respectively, was the same in both normal and diabetic patients. These results suggested that the increased basal phosphorylation of the 94 kD protein in placentas from diabetic patients may be intrinsic to IGF-I and insulin receptor, however, the regulatory mechanisms effecting the increase may not be dependent on IGF-I or insulin.     

Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro

Fresh cytosols extracted from unfertilized amphibian eggs contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca²⁺, and is designated primary CSF (1°CSF). During storage of Ca²⁺-containing cytosols at 2°C, stable CSF activity appears, designated secondary CSF (2°CSF). In Rana pipiens egg cytosols, the development of 2°CSF coincides with the formation of a protein complex with a molecular weight above 2,000 kDa, and this large molecule exhibits a high 2°CSF activity when purified (Shibuya and Masui, 1989: Development 106:799–808). The present study shows that both the formation of 2°CSF protein complex and the development of its activity are inhibited by ethylamine and glycine-ethyl-ester (GEE), both known as potent transglutaminase (TGase) inhibitors. An affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts with an approximately 68-kDa protein in fresh egg cytosols, as well as with the 2°CSF protein complex. In cytosols deprived of transglutaminase by immunoprecipitation, neither the development of 2°CSF activity nor the formation of its protein complex can occur. These results indicate that transglutaminase of Rana pipiens eggs is responsible for the formation of 2°CSF, and that transglutaminase itself is incorporated into 2°CSF molecules. Mol. Reprod. Dev. 47:302–311, 1997. © 1997 Wiley-Liss, Inc.     

Atrial natriuretic peptide binding properties of purified rat glomerular membranes

125I-ANP (3-[125I] iodotyrosyl28) binding studies with purified rat glomerular membranes indicate two types of physiologically relevant hormonal receptors, Types I and II, Kd approximately 5 pM and approximately 2.5 nM, respectively. All preparations were essentially free of capsular and tubular contamination. Binding data indicated that Type I receptors were three times more concentrated than Type II receptors in purified membrane fractions. When purified membranes were cross-linked with 125I-rANP, using disuccinimidyl suberate and separated by SDS-PAGE, approximately 75- and approximately 140-kDa proteins were specifically labeled in a ratio of approximately 3:1, respectively. Thus, in purified renal glomerular membranes, Type I receptors with molecular weight of approximately 75-kDa appeared to predominate and would be detectably saturated at circulating ANP concentrations as low as 15 pg/ml. These findings could account for the exquisite sensitivity of natriuretic response to ANP.