Entrapment of protein protease inhibitors in red blood cells.

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.     

4-Azasteroidal 5 alpha-reductase inhibitors without affinity for the androgen receptor

In efforts to develop potent 5 alpha-reductase inhibitors without affinity for the androgen receptor, synthetic 3-oxo-5 alpha-steroids were tested for their ability to inhibit 5 alpha-reductase, using [14C]testosterone as the substrate, and for their ability to inhibit the binding of [3H]5 alpha-dihydrotestosterone to the androgen receptor of rat prostate cytosol. 2',3' alpha-Tetrahydrofuran-2'-spiro-17-(5 alpha-androstan-3-one) is not an inhibitor of 5 alpha-reductase and has a high affinity for the androgen receptor; substitution of the -CH2- at the 4-position with N-H resulted in a good inhibitor of 5 alpha-reductase. The 4-N-CH3 derivative is even more active, whereas the N-CH2-CH3 derivative is inactive. These 4-aza derivatives have much lower affinity for the androgen receptor than the parent compound. The 4-N-H derivatives of several 3-oxo-5 alpha-steroids were found to be 20-100% as potent as their corresponding 4-N-CH3 analogs as inhibitors of 5 alpha-reductase, whereas their androgen receptor affinities were at least 40-fold lower than their 4-N-CH3 analogs. Their 5 beta-isomers did not inhibit either 5 alpha-reductase or the androgen receptor binding of [3H]5 alpha-dihydrotestosterone. Two of these 4-N-H steroids, 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one and 17 beta-N, N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one, are potent 5 alpha-reductase inhibitors with Ki values equal to 29.2 +/- 1.7 and 12.6 +/- 0.8 nM, respectively, but have little affinity for the androgen receptor. The inhibition of 5 alpha-reductase by both compounds is competitive with testosterone. When [3H]testosterone was incubated with minced rat prostate in the presence of either of these two 4-azasteroids, the nuclear concentration of 5 alpha-dihydrotestosterone decreased and that of testosterone increased. The total nuclear uptake of testosterone plus 5 alpha-dihydrotestosterone was not significantly affected. These 4-azasteroids should be useful for investigating the importance of 5 alpha-reductase in androgen action in vivo.     

Degradation of Rat Brain Cholinergic Muscarinic Receptors In Vitro: Enhancement by Agonists and Inhibition by Antagonists

Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenylmethylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium-dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation.     

Assay and Characterization of Ovarian Testosterone Receptor

Abstract

Ovarian cytosol from immature, hypophysectomized rats was used to characterize the biochemical properties of ovarian androgen receptor and to develop a reliable and convenient assay procedure for its measurement in small quantities of tissue. The results show that Sephadex G-25 columns provide a rapid and reliable method to separate bound from free testosterone for assay of androgen receptor in the ovarian tissue. The binding affinity (K_D=0.6 nM) and sedimentation value (ES) for the ovarian androgen receptor are similar to other steroid receptors and is saturable. It is inactivated by heat and protease digestion and the binding is specific for potent androgens. The ovary contains approximately equal quantities of androgen and estrogen receptor. A physiological role for the presence of androgen receptor in the ovarian tissue is proposed.     

Assay of androgen binding sites by exchange with methyltrienolone (R 1881).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) binds specifically to androgen receptor in rat prostate cytosol where, unlike androstanolone, it is not metabolized. By exchanging bound endogenous hormone in rat prostate cytosol with labelled R 1881, it is possible to measure total (free anc occupied) binding sites. This assay method has also been applied to the measurement of androgen receptor sites in human benign prostatic hypertrophy where R 1881 has the added advantage of not being bound by any contaminating plasma protein (sex hormone binding protein).     

Assay of primate seminiferous tubule androgen receptors using [3H]mibolerone

The synthetic radiolabelled androgen mibolerone (7 alpha, 17 alpha-dimethyl-19-nortestosterone) was used to characterize androgen receptor binding in the seminiferous tubules from Cynomolgus monkey testis. Mibolerone binding was of high affinity (Kd = 0.6-5.4 nM) and limited capacity (37-50 fmol/mg protein), and was androgen specific. Sucrose density gradient centrifugation using a vertical tube rotor permitted the identification of a 9S molybdate-stabilized receptor under low salt conditions. The receptor bound to DEAE-cellulose. Methyltrienolone, but not mibolerone, also bound to a low affinity high capacity binding site in tubule cytosol, which probably represents glucocorticoid receptor binding, since it could be displaced by excess dexamethasone. However, occupancy of this low-affinity binding site by dexamethasone in an androgen receptor assay with [3H]methyltrienolone lead to a 33% underestimation of receptor binding, which appeared to relate to radioactive decomposition. Mibolerone, as well as methyltrienolone, bound to a progestin-binding protein in seminiferous tubule cytosol. These studies provide methods for the study of seminiferous tubule androgen receptors in subhuman primates and indicate that, due to its greater stability and lack of binding to glucocorticoid receptor, mibolerone is a useful new ligand in the study of androgen receptors in testis and its constituent cells.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

The synthesis and testing of E-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone and Z-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone as gamma-emitting ligands for the androgen receptor

Two iodinated steroids, E-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone and Z-17 alpha-(2-iodovinyl)-5 alpha-dihydrotestosterone were synthesized in a search for a gamma-emitting androgen that binds with high affinity to the androgen receptor. Such compounds would be extremely useful research tools for studies of androgen responsive tissues and as in vivo probes of androgen responsive tumors such as prostate cancer. These 17 alpha-iodovinyl steroids were synthesized because many 17 alpha-substituents do not interfere markedly with binding to the androgen receptor and because similar analogs of other steroids, estrogens and progestins, have been shown to have the requisite properties for ligands to those receptors. Both of these potential ligands were tested for their ability to compete with [3H]R1881 for binding to the androgen receptor in cytosols from prostate, hypothalamus and pituitary. The relative binding affinities ranged between 5 and 20%, depending upon the tissue and steroid. In order to test the two ligands directly, they were both synthesized labelled with 125I and tested for binding to the androgen receptor in prostatic cytosol and in vivo for specific concentration in androgen responsive tissues. While there was considerable binding in the prostatic cytosol, it was not specific because 5 alpha-dihydrotestosterone did not compete. Likewise in the in vivo experiment there was no evidence for androgen receptor mediated concentration of the tracers. While on the basis of relative binding affinity, these 2 steroids appeared to be good candidates for androgen receptor ligands, neither were useful for this purpose. These results contribute new information which will be valuable in the design of other gamma-emitting androgens and emphasises that, in this process, other factors such as metabolism and nonspecific binding must be considered.     

Steroid metabolism and binding activity in a murine renal tumor cell line

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.     

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.     

Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis

The DNA-binding form of the calf uterine androgen receptor (AR) was subjected to limited protease digestion using chymotrypsin, trypsin and a rat prostate cytosol protease. The properties of the generated polypeptide fragments were identified and compared with those of the intact AR. Physicochemical characterization was achieved through sedimentation analysis, gel filtration chromatography and DEAE anion exchange chromatography. Intactness of functional binding domains was evaluated by measuring the retention of steroid- and DNA-binding capacity. Under non-denaturing conditions the intact AR is a highly asymmetrical molecule with a Stokes radius (RS) of 45A, a sedimentation coefficient of 4.3S and a relative molecular mass of 80,000 daltons. This form of AR has an intrinsic binding affinity for DNA and was eluted from DNA-cellulose with 9 mM MgCl2. Chymotrypsin produced a more globular polypeptide (RS: 31A; 3.1S; 41,000 daltons) with a decreased net negative charge. This fragment also displayed DNA-binding affinity but required a higher concentration of MgCl2 (14 mM) for DNA-cellulose elution, indicating an increased affinity for DNA. The observed reduction in molecular size upon chymotrypsin treatment was confirmed when analysed by SDS-polyacrylamide gel electrophoresis after covalently labelling of the AR with [3H]R1881. Rat prostate cytosol contains a protease which is very active in generating an AR polypeptide with an increased affinity for DNA, without changing the AR net negative charge (RS: 33A; 3.7S; 51,000 daltons). The specificity of this protease remained unknown since none of a large number of inhibitors was able to inactivate this enzyme. The fragment generated is different from that obtained with chymotrypsin since significant differences in size as well as in charge were measured. Trypsin treatment generated a much smaller polypeptide (RS: 25A; 2.9S; 30,000 daltons) which had lost its DNA-binding capacity, but not its steroid binding site. This form probably represents the so-called meroreceptor. When intact AR was treated sequentially with prostate cytosol and trypsin, a polypeptide fragment with identical properties was obtained, indicating the spatial separation of two of the proteolytic cleavage sites. These studies provide evidence for the distinct nature of the molecular domains for androgen and DNA interaction on the calf uterine AR.     

An androgen receptor in the brain of the green frog Rana esculenta

An androgen receptor (AR) has been detected in both the hypothalamic and extra-hypothalamic area of the brain of the male of the green frog Rana esculenta. 3H-T binding activity was detected separately in the hypothalamic and extra-hypothalamic areas. Experiments of binding assay showed 3H-T binding activity in the nuclear extract but not in the cytosol of both the hypothalamic and the extra-hypothalamic areas. The androgen binding moiety was not strictly specific for androgens, binding also 17 beta-estradiol, although to a lesser extent. 3H-testosterone binding activity fluctuated in both the hypothalamic and the extra-hypothalamic areas throughout the reproductive cycle, and paralleled androgen plasma levels.     

Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity

We report here the continued characterization of a 41-kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin-cleavage activity and an echinoderm-specific collagen-cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10-phenanthroline (a metalloprotease inhibitor), and E-64 (a thiol protease inhibitor) were without effect on the gelatin-cleavage activity of the 41-kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin-cleavage activity. This result was confirmed in a quantitative gelatin-cleavage assay using the water soluble, serine protease inhibitor [4-(2-aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin-cleavage activity. Calcium and magnesium competed for binding to the 41-kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41-kDa species.     

Protease Inhibitors Reduce Effects of Denervation on Muscle End-Plate Acetylcholinesterase

The effects of certain protease inhibitors on end-plate acetylcholinesterase (AChE) activity, as well as on wet weight and total protein, were studied in vivo in intact and denervated anterior gracilis muscles from the rat. A combination of leupeptin, pepstatin, and aprotinin, administered intraarterially, partly prevented the early (24 h) denervation-induced decrease in muscle weight and protein content. In turn, leupeptin and aprotinin, either alone or in combination, markedly reduced the decay of AChE activity in the denervated muscles, whereas pepstatin alone was ineffective. Such effects were additive in that the inhibitors in combination were more effective than when they were used separately. Additional experiments indicated that none of the inhibitors, at the concentrations used, affected AChE activity directly, nor did they have a significant effect during processing of the muscle samples. These findings indicate that the initial decay of AChE activity with denervation was effectively reduced by the inhibitors, probably through inactivation of proteolytic enzymes which, otherwise, would be increase in denervated muscle.     

Identification of an androgen receptor in the adult chicken oviduct

The chicken oviduct androgen receptor was characterized by sucrose density gradient centrifugation, Scatchard analysis, competition studies, and affinity labeled with dihydrotestosterone 17 beta-bromoacetate. A specific 8.5 S peak was seen on 0.01 M KCl sucrose density gradients when the receptor was labeled with [3H]5 alpha-dihydrotestosterone. Specific 4.6 S peaks were seen when receptor labeled with [3H]5 alpha-dihydrotestosterone or [3H]dihydrotestosterone 17 beta-bromoacetate was analyzed on 0.3 M KCl sucrose density gradients. Scatchard analysis of [3H]5 alpha-dihydrotestosterone binding by oviduct cytosol was consistent with two binding sites. A Kd of 0.13 nM was found for the high affinity androgen receptor. Competition studies showed the following order of ligand affinity: 5 alpha-dihydrotestosterone greater than dihydrotestosterone 17 beta-bromoacetate greater than progesterone greater than estradiol. A 61.2 kDa protein was specifically covalently labeled with [3H]dihydrotestosterone 17 beta-bromoacetate. The chicken oviduct androgen receptor possesses characteristics similar to other androgen receptors, and provides a good source of androgen receptor for physicochemical studies of the native receptor protein.     

Intracellular inhibition of chromatin binding and transformation of androgen receptor by 3'-deoxyadenosine

Incubation of minced rat ventral prostate with 3'-deoxyadenosine (3'-dA) prior to labeling with the androgen, tritiated 7 alpha, 17 alpha-dimethyl-19-nortestosterone, reduced the level of androgen receptor bound to chromatin and increased the level of cytosolic androgen receptor and the fraction of cytosolic androgen receptor that did not bind to DNA. This effect was specific for 3'-dA and not mimicked by adenosine, 2'-deoxy-adenosine, cytidine, guanosine, or uridine. Adenosine was a competitive inhibitor of the 3'-dA effect. Labeled cytosolic androgen receptor from 3'-dA-treated prostate had properties that were similar to those exhibited by untransformed androgen receptor from prostate cytosol prepared in the presence of Na2MoO4, an inhibitor of receptor transformation in cell-free systems. Both androgen receptors had sedimentation coefficients of 8-9 S in low-salt gradients, did not bind to DNA tightly, and had a high affinity for DEAE-cellulose. The 3'-dA effect on these properties was not observed if androgen receptor from 3'-dA-treated prostate was isolated on high-salt gradients. These findings show that androgen receptor transformation does take place in intact prostate cells and suggest that 3'-dA inhibits chromatin binding of androgen receptor by interfering with androgen receptor transformation. The transformation process appears to involve removal of components from androgen receptor. Since 3'-dA is a potent inhibitor of the synthesis, polyadenylation, and nucleocytoplasmic transport of RNA, the 3'-dA effect may indicate a role for RNA in the mechanism of receptor transformation in intact target cells.     

A microassay for the measurement of androgen receptors in human prostatic tissue.

A microassay utilizing R 1881 (methyltrienolone) has been developed for the measurement of androgen receptor sites in the cytosol and nuclear extract of human prostatic tissue. Binding of R 1881 to the progesterone binding molecule in cytosol was eliminated by the addition of triamcinolone acetonide. Utilizing a six tube, single point assay, the number of binding sites estimated in nuclear extract averaged 95% of the number measured by a full 7 point Scatchard analysis; the number estimated by the microassay in cytosol averaged 91%. When the single point assay was applied to needle biopsy specimens (200 mg of tissue), the estimated number of binding sites in nuclei averageed 83% of the number measured in bulk tissue (2 grams) utilizing a 7 point Scatchard analysis; the number in cytosol estimated by the microassay on needle biopsy specimens averaged 73%. It is hoped that this technique may be useful in correlating receptor content with hormonal responsiveness in men with metastatic carcinoma of the prostate.     

Concentration and cellular distribution of androgen receptor in human prostatic neoplasia: can estrogen treatment increase androgen receptor content?

The concentration of androgen receptor in cytosol (free and total sites) and nuclear fractions from benign (28 specimens) and malignant prostatic tissue from treated (16 specimens) and untreated patients (10 specimens) were assayed using [3H]methyltrienolone (3H R-1881) as ligand under conditions which stabilize AR and prevent binding of 3H R-1881 to progesterone receptor. It was found that optimum results were obtained when sodium molybdate (10 mM) was added after separation of the nuclear pellet rather than during tissue homogenization; when cytosol and nuclear exchange assays were carried out at 15 degrees C rather than at 0 degrees C; and when hydroxylapatite was used to separate free and bound steroid in the nuclear assay. Although AR values were variable in both BPH and carcinoma tissue, certain patterns of concentration, occupancy, and cellular distribution were observed in different patient groups. In BPH and untreated carcinoma tissue, the mean occupancy of cytosol AR by endogenous androgens was high, but the mean nuclear AR concentration was higher in BPH than in carcinoma tissue. Androgen receptor concentrations in tissue from orchiectomized patients were consistent with the effects of androgen deprivation: total cell AR was depleted, and a higher proportion was present as free cytosol AR. However, in tissue from most patients who had been treated with diethylstilbestrol (DES) on a long-term basis, total cell AR values were high. Although most of the AR was present as free cytosol AR, in three of four patients who had been treated with both orchiectomy and DES, the concentrations of bound cytosol AR and nuclear AR were similar to those in untreated patients.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

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.