Intracellular receptors for 5alpha-dihydrostestosterone in the epididymis of adult rats. Comparison with the androgenic receptors in the ventral prostate and the androgen binding protein (ABP) in the testicular and epididymal fluid

Epididymal cytosol fractions of adult short-time castrated rats contained at least two different androgen protein complexes by experiments $\text{in vivo}$ (Complex I and II).Complex I is probably located intracellularly in the epididymal cells. It was specific for 5α-dihydrotestosterone (DHT) and appeared to be very similar to the cytoplasmic DHT-receptor complexes in rat ventral prostate. By ultracentrifugation on sucrose gradients, it sedimented as heavy aggregates 8–10 S complexes and 3–4 S complexes, which dissociated into 3–4 S complexes at high ionic strength. Complex I was eluted in the void volume from columns of Sephadex G-200.Complex II was also specific for DHT and showed physical properties similar to those of the androgen binding protein (ABP) in the testicular fluid. It was eluted between immunoglobulin G (IgG) (53 Å) and albumin (36 Å) by gel filtration on Sephadex G-200. The sedimentation coefficient was 4.5–5 S (mean 4.6 S_W, 20) at both high and low ionic strength.Complex I and the cytosol receptors for DHT in the rat ventral prostate were both destroyed by heating at 50° C for 30 min, addition of 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS) and charcoal absorption (1 mg/mg protein) overnight, whereas complex II was not influenced by similar treatment.Hemi-castration for 4 weeks caused complex II to disappear completely from the castrated side, confirming the intraluminal localization of this complex. Complex I was not influenced by such treatment, indicating that this protein is located within the epididymal cells. The similarity between complex I and the cytoplasmic DHT-receptor complexes in the ventral prostate also suggests that complex I represents the cytoplasmic receptors for DHT in the epididymis.     

Stability of receptor complexes in the rat ventral prostate and seminal vesicle bound to androgens: thermodynamic study

To examine the behaviour of the receptor-acceptor system of androgen of different biopotencies, we compared the stability of receptor complexes of dihydrotestosterone (DHT), methyltrienolone (R1881) and testosterone (Test) in cytosols, nuclei and nuclear extracts from ventral prostate and seminal vesicle of rats. Liberation of ligand from receptor complexes bound to these ligands followed the first-order kinetics. The rate constant for ligand liberation at 25 degrees C varied with the ligand. The receptor complexes bound to Test were most labile, while the receptor complexes bound to DHT were relatively stable, and intermediate stability was observed in the receptor complexes bound to R1881 under the conditions employed in the present study. Thermodynamic characteristics of the stability of the complexes were also different in these three androgens. The Arrhenius plots of the rate constant for the liberation of ligand from R1881- and DHT-receptor complexes in cytosols and nuclei showed curvilinearities, but the plots for Test-receptor complexes were almost linear. In addition, the stabilizing effect of molybdate on R1881- and DHT-receptor complexes in cytosols was observed in the range of low temperature, while the effect on Test-receptor complexes was significant at the higher temperature. The differences observed in the present study seem to be related to the difference in the biological potency of these androgens.     

Human minimal androgen insensitivity with normal dihydrotestosterone-binding capacity in cultured genital skin fibroblasts: evidence for an androgen-selective qualitative abnormality of the receptor

We have studied a kindred in which two parts of siblings, maternal first cousins, have a form of "minimal" androgen insensitivity that permits morphogenesis of unambiguous male external genitalia, but interferes with normal virilization at puberty. All four had gynecomastia that required reductive surgery. Apart from this common phenotype, they varied considerably in the temporal and regional aspects of their subvirilization and appreciably in their androgenic responsiveness to pharmacological doses of testosterone. The cultured genital skin fibroblasts from one set of siblings have an androgen-receptor activity with the following properties: (1) a normal maximum-binding capacity (Bmax) with 5 alpha-dihydrotestosterone (DHT), or the synthetic androgen, methyltrienolone (MT; R1881) as ligand; (2) a higher than normal apparent equilibrium dissociation constant (Kd) for DHT (0.77 nM) but not for MT; and (3) an elevated dissociation rate (k) of DHT-receptor (0.013 min-1, 37 degrees C), but not MT-receptor, complexes within intact cells. In addition, prolonged incubation with MT, but not DHT, augments the specific androgen-binding activity of the mutant cells as much as that of the controls. Normal cells yield lower values of apparent Kd for DHT (0.1-0.3 nM) after 2- than after 0.5-hr incubation (0.3-1.8 nM), and 1-hr values are intermediate. This occurs despite concurrent catabolic consumption of DHT from the medium and is considered to reflect transformation of initial, low-affinity DHT-receptor complexes to subsequent, higher-affinity states by a process that depends on time and initial ligand concentration. The mutant complexes described here can readily attain the highest state of affinity with MT, but have an impeded, variably expressed ability to do so with DHT. These findings suggest that a structural mutation at the X-linked locus that encodes the androgen-receptor protein is responsible for its androgen-selective dysfunction. Synthetic, nonhepatotoxic androgens, with corrective effects in vitro comparable to those of MT, may be therapeutically useful for these subjects.     

The state transitions of normal and mutant androgen-receptor complexes in human genital skin fibroblasts

We have incubated cells from controls and subjects with receptor-defective androgen resistance with 3H-labelled testosterone (T), methyltrienolone (MT), dihydrotestosterone (DHT) or mibolerone (MB) and studied the temperature dependence of the dissociation rate constants of these various androgen-receptor (A-R) complexes both within cells and after they were extracted from them. In control cells, Arrhenius plots for T-, MT-, DHT- and MB-R complexes were linear and formed a hierarchy of dissociation states with energies of state IV greater than III greater than II, greater than I, respectively. Relative to this hierarchy, the dissociation states of the MB-, DHT- and MT-R complexes in mutant cells were displaced to higher, androgen-inappropriate energies in a mutant-distinctive pattern. When extracted from cells control or mutant T- or MT-R complexes, and mutant (but not control) DHT- or MB-R complexes lowered their respective dissociation rates by undergoing state transitions in conformity with the hierarchy. Hence we propose that different A-R complexes reach different dissociative states by undergoing sequential transitions along a common pathway, and that these transitions are co-regulated both by the chemical characteristics of the bound androgen and by other cellular non-receptor factors.     

Rates of dissociation of sex steroid hormones from human sex hormone-binding globulin: a reassessment

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure the rates of dissociation of dihydrotestosterone (DHT), testosterone (T), and estradiol (E2) from their binding proteins in human serum at 37 degrees C. Because measurements were begun after each hormone had fully (greater than 99%) dissociated from albumin, the observed rates of dissociation correspond to the rates of dissociation of the sex hormone-binding globulin (SHBG)-hormone complexes. The dissociation rate constants of the hormone-SHBG complexes were determined to be 0.016 +/- 0.001, 0.056 +/- 0.002, and 0.083 +/- 0.003 s-1 for DHT, T, and E2, respectively, corresponding to half-times of dissociation (t1/2) of 43, 12 and 8.4 s, respectively. The physiological significance of these findings can best be appreciated by comparing these t1/2 s with the capillary and sinusoidal transit times of various tissues (less than 1 s to approximately 10 s).     

Mechanism of cell-mediated cytotoxicity at the single cell level. II. Evidence for first-order kinetics of T cell-mediated cytolysis and for heterogeneity of lytic rate.

The kinetics and rate of T cell-mediated cytolysis was assessed by measuring the times required for lysis of isolated target cells by single cytotoxic lymphocytes. Single target cell lysis was determined microscopically by observing trypan blue uptake as a function of time of incubation of effector-target conjugates in agarose. Lysis of EL-4 target cells by alloimmune peritoneal exudate lymphocytes was initiated without a lag and was essentially complete at 2 hr. Both zero-order and first-order kinetics equations were analyzed for fit to the 0 to 2 hr lysis values. Statistically, the zero-order kinetic function could be rejected (p greater than 0.05), but the first-order kinetics function (p less than 0.01) could not. This strong evidence for first-order kinetics of T cell-mediated cytolysis implies that within each CTL-target cell population, cytolysis occurs exponentially as a random decay process and that one event in the entire process of cytolysis is rate limiting. The first-order equation was then applied to measurements of the rate of cytolysis in many different individual effector-target cell combinations. Significant differences in the lytic rate were apparent when either the effector or target cell were varied, with the rate constants spanning a 5-fold range. The heterogeneity of lytic rates is consistent with the hypothesis that lytic efficiency is a function of both the effector and target cells used.     

Extracellular correction of the androgen-receptor transformation defect in two families with complete androgen resistance

We have characterized the cellular and extracellular phenotype of the mutant androgen receptor (AR) from two families who have complete androgen resistance despite a normal androgen-binding capacity (Bmax) in their genital skin fibroblasts (GSF). The cellular receptors fail to up-regulate their basal AR activity in response to prolonged incubation with 5 alpha-dihydrotestosterone (DHT), or with two synthetic androgens, methyltrienolone (MT) and mibolerone (MB), and form A-R complexes with increased equilibrium (Kd) and non-equilibrium (k) dissociation constants. In addition, they are thermolabile when recently dissociated, but not in their native state. A-R complexes made in normal or mutant cytosol at 4 degrees C elute from DEAE-Sephacel at approximately 0.25 M KCl (untransformed), with or without prior passage through Sephadex G-25; when made in cells at 37 degrees C, extracted with 0.4 M KCl in a buffer containing 10 mM Na2MoO4, and desalted by G-25, they elute at less than or equal to 0.1 M KCl. Normal KCl-extracted DHT- and MB-R complexes dissociate (37 degrees C) at the same slow, linear rate as their in-cell counterparts (transformed); the mutant ones dissociated more slowly than their rapidly-dissociating in-cell counterparts and, to a variable extent, nonlinearly-an early faster phase, a later slower (transformed). Thus, as judged by two conventional criteria of steroid-R complex transformation, the mutant A-R complexes can transform, possibly in two steps, under certain cell-free conditions. This behavior differentiates a class of structural AR mutations whose molecular definition awaits application of recombinant DNA techniques to the X-linked AR locus.     

Testosterone processing by pituitary cells in culture: an examination of the role of 5 alpha-reduction in androgen action on the gonadotroph

Dispersed rat pituitary cells were exposed to [1,2,6,7-3H]testosterone ([3H]T, 10(-8) M) to assess the role of 5 alpha-reduction in T regulation of gonadotroph secretion. After 4 to 48 hours of exposure, [3H]T metabolites isolated by thin-layer chromatography were characterized in medium and cell homogenates as well as bound to androgen receptors salt-extracted from purified nuclear pellets. Receptor-bound 5 alpha-[3H]dihydrotestosterone ([3H]DHT)/total [3H]androgens rose progressively from 16% at 4 hours to more than 50% at 48 hours. Coincubation with 4-MA (10- to 1,000-fold molar excess) or testosterone-17 beta-carboxylic acid (TCA; 1,000-fold excess) reduced receptor-bound [3H]DHT/[3H]androgen to less than 10% and 20%, respectively, but elevated [3H]T-receptor levels. Despite inhibiting 5 alpha-reductase activity, TCA and 4-MA had no effect on T suppression of gonadotropin-releasing hormone-stimulated luteinizing hormone secretion or T enhancement of total (cell + secreted) follicle-stimulating hormone levels. The results suggest that 5 alpha-reduction to DHT is not essential for the expression of the direct influences of T on gonadotropin synthesis and secretion in rat gonadotrophs.     

Changes in the sedimentation properties of cytosolic androgen receptors associated with activation in vitro and in vivo

In cell-free systems androgen receptor (AR) labeled with (3H)DHT at 0 degrees C in the presence of 50mM molybdate remains unactivated (less than 3% binding to nuclei) and untransformed (7-8S on sucrose density gradients containing 0.4M KCl and 50mM molybdate). In the absence of molybdate, however, these complexes undergo activation and transformation even at 0 degrees C, albeit, very slowly. Incubation of unactivated, untransformed AR complexes at 18 degrees C, or at 0 degrees C in the presence of 0.4M KCl, greatly accelerated both activation and transformation. Activation and transformation are also associated with formation of high affinity (3H)DHT-receptor complexes as indicated by decreased rates of (3H)DHT dissociation from the receptor. Cytosolic AR complexes labeled with (3H)DHT in tissue slices at 37 degrees C, or in vivo, undergo rapid activation, transformation and nuclear translocation. The data suggest that activation and transformation of cytosolic AR in cell-free systems is associated with changes in the physicochemical properties of AR similar to those occurring upon hormone binding in intact cells and in vivo.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105, 000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1, 2-³h]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M ? 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1, 2-α-methylene-6-chloro-pregn-4, 6-diene-17α-o1–3, 20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105,000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1,2-³H]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M — 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1,2-α-methylene-6-chloro-pregn-4, 6-diene-17α-ol-3,20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Kinetic evidence for a unique testosterone-receptor complex in 5α-reductase sufficient genital skin fibroblasts and the effects of 5α-reductase deficiency on its formation

When 5α-reductase-sufficient genital skin fibroblast (GSF) monolayers are incubated with testosterone (T), they first form androgen (A)-receptor (R) complexes that dissociate at a fast rate [k(37°C = 0.024 min⁻¹]. As T is converted to 5α-dihydrotestosterone (DHT), this population of T-R complexes is eventually replaced by one that dissociates much more slowly [k(37°C) = 0.006 min⁻¹], at a rate typical of DHT-R complexes. During the course of T to DHT conversion, one may observe a population of A-R complexes that has a linear (monophasic) intermediate dissociation rate constant [k(37°C) = 0.012 min⁻¹]; this population cannot simply reflect a mixture of T- and DHT-R complexes. The rate at which the complexes are processed from one dissociative form to the next varies with the incubation temperature and the presence or absence of serum in the medium; it also varies within and among GSF strains under apparently constant conditions. To explain these facts, we propose a model that enables the 5α-reductase enzyme to influence the processive dissociative behaviour of T-R complexes by engaging in some sort of coupling with the AR. The proposal is strengthened by a set of observations in cells with constitutive, mendelian or inhibitor-induced 5α-reductase deficiency that preclude a simple quantitative relation between A-R complex processing and the extent of T to DHT conversion.     

Modulation of steroid affinity for the androgen receptor by sucrose

The effects of sucrose on androgen binding to its receptor were investigated. Sucrose decreased the rate of thermal inactivation of unoccupied and occupied androgen receptor (AR) and the rates of [3H]5 alpha-dihydrotestosterone [( 3H]DHT) dissociation from both activated and nonactivated AR complexes. Binding of [3H]DHT to AR in vivo, or in intact cells at 37 degrees C, caused reduction of [3H]DHT dissociation from cytosolic and nuclear complexes, as compared to in vitro labeled receptor complexes. Further, exposure of these complexes to sucrose at 0 degrees C caused an additional reduction of dissociation rates. Thus, the decrease of [3H]DHT dissociation induced by sucrose is independent of the reaction that reduces DHT dissociation from activated and transformed AR. Sucrose also reduced the ability of mersalyl acid to inactivate AR complexes. This effect of sucrose was markedly diminished in the presence of 2M urea. Sucrose did not significantly affect the association rate, sedimentation properties, or nuclear binding ability of AR complexes, but it did decrease the equilibrium dissociation constant. Other monosaccharides and disaccharides also stabilized AR. These data suggest that sucrose induces conformational changes in the steroid binding domain of androgen receptor, thereby reducing the rates of inactivation, steroid dissociation, and the accessibility of sulfhydryl groups to mersalyl.     

Characterization of the cytoplasmic androgen receptor of rat seminal vesicle

Postmitochondrial supernatant (PMS) (1) has been prepared from the homogenate of rat seminal vesicles and the characteristics of the binding reaction of 5-dihydrotestosterone (DHT) to the cytoplasmic androgen receptor have been studied using a charcoal adsorption procedure.

At 0°C apparent equilibrium of binding is reached between 60 and 90 min of incubation but no exchange of bound (³H)DHT can be observed in the presence of a 100-fold excess of unlabelled DHT.

Saturation analysis shows a single class of independent binding sites for DHT with an apparent dissociation constant of 1 nM at 0°C and 2 nM at 25°C. Concentration of binding sites is in the range of 25–80 fmoles/mg protein.

When not occupied by DHT the receptor molecules are inactivated spontaneously following first order reaction kinetics. A rate constant of 0.27 hours⁻¹ at 0°C was determined for the inactivation reaction.

In the (³H)DHT-binding reaction testosterone and 19-nortestosterone are even more efficient competitors than unlabelled DHT, while hydrocortisone does not compete at all. On the other hand significant binding of (³H) testosterone could not be demonstrated.

The (³H)DHT-receptor complex is precipitated from the cytosol by 0 to 33% saturation of ammonium sulphate and sediments as a single, 3.1 S peak in sucrose gradients prepared in 0.4 M NaCl.     

Molecular biology of androgen action: testosterone/dihydrotestosterone receptor and androgen 5 alpha-reductase in the human foreskin

Receptors for testosterone (T) and dihydrotestosterone (DHT) as well as the tissue specific androgen-5 alpha-reductase (A5R) were studied in the foreskin of 52 healthy boys (ages 1-14 years), in order to gain molecular endocrinological data and information about the ontogeny and cytogeny, respectively, of androgen specific target organs. Enzyme determinations were carried out in tissue homogenates by an enzyme kinetic method for the evaluation of Km- and Vmax-values. Reactions velocities were calculated from the turn over rates of T to DHT, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3,beta,17 beta-diol. The precursor (T) was used in increasing concentrations, ranging from 8 to 208 nM. Separation of reaction products was done by thin-layer chromatography and verification of specific radioactivity of metabolites by means of radio gas chromatography on capillary columns. Results of the enzyme analyses: Km = 94.9 +/- 3.5 [nM], and Vmax = 15.8 +/- 1.9 [pmol/mg.h]. Receptors were examined in the cytosolic and nuclear fractions of the tissue specimens. Saturation analyses and calculation of binding data led to specific receptors for T and DHT in the cytosolic (T: Kd = 1.56 +/- 0.12 [nM], Nmax = 122.4 +/- 11.6 [fmol/mg]; DHT: Kd = 1.9 +/- 0.1 [nM], Nmax = 493.3 +/- 77.8 [fmol/mg]) and the nuclear fractions (T: Kd = 1.43 +/- 0.13 [nM], Nmax = 28.7 +/- 3.5 [fmol/mg]; DHT: Kd = 1.37 [nM], Nmax = 196.9 +/- 22.5 [fmol/mg]), Kd-values proved to be quite homogenous (coeff. var. = 0.15-0.21), whereas maximum specific receptor binding activities (Nmax) showed age dependent fluctuations (coeff. var. = 0.35-0.45). Binding capacities of both T- and DHT-receptor, respectively, in cytosolic and nuclear fractions showed peak values in the age group 10-11 years and additional "spikes" of binding rates at age 4-5 years. It is noteworthy that Vmax-values also reached maximum levels in the latter age group. Concerning the ontogeny of the androgen receptor a change of binding properties from the cytosol to the nuclear fraction was observed with the onset of puberty. A comparison of enzyme- and receptor data lead to the theory, that subcellular hormone actions depend on interrelational regulatory mechanisms between androgen receptors (T as well as DHT) and specific enzyme systems (A5R).     

Reaction of c-Type Cytochromes with the Iron Hexacyanides: Mechanistic Implications

The reaction of c-cytochromes with iron hexacyanides is similar in mechanism to the interaction of cytochromes with their physiological oxidants and reductants in that the formation of complexes precedes electron transfer. Analysis of the kinetics of oxidation and reduction of a number of c-cytochromes by solving the simultaneous differential equations defining the mechanism is possible, and allows assignment of all six rate constants describing a minimum three-step mechanism [cyto(Fe(+3)) + Fe(+2) right harpoon over left harpoon cyto (Fe(+3)) - Fe(+2) right harpoon over left harpoon cyto(Fe(+2)) - Fe(+3) right harpoon over left harpoon cyto(Fe(+2)) + Fe(+3)]. We find that the usual steady-state approximations are not valid. Furthermore, the ratio of first-order rate constants for electron transfer was approximately 1.0, and no correlation was found between any of the six rate constants and the differences in oxidation-reduction potential of the iron-hexacyanides and different cytochromes c. However, it was found that the ratio of the rate constants for complex formation between ferricytochrome c and potassium ferrocyanide and ferrocytochrome c and potassium ferricyanide was proportional to the difference in oxidation-reduction potentials. Thus the minimum three-step mechanism given above accurately describes the observed kinetic data. However, this mechanism leads to a number of conceptual difficulties. Specifically, the mechanism requires that the collision complexes formed [cyto(Fe(+3)) - Fe(CN)(6) (-4) and cyto(Fe(+2)) - Fe(CN)(6) (-3)] have very different equilibrium constants, and further requires that formation of the collision complexes be accompanied by "chemistry" to make the intermediates isoenergetic. A more complex five-step mechanism which requires that the reactants [Fe(CN)(6) (-4) and ferricytochrome c or Fe(CN)(6) (-3) and ferrocytochrome c] form a collision complex followed by a first-order process before electron transfer, was found to yield results similar to those of the three-step mechanism. However, describing the formation of the collision complex in terms of a rapid equilibrium circumvents conceptual difficulties and leads to a physically reasonable mechanism. In this mechanism the reactants are in rapid equilibrium with the collision complexes and the rate constants for complex formation are controlled by diffusion and accessibility. The collision complexes then rearrange, possibly through conformational changes and/or solvent reorganization, to yield isoenergetic intermediates that can undergo rapid reversible electron transfer. The five-step mechanism can be described by the same rate constants obtained from the three-step mechanism with the appropriate adjustments to account for rapid equilibrium. This more complex analysis associates the oxidation-reduction potential of a particular cytochrome with the relative magnitude of the first-order conversion of the oxidant and reductant collision complexes to their respective intermediates. Thus the cytochromes c control their oxidation-reduction potential by chemical and/or structural alterations. This mechanism appears to be general in that it is consistent with the observed kinetics of 11 different cytochromes c from a wide variety of sources with a range of oxidation-reduction potentials.     

Non-michaelian behavior of 5 alpha-reductase in human prostate

An in-depth analysis of the kinetics of 5 alpha-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH 5.5, 5 alpha-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this "hysteretic" behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5 alpha-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5 alpha-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.     

Dissociation and auto-oxidation of hemerythrin induced by SH-modification: a kinetic study

Hemerythrin from Siphonosoma cumanense has a trimeric structure consisting of identical subunits, which have no cooperativity nor Bohr effect on oxygen-binding. The trimer was dissociated into its monomers by the modification of the SH group of its cysteines with p-chloromercuriphenylsulfonic acid (PCMPS), which was monitored by stopped-flow of both spectrophotomeric and small angle X-ray scattering methods. The results showed that the process involved sequential modification of the SH groups, dissociation into monomers, and auto-oxidation of ferrous iron in the active center. The modification of the SH groups with PCMPS followed second-order kinetics with a rate constant of 1.8 M-1.s-1. The dissociation and auto-oxidation followed first-order kinetics with rate constants of 4 X 10(-3) s-1 and 5 X 10(-4) s-1, respectively. The obtained rate of auto-oxidation was much faster than that in the native state. These findings lead to the conclusion that the trimeric state of S. cumanense hemerythrin is necessary to prevent auto-oxidation.     

The specific binding of androgens and the subsequent distribution of androgen-receptor complexes within MCF-7 human breast cancer cells

Using a whole cell suspension assay technique, we have examined both the specific binding of testosterone (T) and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) (DHT) and the subsequent distribution of androgen binding activity within MCF-7 human breast cancer cells. We have observed that both androgens are bound with high affinity to the same single class of receptor which is present at a concentration of approximately 8,000 sites per cell. The incubation of cells with either T or DHT was followed by the nuclear accumulation of specifically bound ligand which increased to maximal values within 1 h and then decreased thereafter. However, only about 30% of the total cellular specific binding activity observed with either androgen was localized within the nuclear compartment at any time during a 4 h incubation. Further examination of the extranuclear binding component suggested that a substantial portion of this activity was localized to the particulate fraction of the cytoplasm. The results of these studies suggest that both T and DHT are capable of exerting biological activity within MCF-7 cells, and raise the interesting possibility that androgen-receptor complexes may participate in the direct regulation of mitochondrial and/or microsormal function in this system.     

Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism.

The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21-32, 1989). To determine whether E5 affects EGF:receptor interactions we analyzed the kinetics of 125I-EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I-EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy-independent in both cell lines. The 125I-EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy-dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I-EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy-independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor.