Comparison of the effects of amiodarone and ipodate on the rat heart

Chronic treatment of rats with amiodarone has been shown to produce hypothyroid-like effects such as a reduction in body and heart weight and increased synthesis of the low ATPase V3-cardiac isomyosin (Bagchi, Brown, Schneider and Banerjee 1987). In this report, we have tested the hypothesis that amiodarone causes these effects through the inhibition of intracellular production of triiodothyronine (T3) from thyroxine (T4) by comparing the effects of amiodarone with those of ipodate, a potent inhibitor of T4 to T3 conversion. Separate groups of rats were given dietary ipodate and amiodarone respectively for six weeks. Both agents increased serum T4 and T4/T3 ratios, a finding consistent with the inhibition of peripheral T4 to T3 conversion. However, ipodate failed to produce hypothyroid-like effects on body weight, heart weight and isomyosin transitions similar to those found in the amiodarone group. These data indicate that the hypothyroid-like effects of amiodarone on the rat heart are not due to the inhibition of intracellular generation of T3 from T4.     

Substrate binding to phosphoglycerate kinase monitored by 1-anilino-8-naphthalenesulfonate

The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding.     

Two signal transduction pathways mediate interleukin-1 receptor expression in Balb/c3T3 fibroblasts

Our previous studies showed that platelet-derived growth factor (PDGF) modulated interleukin-1 (IL-1) activity and IL-1 binding to Balb/c3T3 fibroblasts (Bonin, P. D., and Singh, J. P. (1988) J. Biol. Chem. 263, 11052-11055). Subsequent studies have demonstrated an action of PDGF at the level of IL-1 receptor (IL-1R) gene expression. PDGF treatment of Balb/c3T3 cells produces a 10-20-fold stimulation of mRNA for IL-1 receptor. Investigation of the signal transduction pathways shows that activation of either the protein kinase C pathway or the cAMP-mediated pathway leads to the stimulation of IL-1 receptor expression in Balb/c3T3 cells. Treatment of Balb/c3T3 cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, produced an increased 125I-IL-1 binding to cells and stimulation of IL-1R mRNA. Staurosporine, an inhibitor of protein kinase C, blocked the induction of IL-1 binding by PDGF or PMA. Down-regulation of protein kinase C by pretreatment with PMA reduced the subsequent stimulation by PDGF. Chronic treatment with PMA, however, did not produce a complete inhibition of PDGF effect on IL-1R. Further studies showed that the agents that stimulate cAMP accumulation (isobutyl methylxanthine, dibutyryl), directly stimulate adenylate cyclase (forskolin), or activate G protein (choleragen) stimulated 125I-IL-1 binding and IL-1R mRNA accumulation in Balb/c3T3 cells. These studies suggest that potentially two signal transduction pathways mediate IL-1 receptor expression in Balb/c3T3 fibroblasts. Evidence is presented that suggests that stimulation of IL-1R through these two pathways (PMA/PDGF-stimulated and cAMP-stimulated) occurs independent of each other.     

Human aglycosyl-IgG exhibits increased hydrophobicity. Binding/fluorescence studies with 8-anilinonaphthalene-1-sulfonic acid (ANS)

Human monoclonal, aglycosyl-IgG produced in vitro in the presence of tunicamycin, was compared with its native and acid pH-altered counterparts for their respective abilities to bind the fluorescent hydrophobicity probe, 8-anilinonaphthalene sulfonate. A novel technique based on continuous-flow dynamic dialysis (Sparrow et al., 1982, Anal. Biochem. 123:255-264) allowed binding studies under non-equilibrium conditions. While the native IgG conformation exhibits two, weak ANS binding sites (ca. 10(3) l/mol), aglycosyl-IgG has one weak and one moderate affinity (least squares average Ka = 2 X 10(4) l/mol) site, and the acid conformer binds yet another two ANS molecules with moderate affinity (4 X 10(4) l/mol). Increases in affinity and in the number of sites correlate roughly with increased relative percent fluorescence by conventional fluorimetry. The fluorescence lifetime of ANS bound to altered IgGs is about 10% longer (T2 = 15 nsec by time-resolved fluorimetry) than that for native IgG. All populations also exhibit a rapid decay component (T1 = 3 nsec) analogous to that seen for ANS in 50% aqueous dioxane. Results are discussed in relation to structural role(s) for IgG-linked heterosaccharides.     

A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

8‐Anilino‐1‐naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl‐SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl‐SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS. Copyright © 2010 John Wiley & Sons, Ltd.     

The interaction of human glycophorin with 8-anilino-1-naphthalene sulfonate.

Both the sialoglycoprotein of human erythrocyte membranes, glycophorin, and the sialic acid free protein, obtained by treatment of glycophorin with neuraminidase (EC 3.2.1.18), increase the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). Binding of ANS to glycophorin is weak compared with the binding to bovine serum albumin (BSA). equilibrium dialysis gives an apparent binding constant of about 4 X 10(3) M(-1) at neutral pH, but Ka increases 1.75 times when NaCl or CaCl2 are added and 10-fold when the pH is lowered to 3.0. Sialic acid groups do not significantly affect ANS binding, although they have some effect at low ionic strength and neutral pH. Fluorescence studies indicate only one to two binding sites for ANS, with apparent pK = 3.8 +/- 0.2, and located close to aromatic residues in glycophorin. Polarization and quantum efficiency of the fluorescence of ANS associated with glycophorin fail to indicate changes in the vicinity of the binding site when the pH is lowered.     

Amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor

In mammals, sweet taste perception is mediated by the heterodimeric G-protein-coupled receptor, T1R2/T1R3. An interesting characteristic of this sweet taste receptor is that it has multiple ligand binding sites. Although there have been several studies on agonists of sweet taste receptors, little is known about antagonists of these receptors. In this study, we constructed a cell line stably expressing the human sweet taste receptor (hT1R2/hT1R3) and a functional chimeric G-protein (hGα16gust44) using the Flp-In system for measuring the antagonistic activity against the receptor. This constructed cell line responded quite intensely and frequently to the compounds applied for activation of hT1R2/hT1R3. In the presence of 3 mM amiloride, the responses to sweet tastants such as sugar, artificial sweetener, and sweet protein were significantly reduced. The inhibitory activity of amiloride toward 1 mM aspartame was observed in a dose-dependent manner with an IC₅₀ value of 0.87 mM. Our analysis of a cell line expressing hT1R3 mutants (hT1R3-A733V or hT1R3-F778A) made us to conclude that the target site of amiloride is distinct from that of lactisole, a known sweet taste inhibitor. Our results strongly indicate that amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor and also that this receptor has multiple inhibitor binding sites.     

Uridine diphosphate galactose 4-epimerase: nucleotide and 8-anilino-1-naphthalenesulfonate binding properties of the substrate binding site.

Escherichia coli UDP-galactose 4-epimerase in its native form (epimerase.NAD) binds 8-anilino-1-naphthalenesulfonate (ANS) at one tight binding site per dimer with a dissociation constant of 25.9 +/- 2.1 micrometer at pH 8.5 and 27 degrees C. This appears to be the substrate binding site, as indicated by the fact that ANS is a kinetically competitive reversible inhibitor with a Ki of 27.5 micrometer and by the fact that ANS competes with UMP for binding to the enzyme. Upon binding at this site the fluorescence quantum yield of ANS is enhanced 185-fold, and its emission spectrum is blue shifted from a lambdamax of 515 to 470.nm, which suggests that the binding site is shielded from water and probably hydrophobic. Competitive binding experiments with nucleosides and nucleotides indicate that nucleotide binding at this site involves coupled hydrophobic and electrostatic interactions. The reduced form of the enzyme (epimerase.NADH) has no detectable binding affinity for ANS. The marked difference in the affinities of the native and reduced enzymes for ANS is interpreted to be a manifestation of a conformational difference between these enzyme forms.     

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH.

The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.     

Asymmetric membrane expansion and modification of active and passive cation permeability of human red cells by the fluorescent probe 1-anilino-8-napththalene sulfonate.

1. The membrane perturbations induced by the interaction of the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS) with human red blood cells were studied. 2. ANS below 0.5 mM inhibits partially (20% maximum) the ouabain-insensitive Na+ and K+ influx and efflux. Above 0.5 mM ANS increases both Na+ and K+ leak fluxes. The increased cation leaks are larger for Na+ than K+. 3. The (Na+ +K+)-ATPase and ouabain-sensitive Na+ and K+ fluxes are inhibited by ANS. Ouabain-insensitive, Mg2+-dependent ATPase activity of ghosts is stimulated by [ANS] less than 0.3 mM and inhibited by [ANS] greater than 0.3 mM. 4. ANS also inhibits the Na+-dependent, ouabain-insensitive K+ influx that is inhibited by ethacrynic acid and furosemide. 5. Red cells become crenated with [ANS] less than 1 mM and sphere at [ANS] greater than 1 mM. In the former conditions hypotonic hemolysis is decreased whereas the latter increase osmotic fragility. 6. It is suggested that ANS expands the membrane asymmetrically by binding preferentially to the external membrane surface. 7. It is concluded that ANS is a general inhibitor of ion transport, particularly of those processes thought to involve facilitated-diffusion mechanisms. The increased cation leaks observed at high ANS concentrations may be related to prehemolytic membrane disruption. 8. The membrane perturbations caused by ANS are compared to those caused by other reversible inhibitors of anion exchange in red blood cells. Their possible modes of action are discussed.     

Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high-resolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25 degrees C and 37 degrees C. Overall, the affinity of ANS was lower at 37 degrees C compared to 25 degrees C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent-exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.     

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.     

The interaction of 8-anilino-1-naphthalenesulfonate with polylysine and polyarginine

Comparative studies on the interaction of 8-anilino-1-naphthalenesulfonate (ANS) with polylysine and polyarginine have been made by equilibrium dialysis and fluorescence or circular dichroism measurements, to investigate the structural characteristics of the polypeptides. The results are summarized as follows: (i) ANS binds to either of the polypeptides primarily by electrostatic interaction while hydrophobic interaction partially facilitates the dye binding; both interactions are stronger in the polyarginine-dye binding than the polylysine-dye binding. (ii) The fluorescence of ANS is more intensified when the dye binds to polyarginine than to polylysine regardless of the value of r (number of bound dye per amino-acid residue) of polypeptide-dye complexes, although the intensification depends on the r value and becomes maximum at r = 0.25–0.35 for both cases. (iii) The binding of ANS to each polypeptide is cooperative at r < 0.4. (iv) The circular dichroism is more efficiently induced in the spectral region of ANS by binding to polyarginine than to polylysine. From these results, it was concluded that, compared to polylysine, polyarginine suffers some structural change by ANS binding into a more compact molecular configuration having some regularity with a lower dielectric environment.     

The role of gangliosides in the interaction of a growth inhibitor with mouse LM cells

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 micrograms/ml ganglioside GM1 at 0 degrees C for 3 hr sensitized the mouse L-cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I-labeled inhibitor to 3T3 cells was maximal after 60 min at 0 degrees C and saturable at approximately 1 X 10(4) molecules/cell. Furthermore, binding of the inhibitor was dose-dependent, with half-maximal binding at 1.5-2.0 nM and saturation at 8.0-10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 X 10(-9) M and that there are 1 X 10(4) receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0 degrees C and saturation occurred at 5 X 10(3) molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co-receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside-containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one-half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.     

Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis

We have isolated from bovine cerebral cortex cells and purified to homogeneity an 18,000 dalton, pl 3.0 sialoglycopeptide that inhibits protein synthesis and DNA synthesis of nontransformed but not transformed cells without affecting uptake of radiolabeled precursors. In this paper, we examine the relationship between the binding of the sialoglycopeptide inhibitor to 3T3 cells and inhibition of protein synthesis. Binding of the sialoglycopeptide to 3T3 cells was rapid at 37 degrees C and reached a maximum at 30 min; the binding at 37 degrees C was shown to be saturable and specific. Scatchard analysis of the binding indicated that 3T3 cells contained about 2 X 10(4) receptors/cell with a dissociation constant of 1.0-1.5 nM. Several lines of evidence indicated that receptor occupancy on 3T3 cells correlated with the protein synthesis inhibitory activity of the sialoglycopeptide. A comparison of the kinetics of inhibitor binding with the kinetics of protein synthesis inhibition demonstrated that binding directly correlated with the inhibition of protein synthesis, concentration-dependent inhibition of protein synthesis directly correlated with concentration-dependent receptor occupancy, and a direct correlation was also observed between the kinetics of inhibitor dissociation from its specific cell surface receptor and the kinetics of recovery from protein synthesis inhibition.     

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.     

The influence of 4-hydroxy-4-androstene-3,17-dione on androgen metabolism and action in cultured human foreskin fibroblasts

4-hydroxy-4-androstene-3,17-dione (4-OHA) has been shown to be a potent inhibitor of aromatase activity. It is effective in the control of estrogen-dependent processes in female subjects and may potentially be useful in the treatment of estrogen-dependent processes in men. Human foreskin fibroblasts grown in cell culture provide a model to investigate the effects of 4-OHA on extraglandular aromatase activity as well as the ability of the compound to influence androgen receptor binding and the 5 alpha-reduction of testosterone (T). Initial experiments were carried out to determine the potency of 4-OHA in genital skin fibroblasts by incubating cells with 4-OHA over a range of concentrations. When aromatase activity was determined at a substrate concentration close to the apparent Km of the enzyme, a 44% inhibition of enzyme activity occurred at a mean concentration of 5 nM 4-OHA. Enzyme kinetic studies analyzed by Eadie-Hofstee plots demonstrated competitive inhibition by 4-OHA with a mean apparent Ki of 2.7 nM. When 5 alpha-reductase activity was determined in the presence of 200 nM [3H]T, in the absence or presence of 4-OHA, a 50% inhibition of enzyme activity occurred at an inhibitor concentration of 3 microM. In androgen receptor binding studies, 4-OHA possessed 1% of the affinity of dihydrotestosterone (DHT) for [3H]DHT binding sites. In summary: 4-OHA is a potent and specific inhibitor of aromatase activity in human genital skin fibroblasts, the affinity of the enzyme for 4-OHA being greater than its affinity for the substrate, androstenedione. The influence of 4-OHA on 5 alpha-reductase activity and androgen receptor binding is minimal.