Aromatase and sulfatase inhibitors from Lepiota americana.

From an edible mushroom Lepiota americana Pk., (Agaricaceae), 2-aminophenoxazin-3-one that inhibited aromatase at IC50 = 5.7 microM and 3 beta-hydroxy-5,8-epidioxyergosta-6,22-diene that inhibited sulfatase at IC50 = 0.9 microM were isolated. Neither 2-aminophenoxazin-3-one was active against sulfatase nor was 3 beta-hydroxy-5,8-epidioxyergosta-6,22-diene active against aromatase.

Immunoblot analyses of the elicited Sanguinaria canadensis enzyme, dihydrobenzophenanthridine oxidase: evidence for resolution from a polyphenol oxidase isozyme.

In our initial purification of dihydrobenzophenanthridine oxidase from Sanguinaria canadensis plant cell cultures, we reported that our most purified preparations contained a major band at 77 kDa and minor lower Mr bands. Here we present evidence on highly purified dihydrobenzophenanthridine oxidase from elicited S. canadensis cultures to indicate that this enzyme is the 77-kDa protein and that lower Mr bands include an isozyme(s) of the polyphenol oxidase family that copurifies with it. An antibody raised against the 77-kDa protein and an anti-polyphenol oxidase antibody that recognizes a 70-kDa band were used to monitor chromatographic fractions by immunoblot analysis of the oxidases. Oxidase-containing eluates from DEAE-Sephadex, CM, and HiTrap blue were compared to corresponding flow-through fractions. Bands at 77 and 88 kDa were detected with anti-dihydrobenzophenanthridine oxidase antibody in eluates displaying high dihydrobenzophenanthridine oxidase activity. Polyphenol oxidase specific activity and immunoreactivity partitioned both in flow-through and eluate fractions of the CM and HiTrap columns. Estimation of the dihydrobenzophenanthridine oxidase and polyphenol oxidase specific activities for each step showed increasing enrichment of alkaloidal enzyme accompanied by variable dihydrobenzophenanthridine oxidase/polyphenol oxidase activity ratios. Taken together these observations indicate that the dihydrobenzophenanthridine and polyphenol oxidases have Mr values of 77 and 70 kDa, respectively, and the two enzymes are different entities.

Ribozymes: RNA as a therapeutic agent.

RZs, like ASO agents, are several years from approval by the Food and Drug Administration. Although many problems must be solved before they can be used effectively as therapeutic agents, results from a wide variety of in vitro and in vivo experiments are encouraging. The advantages of RZs are their very high specificity of action, their catalytic mechanism, their potential for targeted therapy, and the lack of side effects with their use. These advantages, coupled with the wide applicability of ribozyme technology, will continue to drive the research necessary to develop RZs into useful therapeutic agents.

Testosterone, dihydrotestosterone, trenbolone acetate, and zeranol alter the synthesis of cortisol in bovine adrenocortical cells.

The objective of this study was to examine the effect of anabolic steroids (testosterone, T; dihydrotestosterone, D; trenbolone acetate, B; and zeranol, Z) on cortisol synthesis by cultured bovine adrenocortical cells. Adrenal glands were obtained from slaughter-aged steers (n = 4). Cortical cells were isolated and their steroidogenic capacity was examined. They were plated in multiwell culture plates. At confluence, cells were treated with T, D, B, or Z at 0, 10, 50, 125, or 500 ng/mL (eight wells per treatment). Twenty-four hours after treatment, one-half of each treatment concentration was stimulated with 10(-9) M ACTH. After 8 h of incubation, cortisol concentration in the media was measured using RIA. Cortical cells were removed from the plates using 1 mM EDTA and analyzed for DNA content. Data were subjected to rank transformation and analyzed by randomized complete block design. Adrenocorticotropic hormone stimulated (P < .01) the release of cortisol by more than threefold. Cortisol synthesis was lower (P < .05) in the presence of T, D, and B. Testosterone caused a greater (P < .05) suppression in cortisol production at 50 and 125 ng/mL than did D. The suppression of cortisol synthesis did not differ between B and T or between B and D. Cortisol synthesis was lowered (P < .05) by the presence of T, D, and B in both ACTH-stimulated and nonstimulated cells but was only suppressed in ACTH-stimulated cells of Z-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and properties of sulfhydryl-group-specific reagents containing 125I.

125I-containing compounds that react specifically with sulfhydryl groups were prepared in yields of 30 to 40% on the basis of starting 125I quantity. The synthetic precursors were commercially available heterobifunctional crosslinkers and the peptide L-arginyl-L-tyrosine. Two types of sulfhydryl specific reagents were prepared: 3-(2-pyridyldithio)propionylarginyl-[125I]-monoiodotyrosine, which permits reversible incorporation of 125I at sulfhydryl sites, and 3-maleimidopropionylarginyl- [125I]monoiodotyrosine, an irreversible labeling reagent. These products were isolated in a highly radiochemically pure form by C18 HPLC. The second-order rate constants for the reaction of 3-(2-pyridyldithio)propionylarginylmonoiodotyrosine and 3-maleimidopropionylarginylmonoiodotyrosine with N-acetylcysteine were 28 +/- 3 M-1 s-1 and 154 +/- 4 M-1 s-1, respectively, at pH 7.3. Storage of carrier-free 3-(2-pyridyldithio)propionylarginyl-[125I]monoiodotyrosine and 3-maleimidopropionylarginyl-[125I]monoiodotyrosine at -80 degrees C at a radioactive concentration of 0.4 mCi/ml resulted in conversion of 125I to species that did not react covalently with sulfhydryl groups. This process occurred with first-order kinetics and a t1/2 of 5.7 days for the pyridyldithio compound and 7.5 days for the maleimido compound. No conversion was observed during storage at -80 degrees C at radioactive concentrations of 0.02 mCi/ml or less. The labeling properties of these compounds were examined using red blood cell proteins as a test system. 3-(2-Pyridyldithio)propionylarginyl- [125I]monoiodotyrosine and maleimidopropionylarginyl-[125I]monoiodotyrosine reacted preferentially with membrane - associated sulfhydryl groups when incubated with intact red blood cells.

Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phase high-performance liquid chromatography.

The specificity and rate of cleavage of adrenocorticotrophic hormone (ACTH) peptide bonds by endoproteinase Arg C were analyzed using capillary electrophoresis (CE) and reversed-phase (C18) high-performance liquid chromatography (HPLC). Acidic cleavage products were readily resolved by CE in uncoated capillaries using low ionic strength electrolytes. However, products predicted to have a net positive charge greater than 2 or more than 4 positively charged groups per peptide did not migrate out from the capillary at low ionic strength. Addition of salts and zwitterions to the electrolyte decreased capillary-peptide interactions such that all of the ACTH peptides examined were eluted with high efficiency separation by CE. Commercially obtained endoproteinase Arg C preparations exhibited peptidase activity at Lys-15-Lys16 and at Lys16-Arg17 in addition to the expected cleavage at Arg-X bonds. ACTH peptide bond cleavage rates for Arg8-Trp9, Arg17-Arg-18, Lys15-Lys16, and Lys16-Arg17 were 1.46, 0.096, 0.57, and 0.029 mumol min-1 mg-1 respectively. CE separations generally exhibited better resolution and were accomplished in shorter times than C18 HPLC separations. These properties make CE a particularly appropriate method for kinetic analysis of proteolytic enzyme action on peptide substrates.

Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB).

Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus-1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response.

Effect of chronic administration of nicotine or cocaine on steroidogenesis in rat adrenocortical cells.

Rats were implanted subcutaneously with osmotic mini-pumps containing either 0.9% NaCl, nicotine (1.5 or 4.5 mg kg-1 day-1), or cocaine (30 mg kg-1 day-1), for 14 days. Neither nicotine nor cocaine treatment significantly altered the maximal rate of steroidogenesis in adrenocortical cell preparations from the animals. However, pretreatment with cocaine increased the sensitivity of the preparation to stimulation by ACTH, the ED50 was 5 pM compared with 10 pM from control animals. Addition of nicotine or cocaine at concentrations up to 100 microM to adrenal cell suspensions from naive rats did not stimulate steroidogenesis or increase the sensitivity of cells to ACTH stimulation. These results suggest that the primary chronic effect of nicotine on steroidogenesis is exerted at the level of the hypothalamus and/or pituitary and not directly on adrenocortical cells. On the contrary, pretreatment with cocaine causes persistent changes in adrenocortical cells.

Bivalent ACTH antagonists: influence of peptide and spacer components on potency enhancement.

The antagonist potency of a series of bivalent adrenocorticotropic hormone (ACTH) peptides was examined using suspensions of inner zone rat adrenocortical cells. Bivalent antagonists were prepared by bis(maleimide) covalent cross-linking of carboxyl terminal cysteine sulfhydryl groups of synthetic ACTH peptides, Cys25 ACTH(7-25) and Cys39 ACTH(7-39). Antagonist potency enhancement was defined by shifts in ACTH(1-39) concentration-steroidogenic response curves relative to monovalent antagonist analogs. The EC50 values measured in the presence of 0.5 microM monovalent antagonist were 110 +/- 28 pM for Cys25 ACTH(7-25)-S-N-ethylsuccinimide and 44 +/- 9 pM for Cys39 ACTH(7-39)-S-N-ethylsuccinimide. Some bivalent ACTH antagonists displayed much greater antagonist potency than their monovalent analogs, which supports the findings of Stolz and Fauchere (Helv Chim Acta 71: 1421-1428, 1988). The level of potency enhancement, however, was found to be dependent upon the spacer used to link receptor binding domains and the length of the ACTH peptide used in bivalent antagonist synthesis. The most potent inhibitor, bis(Cys25 ACTH(7-25)-S-succinimidopropionyl)2-hydroxy-1,3-propanediamine, was 28 times more potent than its monovalent analog, Cys25 ACTH(7-25)-S-N-ethylsuccinimide. However, a bivalent Cys25 ACTH(7-25) peptide containing two bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacers that had been linked end-to-end via dithioerythritol showed no potency enhancement. Cys25 ACTH(7-25) based peptides containing one receptor binding domain and having the structure peptide-spacer-cysteine displayed no enhancement in antagonist potency. Bivalent Cys39 ACTH(7-39) linked by bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacer exhibited only 4-fold enhancement in antagonist potency relative to Cys39 ACTH(7-39)-S-N-ethylsuccinimide. We therefore conclude that the potency enhancement observed with bivalent ACTH peptides: (1) is optimal with spacers less than approximately 40 A in length, (2) is not due to direct interactions between the spacer and cell surface, and (3) is dependent on the length of the ACTH peptide component. In addition, these results indicate that electrostatic interaction between bivalent ACTH peptides and plasma membrane lipids does not adequately account for the potency enhancements observed.

Introduction of sulfhydryl groups into proteins at carboxyl sites.

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.

N-iodoacetyltyramine: preparation and use in 125I labeling by alkylation of sulfhydryl groups.

Preparation and use of N-iodoacetyltyramine in generation of 125I-labeled compounds is described. The kinetics of alkylation of N-acetylcysteine by N-iodoacetyltyramine (k2 = 3.0 M-1 s-1) and N-chloroacetyltyramine (k2 = 0.12 M-1 s-1) indicate that N-iodoacetyltyramine is more useful for labeling sulfhydryl-containing compounds to high specific activity with 125I. Conditions for preparation of carrier-free 125I-labeled N-iodoacetyl-3-monoiodotyramine in 50% yield based on starting iodide are described. The high degree of group specificity of N-iodoacetyl-3-monoiodotyramine reaction with sulfhydryl groups is demonstrated by the high reactivity toward sulfhydryl-containing bovine serum albumin and low reactivity toward N-ethylmaleimide-blocked bovine serum albumin and IgG. 125I-labeled N-iodoacetyl-3-monoiodotyramine was also used to prepare an 125I-labeled ACTH derivative that retains full biological activity, further demonstrating the selectivity toward reactions with sulfhydryl groups.

Evidence for the involvement of a labile protein in stimulation of adrenal steroidogenesis under conditions not inhibitory to protein synthesis.

Evidence is presented to support the hypothesis that synthesis of a labile protein is required for stimulation of steroidogenesis in rat adrenocortical cells. Amino acids L-canavanine and L-S-aminoethylcysteine, at concentrations as high as 5 mM, each inhibited steroidogenesis to a much greater extent than they inhibited protein synthesis. S-Aminoethylcysteine caused a 50% decrease in the stimulated rate of corticosterone production under conditions where incorporation of [35S]methionine into protein was unchanged. Both amino acids block stimulation of steroid synthesis at a step subsequent to the formation of cAMP and before the synthesis of progesterone. The onset of this effect, after the addition of the amino acids, on corticosterone production is quite rapid. These results provide support, that is not dependent on inhibition of protein synthesis, for the hypothesis that a labile protein mediates stimulation of steroidogenesis. Reversal of canavanine and S-aminoethylcysteine inhibition of steroidogenesis by arginine and lysine, respectively, suggests that the inhibitors are functioning as amino acid analogs. S-Aminoethylcysteine inhibits the incorporation of [3H]lysine into protein as well as inhibits steroidogenesis; further, [3H]S-aminoethylcysteine is incorporated into protein that is nonstimulatory. These results suggest that lysine residues play an essential role in the function of the labile protein or that the labile protein contains a large number of lysine residues.

Effect of P-450scc inhibitors on corticosterone production by rat adrenal cells.

Suspensions of rat adrenocortical cells produce corticosterone as the major glucocorticoid. Cholesterol side-chain cleavage, the initial and rate-limiting step in the glucocorticoid biosynthetic pathway, is catalyzed by P-450scc. We have examined the effect of a variety of P-450scc inhibitors on corticosterone production by isolated rat adrenocortical cells. These inhibitors include reversible, noncovalently interacting inhibitors as well as mechanism-based inhibitors which irreversibly inactivate P-450scc in vitro. (20S)-22-nor-22-thiacholesterol and (22R)-22-aminocholesterol cause 50% inhibition of corticosterone production at 4 microM and 30 nM, respectively. Inhibition by these compounds was essentially not time-dependent. (20R)-20-(1-hexynyl)-pregn-5-en-3 beta, 20-diol and (20R)-20-(1,5-hexdiynyl)-pregn-5-en-3 beta, 20-diol at 10 microM inhibited corticosterone production in a time-dependent manner, resulting in 30% inhibition of corticosterone production during a 100-min incubation. (20S)-20-(2-trimethylsilyl ethyl)-pregn-5-en-3 beta, 20-diol inhibited in a strongly time-dependent manner. At 10 microM this compound irreversibly inhibited more than 90% of the side-chain cleavage capacity of the cell during a 40-min incubation. Cells treated with this steroid did not regain their capacity for side-chain cleavage after removal of free steroid. None of the inhibitors described above inhibited production of corticosterone by cells supplied with pregnenolone, the product of the P-450scc reaction. We suggest that the only significant effect of these compounds under these conditions is inhibition of the side-chain cleavage enzyme.

Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. Discovery of a rapidly induced protein.

Two-dimensional electrophoretic techniques were used to identify and characterize a protein that is not produced in quiescent isolated rat adrenal cells but is produced in response to acute stimulation by adrenocorticotropic hormone (ACTH) or dibutyryl cAMP. The molecular weight of this protein is 28,000 (sodium dodecyl sulfate electrophoresis), and its isoelectric point is 6.5 (isoelectric focusing). Mapping of proteolytic peptides suggests that this induced protein (i) is quite similar in primary structure to another protein (p), which is produced only in nonstimulated adrenal cells. The time course of formation of protein i and its ACTH dose response closely parallel the increase of corticosteroid production in stimulated cells. The possibility that protein i is produced in response to increased levels of some steroid of the glucocorticoid pathway is precluded by the observation that inhibition of corticosteroid synthesis by aminoglutethimide does not alter the rate of production of i. Addition of cycloheximide before ACTH, which prevents stimulation of corticosteroidogenesis, also prevents formation of protein i implying that the production of protein i depends on continuing protein synthesis. [35S/32S]Methionine pulse-chase experiments, i.e. addition of excess [32S] methionine and ACTH after prelabeling with [35S]methionine, show that protein i is not produced from pre-existing protein p or other pre-existing proteins even if protein synthesis (and increased steroid production) is not inhibited. These findings exclude post-translational modification as a mechanism for the production of i but are consistent with p and i being related by cotranslational modification. Addition of cycloheximide after stimulation causes the formation of protein i to cease, but the amount of the protein does not decrease with the same kinetics as the return of corticosteroid production to its unstimulated level. [35S/32S]Methionine pulse-chase experiments imply protein i, even under conditions of ongoing ACTH stimulation and protein synthesis, is degraded with approximately the same kinetics as after cycloheximide inhibition. The close concurrence under a wide variety of experimental conditions between the appearance of protein i and the increase in adrenal corticosteroid production, coupled with the fact that the former does not occur as a result of the latter, make protein i a likely candidate for the postulated corticosteroidogenic stimulatory protein (Ferguson, J.J. (1962) Biochim. Biophys. Acta 57, 616-617). The fact that stimulation of steroidogenesis may occur via co-translational modification of a regulatory protein is an intriguing possibility which readily explains both the observed rapid and protein synthesis-dependent stimulation and the lack of dependence of stimulation on transcription (Schulster, D. (1974) Mol. Cell. Endocr. 1, 55-64).

Cytodifferentiation and alkaloid accumulation in cultured cells of Papaver bracteatum.

Cultured Papaver bracteatum cells accumulate large amounts of dopamine (0.1-4 mg/g fresh wt.) and much lower levels of the alkaloids, thebaine (0-6 µg/g fresh wt.) and sanguinarine (10-500 µg/g fresh wt.). Upon removal of hormone from their culture media, organogenesis and an elevation in morphinan alkaloid concentration were observed. Light and electron microscopic studies reveal that a correlation exists between the appearance of laticifer-like cells and thebaine in these cells.

Evidence for siroheme-Fe4S4 interaction in spinach ferredoxin-sulfite reductase.

Spinach ferredoxin-sulfite reductase (SiR) contains one siroheme and one Fe4S4 center per polypeptide subunit. The heme is entirely in the high-spin Fe3+ state in the oxidized enzyme. When SiR is photochemically reduced with ethylenediaminetetraacetate (EDTA)-deazaflavin, the free enzyme and its CN- and CO complexes show changes in absorption spectra associated with the heme even after the heme has been reduced from the Fe3+ to the Fe2+ state. With CO- or CN--SiR, these spectral changes are associated with the appearance of a classical "g = 1.94" type of EPR spectrum characteristic of reduced Fe4S4 centers. The line shapes and exact g values of the g = 1.94 EPR spectra vary with the nature of the ligand bound to the heme Fe. Photoreduction of free SiR results in production of a novel type of EPR signal, with g = 2.48, 2.34, and 2.08 in the fully reduced enzyme; this signal accounts for 0.6 spin per heme. (A small g = 1.94 type EPR signal, representing 0.2 spin per heme, is also found.) These data suggest the presence of a strong magnetic interaction between the siroheme and Fe4S4 centers in spinach SiR, this interaction giving rise to different EPR signals depending on the spin state of the heme Fe in the reduced enzyme.

Spinach siroheme enzymes: Isolation and characterization of ferredoxin-sulfite reductase and comparison of properties with ferredoxin-nitrite reductase.

Sulfite reductase (SiR) has been purified to homogeneity from spinach leaves. Two forms of the enzyme were separated by hydroxylapatite chromatography. One, with subunit Mr 69 000 appears to be proteolytically cleaved to give rise to the other, with subunit Mr 63 000, during the purification procedure. The two species have identical catalytic activities (on a per heme basis) when reduced methylviologen (MV+) or ferredoxin (Fdr) is used as electron donor for sulfite reduction, and they exhibit nearly identical optical and EPR spectra. Both enzyme forms exist in 50 mM phosphate buffer (pH 7.7) primarily as dimers at 20 degrees C. Spinach SiR contains 1 mol of siroheme and one Fe4S4 center per subunit. The heme iron is the high spin Fe3+ state in the enzyme as isolated. Near quantitative reduction of the Fe4S4 center by dithionite could be achieved if SiR was either converted to the CO complex or treated with 80% dimethyl sulfoxide. Spinach SiR and nitrite reductase (NiR) both catalyze Fdr-or MV+-de-pendent six-electron reductions of SO3(2)- and NO2-, as well as the two electron reduction of NH2OH. Vmax values are highest with the nitrogenous substrates. However, the Km of SiR for So3(2-), and of NiR for NO2-, is at least 2 orders of magnitude less than with either of the other substrates. Rates of reduction with Fdr as electron donor are greater than with MV+ as donor, No immunological cross-reaction could be detected between spinach SiR and Escherichia coli SiR or between spinach SiR and NiR.

Escherichia coli sulfite reductase hemoprotein subunit. Prosthetic groups, catalytic parameters, and ligand complexes.

Escherichia coli NADPH-sulfite reductase can be dissociated into an oligomeric flavoprotein and a monomeric hemoprotein (HP) subunit in 4 M urea. HP catalyzes stoichiometric 6-electron reductions of SO32- (to S2-) and of NO2-, as well as 2-electron reduction of NH2OH, with reduced methyl viologen (MV+) as reductant. While Vmax values are highest with the nitrogenous substrates, Km for SO32- is 2 to 3 orders of magnitude less than the Km for NO2- or NH2OH. EPR spectroscopic and chemical analyses show that HP contains one siroheme and one Fe4S4 center per polypeptide. The heme is in the high spin Fe3+ state in HP as isolated. Near-quantitative reduction of the Fe4S4 center to a state yielding a g = 1.94 type of EPR spectrum by S2O42- and/or MV+ could be achieved if HP was converted to either the CN- or CO complex or treated with 80% dimethyl sulfoxide. HP binds one SO32- or CN- per peptide. Binding of these ligands, as well as CO, appears to be mutually exclusive and to involve the heme. The heme Fe3+/Fe2+ potential is shifted from -340 mV in the free HP to -155 mV in the HP-CN- complex. The potential of the Fe4S4 center is approximately 70 mV more negative in the CN- as opposed to the CO-ligated HP (-420 mV), a result which indicates the presence of heme-Fe4S4-ligand interaction in the HP complexes.