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Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sativus (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30% (v/v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gel-filtration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS/PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V(max) and K(m) values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 micromol/min per mg of protein and 23 microM in the absence of ascorbate and 280 micromol/min per mg of protein and 250 microM in the presence of 500 microM ascorbate, respectively. As the ascorbate concentration was increased from 50 to 500 microM, the V(max) and K(m) values increased in parallel, and thus the V(max)/K(m) ratio remained constant. Similarly, raising the concentrations of sinigrin increased the concentration of ascorbic acid required for half-maximal activation (K(a)). At a sinigrin concentration of 250 microM, the K(a) for ascorbic acid was 55 microM. Sulphate, a reaction product, was a competitive inhibitor of activity, having a K(i) of 60 mM with respect to sinigrin and of 27 mM with respect to ascorbate. Thus activation of myrosinase from R. sativus by ascorbic acid exemplifies an unusual and possibly unique example of linear 'uncompetitive activation' (i.e. a proportionate increase in V(max) and K(m)) of an enzyme. The enzyme also had beta-glucosidase activity and hydrolysed p-nitrophenyl-beta-d-glucopyranoside.

A mammalian cytosolic FAD-dependent enzyme that catalyzes the reduction of quinones by N-ribosyl- and N-alkyldihydronicotinamides, but not by NADH, NADPH, or NMNH (reduced nicotinamide mononucleotide), was isolated from bovine kidney more than 30 years ago [S. Liao, J. T. Dulaney and H. G. Williams-Ashman (1962) J. Biol. Chem. 237, 2981-2987]. This enzyme is designated here as quinone reductase type 2 (QR2). Bovine QR2 is a homodimer that migrates on SDS/PAGE at approximately 22 kDa. Three tryptic peptides of bovine QR2 (representing 39 amino acids) showed 43% identity to human NAD(P)H:quinone reductase (DT-diaphorase; EC 1.6.99.2), here designated QR1 and 82% identity to a related human cDNA clone [called hNQO2 by A. K. Jaiswal, P. Burnett, M. Adesnik and O. W. McBride (1990) Biochemistry 29, 1899-1906], and designated here as hQR2. The protein encoded by the latter cDNA did not show QR activity when tested with conventional nicotinamide nucleotides. The unexpected high homology between the old flavoenzyme and hQR2 prompted us to clone and overexpress hQR2. The properties of hQR2 were identical to those of the flavoenzyme described by S. Liao and H. G. Williams-Ashman, thus establishing their genetic identity. Recombinant human QR2: (i) reacts with N-ribosyl- and N-alkyldihydronicotinamides, but not with NADH, NADPH, or NMNH; (ii) is very weakly inhibited by dicumarol or Cibacron blue; (iii) is very potently inhibited by benzo[a]pyrene. The x-ray crystal structure of rat QR1 shows that the 43 amino acid C-terminal tail of QR1 provides the binding site for the hydrophilic portions of NADH and NADPH. In the absence of this binding site in QR2, the enzyme retains the essential catalytic machinery, including affinity for FAD, but cannot bind phosphorylated hydride donors.

Much effort has been devoted to developing methods for the efficient isolation and identification of glucosinolates. Existing methods for separation involve ion exchange, GLC, and HPLC (mostly after chemical modification by enzymatic sulfate removal and/or silylation). We demonstrate a simple and direct strategy for analyzing the glucosinolate content of plant extracts, made possible by a new combination of widely available techniques: (a) reverse-phase paired-ion chromatography (PIC) of plant extracts, (b) hydrolysis of glucosinolates by myrosinase and quantitation of resulting isothiocyanates by cyclocondensation with 1, 2-benzenedithiol; (c) a novel method for replacing the PIC counterions by ammonium ions, permitting direct bioassay, mass, and 1H NMR spectrometry; (d) mass spectrometric analysis of ammonium salts by negative-ion fast atom bombardment (FAB) to determine m/z of the [M - H]- ion, and by chemical ionization (CI) in ammonia to obtain accurate masses of characteristic fragment ions, principally [R-CN:NH4]+, [R-CH=NOH:H]+ and [R-CH=NOH:NH4]+; and (e) high-resolution 1H NMR spectroscopy of intact glucosinolates. FAB and CI mass spectra, as well as high-resolution 1H NMR spectra were obtained for a variety of glucosinolate standards. The results provide guidance for the isolation and characterization of unknown glucosinolates from plants. These combined procedures were applied to a sample of broccoli (cultivar SAGA), in order to resolve and identify its major glucosinolates: 4-methylsulfinylbutyl glucosinolate (glucoraphanin) and 4-methylthiobutyl glucosinolate (glucoerucin). Thus, this analytical strategy provides a powerful technique for identifying and quantitating glucosinolates in plant extracts without resorting to derivatization.

The induction of NAD(P)H:quinone reductase (EC 1.6.99.2; QR) in Hepa 1c1c7 murine hepatoma cells provides a versatile quantitative model for measuring the potencies of inducers for Phase 2 detoxication enzymes. Since many inducers of these enzymes also protect animals and their cells against the toxic and neoplastic effects of carcinogens, understanding the mechanisms of induction of Phase 2 enzymes is important. Both HgCl2 and 2,3-dimercaptopropanol (BAL) are inducers of QR in these cells, and paradoxically BAL (which is about 30 times less potent than HgCl2) enhances the inducer potency of HgCl2 substantially. This synergism depends on the presence of two thiol groups on adjacent carbon atoms. Since nonchelated mercury(II)-thiol compounds did not show synergism, the formation of very high affinity bidentate chelates appears to be essential for such synergism. A major mechanism for the augmentation of the inducer potency of mercury(II) by BAL is the more rapid cellular uptake and the accumulation of higher intracellular concentrations of mercury. It is also possible that BAL-mercury chelates are intrinsically more potent as inducers. Although equimolar mixtures of BAL and HgCl2 and the synthetic chelate isolated from such mixtures were more potent inducers than HgCl2 alone, the presence of excess BAL increased this inducer synergism even further. By chromatography we showed the reversible formation of higher order complexes between BAL and mercury(II). Such complexes are transported into cells more efficiently and appear to be more potent than free HgCl2 or the chelate obtained from equimolar mixtures of BAL and HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to conveniently and rapidly isolate by group both conjugated and unconjugated serum androgens, a scheme has been devised for their differential extraction from commercially available, disposable octadecylsilane cartridges (Sep-Pak C18). Using added radioactive steroid standards and detection of endogenous serum steroids by group-specific enzymatic assays, the quantitative recovery of steroid glucuronides and sulfates in the 47% methanol fraction and of unconjugated steroids in the 100% methanol fraction was observed. Maximum recovery of serum protein-bound steroids (e.g. testosterone) was achieved with serum denatured by urea and heat. In order to separate glucuronides from sulfates, sequential hydrolysis of the conjugated fraction (47% methanol) by enzymatic hydrolysis and then organic solvolysis as well as an additional Sep-Pak cartridge extraction step was required. Groups of extracted steroids may be further separated and assayed by any appropriate method(s). An application is given which employs HPLC and an enzymatic assay for 17 beta-hydroxy- and 17-oxo-steroids to provide separate profiles of unconjugated, glucuronidated, and sulfated androgens in human, male serum.

It is the objective of the in vitro studies reported herein to further evaluate the role of insulin in the regulation of ovarian androgen biosynthesis, to assess its dose requirements, and to elucidate the cellular mechanism(s) underlying its high dose action. To this end, use was made of recently developed primary culture systems of ovarian androgen-producing cells, the differentiation of which is subject to regulation by gonadotropic and insulinotropic signaling. Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) thecal-interstitial cells from immature rats with insulin (1 microgram/ml) or hCG (1 ng/ml), resulted in 1.5- and 2.6-fold increments in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstan-17-one), the main androgenic steroid identified in culture medium by HPLC. However, combined treatment with both agents unmasked a synergistic interaction resulting in 5.7-fold amplification of hCG action, the increase in androsterone accumulation representing enhanced biosynthesis rather than diminished degradation. Unaccounted for by cellular growth and independent of the cellular density of plating (1 X 10(4)-1 X 10(6) viable cells/culture) or the hCG dose (0.1-10 ng/ml) employed, the insulin effect proved time and dose dependent with a minimal time requirement of 72 h. [125I-TyrA14]Iodoinsulin binding to untreated highly enriched thecal-interstitial cells proved highly specific, saturable, and reversible, displaying a single class (Hill coefficient = 0.93 +/- 0.07) of high affinity (Kd = 1.7 X 10(-10) M), low capacity (4746 +/- 283 sites/cell) binding sites. Treatment with physiological concentrations (10 ng/ml) of insulin produced limited, albeit measureable, down-regulation of the insulin receptor. In contrast, provision of relatively high concentrations (1 microgram/ml) of insulin resulted (despite marked adsorption/degradation) in substantial (greater than 60%) down-regulation of the insulin receptor, but not the type I insulin-like growth factor receptor, the ligand of which has also been shown to amplify hCG-supported androgen biosynthesis. These findings suggest that the thecal-interstitial cell is a site of insulin reception and action, that physiological concentrations of insulin are capable of participating in the regulation of ovarian androgen biosynthesis, and that this effect is probably mediated via high affinity insulin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

An assay system was developed utilizing microsomal 7-ethoxycoumarin O-deethylase to monitor the formation and efflux of malate from mitochondria in vitro. Malate was coupled by means of malic enzyme, NADP+, and the microsomal mixed-function oxidase system to the production of highly fluorescent 7-hydroxycoumarin. Under conditions of the assay, steady-state concentrations of NADPH could be maintained, and half-maximal rates of microsomal 7-ethoxycoumarin O-deethylase were observed with about 0.2 microM NADPH and 250 microM malate. Addition of succinate to mixtures of intact mitochondria and microsomes enhanced rates of 7-hydroxycoumarin formation by facilitating the generation and transport of malate from mitochondria (half-maximal stimulation was observed with 0.4 mM succinate). Stimulation of 7-hydroxycoumarin formation by succinate, under conditions where concentrations of cofactors and substrate were adjusted to resemble those in the intact cell, provides further evidence that reducing equivalents for mixed-function oxidation may arise via malate efflux from mitochondria.

To characterize and clarify the time-related pattern of steroidogenesis in primary testicular cultures from adult hypophysectomized rats, we have determined the pattern of C19 and C21 steroids using novel enzymatic assay techniques that rely on highly specific bacterial hydroxysteroid dehydrogenases. Steroids contained in culture media were separated in a standardized high performance liquid chromatography system and the 17 beta-hydroxy- and 17-oxosteroids were quantified by a transydrogenase assay. The individual 3 alpha-, 3 beta-, 17 beta-, and 20 alpha-hydroxysteroids were in turn measured by enzymatic oxidation. Presumptive steroid identities were confirmed by enzymatic oxidation or reduction to products that were rechromatographed and identified by co-elution with standards. Although human chorionic gonadotropin stimulated an increase in the "adult" hormones, testosterone and 4-androstene-3, 17-dione, on both Days 1 and 11 of culture, the majority of the steroids found, even on Day 1, were 3 alpha-hydroxy-5 alpha-dihydrosteroids rather than delta 4-3-oxosteroids. A specific 5 alpha-reduced, C21 steroid: 5 alpha-pregnane-3 alpha, 20 alpha-diol, increased over time and became the most abundant gonodotropin-stimulated steroid (about 5-fold in excess of testosterone) by Day 11. In contrast, testosterone was the identifying steroid of nondispersed testes from both intact and hypophysectomized rats. Studies with tracer quantities of [3H]pregnenolone in culture confirmed the initial (Day 1) preponderance of 3 alpha-hydroxy-5 alpha-dihydrosteroids, as well as the accumulation with time of 20 alpha-hydroxysteroids. These findings suggest that contrary to expectation, cultured testicular cells from young adult hypophysectomized rats display a relatively atypical steroidogenic pattern. Although the cellular mechanisms underlying the time-dependent accumulation of C21 steroids remain uncertain, these patterns suggest either regressive changes in the original parent cells or the emergence of a population of latent cells. Although of limited utility as a model for examining adult testicular physiology, primary cultures of dispersed whole testes should prove useful in studies of culture-induced phenotypic regression and the attendant alteration at the level of gene expression.

Dehydroepiandrosterone sulfate (DHEAS) and unconjugated dehydroepiandrosterone (DHEA) are secretory products of the adrenal cortex. Measurement of serum levels of these steroids is of increasing epidemiologic interest, since low serum concentrations of DHEAS or DHEA have been associated with an increased risk of dying of cardiovascular disease or of developing cancer. Radioimmunoassays (RIAs) are the most convenient systems for the measurement of serum DHEAS concentrations in multiple samples. However, using sera from four individuals we show that different RIA kits provide quite different estimates of serum DHEAS concentrations. Moreover, these results do not always agree with the serum concentrations determined by an independent chromatographic and enzymatic reference method. The results highlight the need for an independent method of determining DHEAS levels in sera that can provide guidance in selecting an appropriate RIA, and in interpreting the results.