Max App Research Articles

Functional characterization of ent-kaurene oxidase, MtKO, from Montanoa tomentosa (Zoapatle)

Kaurene oxidases are P450 proteins that catalyze the conversion of ent-kaurene into kaurenoic acid, the final enzymatic product with a wide range of pharmacological properties. We describe the functional characterization of an ent-kaurene oxidase (EC 1.14.13.78) isolated from Montanoa tomentosa after heterologous expression in Saccharomyces cerevisiae, as well as the detection of the enzymatic activity in the plant itself. In the presence of NADPH and FAD, the microsomal fraction from transformed INVSc1 cells, ent-kaurene produced ent-kaurenoic acid, which was confirmed by GC-MS analyses. The kinetic parameters for ent-kaurene using 0.5 mg of microsomal protein were Km app= 80.63±1.2 μM and V max app= 31.80±1.8 μmol-1mg-1h-1. Optimal temperature and pH were 30°C and 7.6, respectively. Similar kinetic parameters were observed when leaf microsomes from M. tomentosa were assayed under the same conditions as for yeast microsomes. This result strongly suggests that ent-kaurene oxidase activity is present in leaf microsomes. The enzymatic activity was competitively inhibited by paclobutrazol, with IC50=43.9 μM, implying that MtKO is resistant to inhibition by azolic-type compounds. This study confirmed the biochemical detection of ent-kaurene oxidase activity in the plant, and the heterologous functionality of a cDNA with an ent-kaurene oxidase identity from M. tomentosa (zoapatle).

In vitro metabolism of the anti-androgenic fungicide vinclozolin by rat liver microsomes

Vinclozolin (V) is a fungicide used in agricultural settings. V administered to rats is hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2). V, M1 and M2 have antiandrogenic properties by interacting with the androgen receptor. Data on V, M1 and M2 biotransformation are limited. Our objective was to characterize V metabolism by rat liver microsomes. V was incubated with non-treated adult male Long-Evans rat liver microsomes and NADPH. Several metabolites were detected following the extraction of incubate with acetonitrile and analysis by HPLC/DAD/MSD. One metabolite was identified as [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4), which was gradually converted to 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (M5). Both co-eluted in the same HPLC peak. Another metabolite ([M7]) was detected by UV but was unstable for mass spectral analysis. The K(M app) for co-eluted M4/M5 and [M7] was 53.7 and 135.4 μM, the V(max app) was 0.812 and 0.669 nmoles/min/mg protein, and CL(int) was 15.1 and 4.9 ml/min/g protein, respectively. Pilocarpine, orphenadrine and proadifen and anti-rat cytochrome P450 (CYP)2A, 2B and 3A antibodies inhibited M4/M5 and [M7] formation. These results indicate that V is efficiently metabolized by CYP. Determination of the metabolites of V will provide further insight into the relationship between toxicity and tissue dose of V and its metabolites.

Automatic flow methodology for kinetic and inhibition studies of reactions with poorly water-soluble substrates in ionic liquid systems

In the present work an automatic generic tool, based on sequential injection analysis (SIA) for kinetic and inhibition studies of reactions with poorly water-soluble compounds in ionic liquid (IL)-containing systems, is described. The oxidation of the poorly water-soluble phenolic compound, caffeic acid, catalyzed by the mushroom tyrosinase, in different 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF 4])/buffer mixtures as reaction media, was investigated. This determination was based on measuring depletion rate of the substrate caffeic acid at its maximum wavelength ( λ max 311 nm). The influence of several parameters such as substrate and enzyme concentration, temperature, pH, delay times and measurement periods on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. The obtained results showed that tyrosinase was active in oxidising caffeic acid in this water-miscible IL and the presence of an impaired tyrosinase activity with increase in [bmim][BF 4] concentration as an increase in the apparent Michaelis–Menten constant ( K M app ) was observed while the maximum reaction rate ( V max app ) remained fairly constant. The results were compared to those obtained when the assay was performed in water/methanol mixtures under the same conditions to substantiate [bmim][BF 4] as an alternative to conventional organic solvents. Additionally, it was shown that tyrosinase is effectively inhibited by the substrate analogues tested ( trans-cinnamic acid and 3,4-dihydroxybenzoic acid) in the IL-containing aqueous system used.

Immobilization of mushroom tyrosinase on controlled pore glass: Effect of chemical modification

Mushroom tyrosinase immobilization on controlled pore glass with three different functional groups like alkylamino, carboxyl and arylamino is performed. The effect of the alkylamino-CPG arm extension on enzymatic stability and catalytic activity is also investigated. The results expressed in terms of ligand coupling, % yield and kinetic parameters, like the apparent K m app and V max app and the inherent K m inh and V max inh values demonstrate the best performance of tyrosinase immobilized on alkylammino-CPGs.

Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase

NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis–Menten constant ( K m app ) was ∼0.3 nM and the apparent maximum velocity ( V max app ) was 16 U mg −1. Substrate inhibition was observed above 5 nM. NADH was the electron donor, K m app = 340 μ M and V max app = 46 U mg - 1 . FAD was also a cofactor with a K m app of 3.1 μM and V max app of 89 U mg −1. The turnover number for NADH oxidation was 25 s −1. Possible physiological roles of the Fe(III) reduction by this enzyme are discussed.

Characterization of reversible and pseudo-irreversible acetylcholinesterase inhibitors by means of an immobilized enzyme reactor

The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver–Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak area. AChE-CIM-IMER kinetic parameters ( K m app and v max app ) were derived as well as inhibitory constants ( K i app ) of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylation steps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials.

Parallel synthesis of libraries of anodic and cathodic functionalized electrodeposition paints as immobilization matrix for amperometric biosensors

The integration of flexible anchoring groups bearing imidazolyl or pyridyl substituents into the structure of electrodeposition paints (EDP) is the basis for the parallel synthesis of a library containing 107 members of different cathodic and anodic EDPs with a high variation in polymer properties. The obtained EDPs were used as immobilization matrix for biosensor fabrication using glucose oxidase as a model enzyme. Amperometric glucose sensors based on the different EDPs showed a wide variation in their sensor characteristics with respect to the apparent Michaelis–Menten constant ( K M app) representing the linear measuring range and the maximum current ( I max app). Based on these results first assumptions concerning the impact of different side chains in the EDP on the expected biosensor properties could be obtained allowing for an improved rational optimization of EDPs used as immobilization matrix in amperometric biosensors.

Novel Mechanism of Inhibition of Nuclear Factor-κB DNA-Binding Activity by Diterpenoids Isolated from Isodon rubescens

The development of specific inhibitors that can block nuclear factor-kappaB (NF-kappaB) activation is an approach for the treatment of cancer, autoimmune, and inflammatory diseases. Several diterpenoids, oridonin, ponicidin, xindongnin A, and xindongnin B were isolated from the herb Isodon rubescens. These compounds were found to be potent inhibitors of NF-kappaB transcription activity and the expression of its downstream targets, cyclooxygenase-2 and inducible nitric-oxide synthase. The mechanisms of action of the diterpenoids against NF-kappaB are similar, but significant differences were also identified. All of the diterpenoids directly interfere with the DNA-binding activity of NF-kappaB to its response DNA sequence. Oridonin and ponicidin have an additional impact on the translocation of NF-kappaB from the cytoplasm to nuclei without affecting IkappaB-alpha phosphorylation and degradation. The effect of these compounds on the interaction of NF-kappaB with consensus DNA sequences is unique. Different inhibitory effects were observed when NF-kappaB bound to various DNA sequences. Both p65/p65 and p50/p50 homodimers, as well as p65/p50 heterodimer association with their responsive DNA, were inhibited. Kinetic studies on NF-kappaB-DNA interaction indicate that the diterpenoids decrease the B(max app) but have no effect on K(d app). This suggests that this class of compounds interacts with both p65 and p50 subunits at a site other than the DNA binding site and subsequently modulates the binding affinity of the transcription factor toward DNA with different NF-kappaB binding sequences. The diterpenoid structure could therefore serve as a scaffold for the development of more potent and selective NF-kappaB inhibitors that target regulated gene transcription.

Inhibition of diethyl ether degradation in Rhodococcus sp. strain DEE5151 by glutaraldehyde and ethyl vinyl ether

Alkyl ether-degrading Rhodococcus sp. strain DEE5151, isolated from activated sewage sludge, has an activity for the oxidation of a variety of alkyl ethers, aralkyl ethers and dibenzyl ether. The whole cell activity for diethyl ether oxidation was effectively inhibited by 2,3-dihydrofurane, ethyl vinyl ether and glutaraldehyde. Glutaraldehyde of less than 30 μM inhibited the activity by a competitive manner with the inhibition constant, K I of 7.07 ± 1.36 μM. The inhibition type became mixed at higher glutaraldehyde concentrations >30 μM, probably due to the inactivation of the cell activity by the Schiff-base formation. Structurally analogous ethyl vinyl ether inhibited the diethyl ether oxidation activity in a mixed manner with decreasing the apparent maximum oxidation rate, v max app , and increasing the apparent Michaelis–Menten constant, K M app . The mixed type inhibition by ethyl vinyl ether seemed to be introduced not only by the structure similarity with diethyl ether, but also by the reactivity of the vinyl ether with cellular components in the whole cell system.

Formaldehyde degradation in an anaerobic packed-bed bioreactor

The development of appropriate technologies for the treatment of formaldehyde discharged into the environment is important to minimize its impact. Aerobic systems have been employed, although alternative anaerobic treatments have also been widely studied, mainly due to their low energy consumption and sludge production. However, toxic substances can lead to disturbances in anaerobic reactors. Some research has already been developed on formaldehyde anaerobic biological treatment, but no consensus has yet been reached about its behavior nor has the most efficient system been identified. Aiming at finding supporting evidence for this issue, therefore, this study investigated the degradation and toxicity of formaldehyde in a Horizontal-Flow Anaerobic Immobilized Sludge Reactor. Formaldehyde concentrations of 26.2–1158.6 mg HCHO/L were applied in the reactor, resulting in formaldehyde and chemical oxygen demand removal efficiencies of 99.7% and 92%, respectively. Volatile fatty acids with up to five carbons, found during the degradation of formaldehyde, are believed to indicate that the degradation followed routes unlike those suggested in the literature, which reports the formation of intermediates such as methanol and formic acid. The Monod kinetic model adhered to the experimental data well, with apparent kinetic parameters estimated as r max app=2.79×10 −3 mg HCHO/mg SSV h and K s app=242.8 mg HCHO/L.

Effects of plasma apolipoproteins on lipoprotein lipase-mediated lipolysis of small and large lipid emulsions

Large (ca. 120 nm) and small (ca. 35 nm) emulsions consisting of triolein (TO) and phosphatidylcholine (PC) were prepared as the primary protein-free models of chylomicrons and their remnants, respectively. Lipoprotein lipase (LPL)-mediated lipolysis of emulsion TO was retarded in chylomicron-free human plasma compared with the hydrolysis activated by isolated apolipoprotein C-II (apoC-II). In 30% plasma, free fatty acid (FFA) release rate was higher for large emulsions than for small ones, while both emulsions were hydrolyzed at similar rates in the presence of isolated apoC-II. Isolated apolipoprotein C-III (apoC-III) or apolipoprotein E (apoE) worked as LPL-inhibitor of the lipolysis activated by apoC-II. It was also observed that apolipoprotein A-I (apoA-I) showed distinct inhibitory effects on the lipolysis of large and small emulsions: more effective inhibition for small emulsions. Kinetic analyses showed that K m app and V max app for the lipolysis of emulsions were lower in the presence of 30% plasma than isolated apoC-II. ApoA-I also markedly decreased K m app and V max app for LPL-catalyzed hydrolysis of both emulsions. In chylomicron-free serum, the density of bound apoA-I at small emulsion surfaces was about three fold greater than large emulsion surfaces, but the binding densities of apoC-II, apoC-III and apoE were less for small emulsion surfaces than for large ones, suggesting that apoA-I preferentially binds to small particles and displaces other exchangeable apolipoproteins from particle surfaces. These results indicate that, in addition to the well known inhibitory effects of apoC-III and apoE, apoA-I in plasma regulates the lipolysis of triglyceride (TG)-rich emulsions and lipoproteins in a size-dependent manner.

Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and medium-chain fatty acids and xenobiotic carboxylic acids

Two distinct forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) were isolated from human liver mitochondria. They were referred to as HXM-A and HXM-B based on their order of elution from a DEAE-cellulose column. Activity of the two ligases was determined toward 15 different carboxylic acids. HXM-A represented 60–80% of the benzoate activity in the lysate, and kinetic analysis revealed that benzoate was the best substrate (highest V max/ K m). The enzyme also had medium-chain fatty acid:CoA ligase activity. HXM-B had the majority of the hexanoate activity and hexanoate was its best substrate. It was, however, also active toward many xenobiotic carboxylic acids. Comparison of these two human XM-ligases with the previously characterized bovine XM-ligases indicated that they were kinetically distinct. When assayed with benzoic acid as substrate, both HXM-A and HXM-B had an absolute dependence on either Mg 2+ or Mn 2+ for activity. Further, addition of monovalent cation (K +, Rb +, or NH 4 +) stimulated HXM-A activity by >30-fold and HXM-B activity by 4-fold. For both forms, activity toward straight-chain fatty acids was stimulated less by K + than was activity toward benzoate or phenylacetate. A 60 kDa short-chain fatty acid:CoA ligase was also isolated. It had activity toward propionate and butyrate, but not acetate, hexanoate or benzoate. The K m app values were high but similar for propionate and butyrate (285 μM and 250 μM, respectively) but the V max app was nearly 6-fold greater with propionate as substrate. While the K m values are somewhat high, the enzyme is still more efficient with these substrates than either of the XM-ligases.

Purification of hydroperoxide lyase from cucumbers

Hydroperoxide lyase (HPL) was extracted from cucumber fruit ( Cucumis sativus) and purified by centrifugation, solubilization with detergent, ion-exchange chromatography and hydroxyapatite chromatography. 9-Hydroperoxy-linoleic acid and 13-hydroperoxy-linoleic acid lysing activities were purified 88-fold and 82-fold, respectively. The purified HPL preparation consisted of a single major band following SDS-electrophoresis with a molecular weight of about 55 000 Da; pH 6 was optimum for the lysis of both 9-hydroperoxy-linoleic acid and 13-hydroperoxy-linoleic acid substrates. The enzyme was relatively stable and retained more than two thirds of original activity after 3 weeks at 4°C, but lost half of its activity after 2 min at 50°C. Apparent K m values for 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 13-hydroperoxy-linoleic acid and 13-hydroperoxy-linolenic acid were 6.76, 6.02, 5.46 and 12.4 μM respectively. Corresponding V max values were 19.3, 12.0, 7.58 and 11.4 μmol min. The V max app/ K m app values for 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 13-hydroperoxy-linoleic acid and 13-hydroperoxy-linolenic acid were 2.86, 1.99, 1.39 and 0.92, respectively. It is suggested that cucumber mesocarp contains only one type of HPL which is able to more efficiently catalyse the lysis of the 9-acyl hydroperoxides and especially 9-hydroperoxy-linoleic acid.©

Catalytic behavior of Pseudomonas cepacia lipase in w/o microemulsions

The activity of purified Pseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT). Kinetic studies showed that the reaction follows a ping-pong bi-bi mechanism with inhibition by both substrates. The apparent kinetic parameters of the reaction were found to be K(m octanol) = 310 mM, K(m lauric acid) = 78 mM, and V(max) = 250 mumol min(-1) mg(-1). The same system was used for the synthesis of mono- and diglycerides from glycerol and lauric acid, which was successful at very low w(o) values. The catalytic behavior of P. cepacia lipase was also studied in esterification reactions performed in a nonionic microemulsion system formulated by tetraethyleneglycoldodecylether (C(12)E(4)). The optimum activity was found at about w(o) = 8. The apparent values of V(max app) and K(m app) for octanol were calculated and found to be 100 mumol min(-1) mg(-1) and 76 mM, respectively. (c) 1995 John Wiley & Sons, Inc.

Kinetic evidence for separate 3β-hydroxysteroid dehydrogenase-isomerases in androgen and 16-androstene biosynthetic pathways in the pig testis

The microsomal fraction from the testes of immature pigs (<1 week old) contains 3β-hydroxysteroid dehydrogenase-isomerase (3β-HSD-isomerase) activities that convert dehydroepiandrosterone (DHA) to 4-androstenedione and 5,16-androstadien-3β-ol (andien-β) to 4,16-androstadien-3-one (dienone). These reactions are necessary for the biosynthesis of hormonally and pheromonally active steroids. Kinetic analyses of these activities were done to determine whether they are catalysed by a single enzyme or if there is any interaction between the substrates and products of one reaction on the activity of the other enzyme. Kinetic parameters were determined and the affinities for steroid substrate were similar (7–9 μmol/l) but the V max app value for the conversion of andien-β to dienone was 10-fold that of the DHA to 4-androstenedione reaction. In analyses of the conversion of DHA to 4-androstenedione, neither andien-β nor dienone inhibited the reaction and especially, no effect on the K m app for DHA was observed which would have indicated competition between DHA and andien-β for the same active site ( K i app from slope and intercept replots were between 3 and 80 times the values of the kinetic constants). Similarly, DHA and 4-androstenedione had minor or negligible effects on the conversion of andien-β to dienone ( K i app from slope replots were the same as the K m app but the K i app from the intercept replot was 12 to 25% of the V max app ). It is concluded that substrate specific 3β-HSD-isomerases for andien-β and DHA exist in the immature pig testis and there is little, if any interaction between these enzymes.

The effect of the UL42 protein on the DNA polymerase activity of the catalytic subunit of the DNA polymerase encoded by herpes simplex virus type 1

The effect that the UL42 protein of herpes simplex virus type 1 has on the DNA polymerase activity of the DNA polymerase catalytic subunit (Pol) of the same virus has been investigated. The observed effects are critically dependent on the salt used and its concentration, such that the UL42 protein may inhibit, have little or no effect on, or activate the Pol activity, depending on the conditions used. The observed effects are due to the values for K m app for activated DNA and V max app for Pol and the Pol-UL42 protein complex differently varying with salt concentration.

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K(m)-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V(max app) was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 x 10(6) vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H(+)-translocating enzyme activities revealed K(m app) values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Delta muH(+) while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.

Properties of NAD (P) H-linked aldose reductase from Crytococcus lactativorus

A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus. The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d-xylose. l-Sorbose and d-fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The K m app value for d-xylose was 8.6 mM and the V max app was 20.8 units/mg NADH was used as a coenzyme. The K m app values for NADPH and NADH were 6μM and 170 μM, respectively, when d-glucose was used as a substrate.

Comparison of 59Fe 3+ uptake in vitro and in vivo by mouse duodenum

Initial rates of 59Fe 3+ uptake by mouse duodenal fragments (in vitro) and tied-off duodenal segments (in vivo) have been characterised for control and hypoxic animals. 59Fe 3+ uptake by duodenal fragments was rapid, selective and dependent on medium Fe 3+-nitrilotriacetate concentration. Most of the 59Fe 3+ uptake (70–75%) occurred via the mucosal route and was dependent on the metabolic state of the tissue. Mucosal uptake showed an adaptive increase following exposure of animals to 3 days hypoxia; the enhancement was due to a 2–3-fold increase in V max app, without any significant changes in the K m app. Studies of upper small intestine transit times showed a mean residence time of 4–5 min for 59Fe-labelled mouse chow, emphasising the importance of initial uptake measurements. Time courses for in vivo total mucosal uptake exhibited linearity over a wide variety of absorption rates after correction for the permeation by intact metal-chelate complex. The corrected uptake showed a hyperbolic dependence on medium Fe 3+-nitrilotriacetate concentration. Kinetic studies revealed a 2–3-fold increase in total mucosal uptake in hypoxia. Mucosa-to-carcass transfer of 59Fe was also markedly increased by chronic hypoxia. The in vitro system exhibits similar qualitative and quantitative kinetics for Fe 3+ transport via the mucosal membrane to those obtained in vivo. The results observed in vitro are thus valid and provide a convenient method for further studies on Fe 3+ transport in animals and in man.

The effects of diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxamide (4-ma) and (4r)-5,10-SECO-19-norpregna-4,5-diene-3,10,20-trione (SECO) on androgen biosynthesis in the rat testis and epididymis

We have investigated the effects of two 4-ene-steroid 5α-reductase inhibitors, diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxamide (4-MA) and (4R)-5,10-seco-19-norpregna-4,5-diene-3, 10,20-trione (SECO), on testicular and epididymal androgen biosynthesis. Kinetic analyses revealed that both compounds inhibited epididymal DHT biosynthesis. 4-MA was a competitive inhibitor of epididymal nuclear and microsomal 4-ene-steroid 5α-reductases (3-oxo-5α -steroid: NADP 4-ene-oxidoreductase EC 1.3.1.22) with K i app values of 12.8 and 15.1 nmol/1 compared to the respective K m app values of 185 and 240 nmol/l. Values for the V max app were always within 70–130% of the control. SECO at 1.0 μmol/l, also inhibited epididymal nuclear and microsomal 4-ene-steroid-5α-reductases, causing respectively 2.9 and 5.2-fold increases in K m app . The V V app values were unchanged. However, SECO concentrations of 5 and 25 μmol/l abolished 4-ene-steroid 5α-reductase activity at all testosterone concentrations. To examine the specificity of these compounds, we investigated their effects on the enzymes that convert pregnenolone to testosterone. Rat testis microsomes converted pregnenolone to testosterone via the 4-ene-3-oxo pathway, with the major metabolites being progesterone, 17-hydroxyprogesterone, 4-androstenedione and testosterone; some 17-hydroxypregnenolone was also formed. Very small amounts of dehydroepiandrosterone (DHA) and 5-androstenediol were detected. SECO, at a concentration that completely inhibited epididymal 4-ene-steroid 5α-reductase activity, did not alter the metabolic profile of pregnenolone metabolism. However, 4-MA prevented the appearance of 4-ene steroids, and large quantities of 17-hydroxypregnenolone and DHA accumulated, suggesting that inhibition of the 3β-hydroxysteroid: NAD(P) + oxidoreductase (EC 1.1.1.51) and 3-oxosteroid 5-ene-4-ene-isomerase (EC 5.3.3.1) [3β-hydroxysteroid dehydrogenase-isomerase] was occurring. Optimal conditions for the microsomal conversion of DHA to 4-androstenedione were determined; kinetic analyses of the 3β -hydroxysteroid dehydrogenase-isomerase activity revealed that 4-MA inhibited this reaction non-competitively, reducing V max app values to 25% of the control. The K i app determined from the intercept replot, was 121 nmol/1, and the V m app was always between 90 and 130% of the control value. It is concluded that SECO is more specific than 4-MA in its effects on androgen biosynthesis in the testis and epididymis and that both these drugs should provide useful tools in assessments of the relative contributions of 5α-reduced androgens to androgen dependent processes.