Synthesis and characterization of 5-alkoxycarbonyl-4-hydroxymethyl-5-alkyl-pyrroline N-oxide derivatives

The syntheses, analytical properties, and spin trapping behavior of four novel EMPO derivatives, namely 5-ethoxycarbonyl-4-hydroxymethyl-5-methyl-pyrroline N-oxide (EHMPO), 5-ethoxycarbonyl-5-ethyl-4-hydroxymethyl-pyrroline N-oxide (EEHPO), 4-hydroxymethyl-5-methyl-5-propoxycarbonyl-pyrroline N-oxide (HMPPO), and 4-hydroxymethyl-5-methyl-5-iso-propoxycarbonyl-pyrroline N-oxide (HMiPPO), towards different oxygen- and carbon-centered radicals are described.     

Synthesis and characterization of several carbamoyl- and methylcarbamoyl-substituted EMPO derivatives

The spin trapping behavior of four novel carbamoyl-substituted EMPO derivatives, namely 5-carbamoyl-3,5-dimethyl-pyrroline N-oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), and 3-ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), towards different oxygen- and carbon-centered radicals is described, the half lives of the respective superoxide adducts ranging from about 10 to 20 min. The most characteristic adducts were, however, formed from methyl, hydroxymethyl, hydroxyethyl, and carbon dioxide anion radicals.     

Free radical trapping properties of several ethyl-substituted derivatives of 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO)

The spin trapping behavior of several ethyl-substituted EMPO derivatives, cis- and trans-5-ethoxycarbonyl-3-ethyl-5-methyl-pyrroline N-oxide (3,5-EEMPO), 5-ethoxycarbonyl-4-ethyl-5-methyl-pyrroline N-oxide (4,5-EEMPO), cis- and trans-5-ethoxycarbonyl-5-ethyl-3-methyl-pyrroline N-oxide (5,3-EEMPO), and 5-ethoxycarbonyl-5-ethyl-4-methyl-pyrroline N-oxide (5,4-EEMPO), toward a series of different oxygen- and carbon-centered radicals, is described. Considerably different stabilities of the superoxide adducts (ranging from about 12 to 55 min) as well as the formation of other radical adducts were observed.     

Evaluation of spin trapping agents and trapping conditions for detection of cell-generated reactive oxygen species

Electron paramagnetic resonance with spin trapping is a useful technique to detect reactive oxygen species, such as superoxide radical anion (O2*-), a key species in many biological processes. We evaluated the abilities of four spin traps in trapping cell-generated O2*-: 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), 2-diethoxyphosphoryl-2-phenethyl-3,4-dihydro-2H-pyrrole N-oxide (DEPPEPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Optimal experimental conditions for obtaining maximal signal intensity of O2*- adduct in a cellular system were first studied. The maximal intensities of BMPO, DEPMPO, and DMPO adducts were similar while DEPPEPO did not trap cell-generated O2*- induced by 1,6-benzo[a]pyrene quinone in a human mammary epithelial cell line (MCF-10A). BMPO and DEPMPO adducts were more stable, considering the stability of their maximal signal, than DMPO adduct in the tested cellular systems. In addition, we observed that O2*- spin adducts were reduced to their corresponding hydroxyl adducts in the cellular system. The selection of optimal spin trap in trapping cell-generated O2*- is discussed.     

Biocatalytic ammonolysis of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester: preparation of an intermediate to the dipeptidyl peptidase IV inhibitor Saxagliptin

An efficient biocatalytic method has been developed for the conversion of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester (1) into the corresponding amide (5S)-5-aminocarbonyl-4,5-dihydro-1H-pyrrole-1-carboxylic acid, 1-(1,1-dimethylethyl)ester (2), which is a critical intermediate in the synthesis of the dipeptidyl peptidase IV (DPP4) inhibitor Saxagliptin (3). Candida antartica lipase B mediates ammonolysis of the ester with ammonium carbamate as ammonia donor to yield up to 71% of the amide. The inclusion of Ascarite and calcium chloride as adsorbents for carbon dioxide and ethanol byproducts, respectively, increases the yield to 98%, thereby offering an efficient and practical alternative to chemical routes which yield 57-64%.     

Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([(15)N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min(-1)) and NADPH/calcium-calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO-OOH), the superoxide adduct of EMPO (EMPO-OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide is more persistent than EMPO-OOH, the electron spin resonance spectra of [(14)N]EMPO-OOH and [(15)N]EMPO-OOH are less complex and easier to interpret. Potential uses of [(15)N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.     

Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.     

Determination of clozapine,and its metabolites,N-desmethylclozapine and clozapine N-oxide in dog plasma using high-performance liquid chromatography

Clozapine and its two major metabolites, N-desmethylclozapine and clozapine N-oxide were quantified using a high-performance liquid chromatographic method with UV detection in dog plasma following a single dose of clozapine. The analysis was performed on a 5-micrometer Hypersil CN (CPS-1; 250x4.6 mm) column. The mobile phase consisted of acetonitrile-water-1 M ammonium acetate (50:49:1, v/v/v), which was adjusted to pH 5.0 with acetic acid. The detection wavelength was 254 nm. A liquid-liquid extraction technique was used to extract clozapine and its metabolites from dog plasma. The recovery rates for clozapine, N-desmethylclozapine, and the internal standard (I.S.) were close to 100% using this method. The recovery rate for clozapine N-oxide (62-66%) was lower as expected because it is more polar. The quantitation limits for clozapine, clozapine N-oxide, and N-desmethylclozapine were 0.11, 0.05 and 0.05 microM, respectively. Intra-day reproducibility for concentrations of 0.1, 1.0 and 5.0 microM were 10.0, 4.4 and 4.2%, respectively, for N-oxide; 11.2, 4.3 and 4.9%, respectively, for N-desmethylclozapine; and 10.8, 2.2 and 4.9%, respectively, for clozapine. Inter-day reproducibility was <15% for clozapine N-oxide, <8% for N-desmethylclozapine and <19% for clozapine. This simple method was applied to determine the plasma concentration profiles of clozapine, N-desmethylclozapine and clozapine N-oxide in dog following administration of a 10 mg/kg oral dose of clozapine.     

Studies on the Chemical Constituents of Fritillaria pallidiflora Schrenk

Six steroid alkaloids, imperialine (Ⅰ), imperialine-3β-D-glucoside (Ⅱ), peimissine (Ⅲ), imperialine N-oxide (Ⅳ), cycloparnine (Ⅴ), and cycloposine (Ⅵ) were isolated from the bulbs of Fritillaria pallidiflora Schrenk. Their structures were determined from spectral data and chemical evidences. Imperialine N-oxide was first obtained from nature.     

Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7-11 min.). In model experiments using Fe(3+)-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe(2+) complexes with phosphate or DTPA or inactivation of iron by addition of "Desferal" (Novarti's Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe(2+) under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.     

Design, synthesis, and biological activity of non-amidine factor Xa inhibitors containing pyridine N-oxide and 2-carbamoylthiazole units

Based on the both of results for X-ray studies of tetrahydrothiazolopyridine derivative 1c and FXV673, we synthesized a series of thiazol-5-ylpyridine derivatives containing pyridine N-oxide and 2-carbamoylthiazole units to optimize the S4 binding element. N-Oxidation of thiazol-5-ylpyridine increased the anti-fXa activity more than 10-fold independent on the position of N-oxide. The 4-pyridine N-oxide derivatives 3a and 3d excelled over the tetrahydrothiazolopyridine 1b in potency. 2-Methylpyridine N-oxide 3d exhibited 49-fold selectivity over thrombin. Our modeling study proposed a binding mode that the pyridine N-oxide ring of 3a stuck into the "cation hole" , and the oxide anion of 3a occupied in the almost same space to that of FXV673. From observations of the SAR and modeling studies, we suggested the possibilities that the formation of hydrogen bond with the oxide anion in the "cation hole" and the affinity of cationic pyridine ring to S4 subsite were responsible for increase in anti-fXa activity.     

Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis

The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the superoxide radical adduct, as has been previously suggested [correction]. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M(-1)s(-1)), DEPMPO (0.53 M(-1)s(-1)), and BMPO (0.24 M(-1)s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20 degrees C.     

Purification and immobilization of rabbit liver aldehyde oxidase

Aldehyde oxidase (E.C. 1.2.3.1) was isolated from rabbit liver and two potential bioaffinity ligands, i.e., 3-aminocarbonyl-1-benzyl-6-methylpyridinium bromide and 3-aminocarbonyl-1-benzyl-4,6-dimethylpyridinium chloride, were tested for their applicability in a purification procedure for this enzyme. Various supports and different coupling methods were investigated for the immobilization of aldehyde oxidase. Adsorption to n-hexyl- and n-octylamine-substituted Sepharose 4B and DEAE Sepharose 6B gave the best retention of aldehyde oxidase activity. The storage stability of free enzyme and enzyme immobilized to n-octylamine-substituted Sepharose 4B was studied in several buffers at pH 7.8 and 9.0. This showed that the stability of immobilized enzyme was much less than that of free enzyme. The apparent operational stability of the immobilized enzyme preparation, however, improved substantially compared to soluble enzyme, although the corresponding product yield is still very poor. Coimmobilization of catalase and/or superoxide dismutase provided no significant increase of the apparent operational stability and product yield. A positive effect on both parameters was found for aldehyde oxidase-n-alkylamine Sepharose 4B preparations by increasing the amount of enzyme adsorbed per unit weight of support, whereas the productivity of these preparations remained about constant.     

Development of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as orally active human chymase inhibitors

A novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones was designed, synthesized, and evaluated as human chymase inhibitors. From this series, we identified several compounds which were effective, via oral administration, in a mouse model of chronic dermatitis.     

3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 1: Discovery and exploration of the carboxyamide side chain

A series of 3-(2-aminocarbonyl-4-phenoxymethylphenyl)propanoic acid analogs were synthesized and evaluated for their EP3 antagonist activity in the presence of additive serum albumin. Several compounds were biologically evaluated for their in vivo efficacy with respect to the PGE₂-induced uterine contraction in pregnant rats as well as their pharmacokinetics. The discovery process of these potent and selective EP3 antagonists and their structure activity relationship are also presented.     

The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides: the mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver

Nonstereospecific addition of free radicals to chiral nitrones yields cis/trans diastereoisomeric nitroxides often displaying different electron spin resonance (ESR) characteristics. Glutathione peroxidase-glutathione (GPx-GSH) reaction was applied to reduce the superoxide adducts (nitrone/*OOH) to the corresponding hydroxyl radical (HO*) adducts (nitrone/*OH) of two nitrones increasingly used in biological spin trapping, namely 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide, and of 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO), a sterically hindered DEPMPO analogue. The method offered improved conditions to record highly resolved ESR spectra and by accurate simulation of line asymmetry we obtained clear evidence for the existence of previously unrecognized isomer pairs of cis- and trans-[DEPMPO/*OH] and [DIPPMPO/*OH]. Additional nitrone/*OH generation methods were used, i.e. photolysis of hydrogen peroxide and the Fenton reaction. We developed a kinetic model involving first- and second-order decay and a secondary conversion of trans to cis isomer to fully account for the strongly configuration-dependent behavior of nitrone/*OH. In the reductive system and, to a lower extent, in the Fenton or photolytic systems cis-nitrone/*OH was the more stable diastereoisomer. In various biologically relevant milieu, we found that the cis:trans-nitrone/*OH ratio determined right after the spin adduct formation significantly differed upon the GPx-GSH vs (Fenton or photolytic) systems of formation. This new mechanistic ESR index consistently showed for all nitrones that nitrone/*OH signals detected in the postischemic effluents of ischemic isolated rat livers are the reduction products of primary nitrone/*OOH. Thus, ESR deconvolution of cis/trans diastereoisomers is of great interest in the study of HO* formation in biological systems.     

Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans.

When tested as a microbial model for mammalian drug metabolism, the filamentous fungus Cunninghamella elegans metabolized chlorpromazine and methdilazine within 72 h. The metabolites were extracted by chloroform, separated by high-performance liquid chromatography, and characterized by proton nuclear magnetic resonance, mass, and UV spectroscopic analyses. The major metabolites of chlorpromazine were chlorpromazine sulfoxide (36%), N-desmethylchlorpromazine (11%), N-desmethyl-7-hydroxychlorpromazine (6%), 7-hydroxychlorpromazine sulfoxide (36%), N-hydroxychlorpromazine (11%), 7-hydroxychlorpromazine sulfoxide (5%), and chlorpromazine N-oxide (2%), all of which have been found in animal studies. The major metabolites of methdilazine were 3-hydroxymethdilazine (3%). (18)O(2) labeling experiments indicated that the oxygen atoms in methdilazine sulfoxide, methdilazine N-oxide, and 3-hydroxymethdilazine were all derived from molecular oxygen. The production of methdilazine sulfoxide and 3-hydroxymethdilazine was inhibited by the cytochrome P-450 inhibitors metyrapone and proadifen. An enzyme activity for the sulfoxidation of methdilazine was found in microsomal preparations of C. elegans. These experiments suggest that the sulfoxidation and hydroxylation of methdilazine and chlorpromazine by C. elegans are catalyzed by cytochrome P-450.     

Biological invasion alters regional nitrogen-oxide emissions from tropical rainforests

We explored whether the invasion of an exotic, nitrogen (N) fixing tree into native Hawaiian tropical forests has altered regional emissions of nitrous oxide (N₂O) and nitric oxide (NO), two atmospherically important trace gases produced by microorganisms in soils. Ecosystem processes, including nitrification and N-oxide emissions, were not affected by Morella faya (formerly Myrica faya ) invasion until it dominated the community with few native species in the overstory or understory. Remote-sensing estimates of upper-canopy leaf N concentration were strongly correlated to N-oxide emissions in ecosystems at the mesic-wet end of a precipitation gradient, where temperatures are warm, relatively constant, and N limits biological processes. In contrast, remotely sensed and field-based canopy chemistry was not related to N-oxide emissions in dry forest ecosystems where the seasonality of temperature and moisture exerted stronger control over soil gas fluxes. Thus, remote sensing of canopy N was useful for estimating the impact of M. faya on regional N-oxide emissions only in regions receiving >1800 mm rainfall annually. Our estimates of N-oxide emissions from M. faya are half as large and 35 times more precise than those made using traditional, plot-level methods of extrapolation. Over the 40 years since its first occurrence in wet forests of Hawai'i Volcanoes National Park, M. faya has increased N-oxide emissions 16-fold, with its effects most pronounced in summer and at the N-rich centers of dense, monospecific stands.     

Detoxification of 6-hydroxydopamine-induced Parkinsonian neurodegeneration by G-CYPMPO, a novel radical trapper

2-(5,5-Dimethyl-2-oxo-2-λ(5)-[1,3,2]dioxaphosphinan-2-yl)-2-methyl-3,4-dihydro-2H-pyrroline N-oxide {2-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinan-2-yl)-3,4-dihydro-2-methyl-2H-pyrrole N-oxide, G-CYPMPO} as the stable crystals having gauche conformation was successfully synthesized as a novel 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO)-type spin trap agent. However, the function of G-CYPMPO in vivo is still unclear. Thus, the purpose of this study was to evaluate the effects of G-CYPMPO in an in vivo model of Parkinson's disease (PD). Rats were microinjected with 6-hydroxydopamine (6-OHDA, 32nmol) in the presence or absence of G-CYPMPO (0.4, 1.2, 4nmol). We investigated behavioral and histochemical parameters in this rat model of PD. In addition, to examine the effects of G-CYPMPO against oxidative stress, we used electron spin resonance (ESR) spectrometry. Intranigral injection of 6-OHDA alone induced a massive loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc). Co-microinjection of G-CYPMPO significantly prevented 6-OHDA-induced dopaminergic neurodegeneration and behavioral impairments. Immunoreactivities for glial markers, such as cluster of differentiation antigen-11b (CD11b) and glial fibrillary acidic protein (GFAP), were notably detected in the SNpc of rats injected with 6-OHDA alone. These immunoreactivities were markedly suppressed by the co-microinjection of G-CYPMPO, similar to the results in vehicle-treated rats. In addition, G-CYPMPO directly trapped hydroxyl radical (OH) generated from 6-OHDA and Fe(2+) in a concentration-dependent manner. These results suggest that G-CYPMPO attenuates 6-OHDA-induced dopaminergic neurodegeneration in a rat model of PD, and is a useful tool for biological research.     

Properties of purified squalene-hopene cyclase from Bacillus acidocaldarius

The squalene-hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X-100, chromatography on DEAE-cellulose, phenyl Sepharose and two gel-filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n-alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate, dodecyldimethylamine N-oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate and dodecyldimethylamine N-oxide was demonstrated by Lineweaver-Burk plots.