Metabolism of territrem a in liver microsomes from wistar rats: 2. Sex differences and regulation with gonadal hormones and phenobarbital.

The aim of the present study was to assess the effects of sex difference on metabolism of territrem A (TRA) by liver microsomes from 7-wk-old Wistar rats. Metabolism of TRA to 2-dihydro-4beta-demethylterritrem A (MA2) through 4beta-hydroxymethyl-4beta-demethylterritrem A (MA1) and 4beta-oxo-4beta-demethylterritrem A (MAX) was observed in intact male rats. However, in intact female rats only MA1 was formed, although the amount of MA, formed in females was much less than in males. Phenobarbital pretreatment enhanced this step and was not affected by gonadectomy. In the gonadectomized rats of both sexes, MA2 was formed from TRA when the animals were further treated by testosterone and was significantly enhanced by treatment with phenobarbital. However, estradiol treatment or estradiol in combination with phenobarbital treatment did not affect MA2 formation from TRA in gonadectomized rats.

Metabolism of territrem a by liver microsomes of Wistar rats: identification of the metabolites and their metabolic sequence.

The metabolism of territrem A (TRA) was investigated in liver microsomes of male Wistar rats. The results indicated that three metabolites were produced from TRA and these metabolic reactions were inhibited by metyrapone, an inhibitor of cytochrome P-450. Based on analysis by high-performance liquid chromatography (HPLC), mass, and nuclear magnetic resonance (NMR) spectroscopic techniques, the structure of these metabolites were identified as 4beta-hydroxymethyl-4beta-demethylterritrem A (MA1), 4beta-oxo-4beta-demethylterritrem A (MAX), and 2-dihydro-4beta-demethylterritrem A (MA2). It was proposed that reactions proceeded by three sequential oxidative reactions in the pyran moiety of TRA: first, hydroxylation at the 4beta-C methyl group of TRA to form MA1; second, oxidation at the 4beta hydroxyl group of MA, to form MAX; and third, decarbonylation at the 4beta-C oxo group of MAX to form MA2.

Territrem B, a tremorgenic mycotoxin that inhibits acetylcholinesterase with a noncovalent yet irreversible binding mechanism.

Territrem B (TRB) is a fungal metabolite isolated from Aspergillus terreus shown previously to be a potent and irreversible inhibitor of acetylcholinesterase (AChE). In the present study, a number of binding and inhibition assays were carried out to further characterize the inhibitory effect of TRB. The results indicate that the binding of TRB (a) is much more selective than a well characterized selective inhibitor of AChE, BW284C51, (b) adopts a one-to-one stoichiometry with the enzyme, (c) cannot be undone by an AChE-regenerating oxime agent, which contrasts the ability of 8 M urea to release AChE-bound TRB, (d) is enhanced by high concentration NaCl but prevented, unless preincubated, by Triton X-100, and (e) exhibits quasi-first order kinetics with an overall inhibition constant of 0.01 nM(-1) min(-1). Together these results suggest a very different irreversible binding (a noncovalent type) from that of the covalent type, which involves typical irreversible AChE inhibitors such as diisopropylfluorophosphate and neostigmine. According to the prediction of a molecular modeling study, the distinct AChE inhibitory characteristics of TRB may arise from the inhibitor being noncovalently trapped within a unique active-site gorge structure of the enzyme. It was predicted that an optimal TRB. AChE binding would position a narrowing connection of the TRB structure at a constricted area near the entrance of the gorge, thereby providing a structural basis for the observed irreversible binding.

Structure and anti-acetylcholinesterase activity of 4 alpha-(hydroxymethyl)-4 alpha-demethylterritrem B.

The structure of a metabolite produced on incubation of territrem B (1) with rat liver microsomes has been established to be 4 alpha-(hydroxymethyl)-4 alpha-demethylterritrem B (5). Compound 5 was a potent inhibitor of electric eel acetylcholinesterase (AChE) (E.C. 3.1.1.7).

Synthesis, structure and anti-acetylcholinesterase activity of 4beta-methoxymethyl-4beta-demethyl territrem B.

4beta-Methoxymethyl-4beta-demethyl territrem B [5] was synthesized from 4beta-hydroxymethyl-beta-demethyl territrem B [4] by treatment with dimethyl sulfate in methanolic NaOH. The structure of 5 was elucidated by uv, nmr and mass spectra. The IC50 of 5 on acetylcholinesterase (AChE) was 6.30 x 10(-5) M, which indicated 0.4% of the anti-AChE activity of territrem B [2].

Acetylcholinesterase inhibition by territrem B derivatives.

Five derivatives of territrem B [2], a potent acetylcholinesterase inhibitor isolated from a rice culture of Aspergillus terreus, were prepared from 2 as well as from its major metabolite, 4 beta-hydroxymethyl-4 beta-demethylterritrem B [4]. The inhibitory activities of these derivatives against electric eel acetylcholinesterase (E.C. 3.1.1.7) were tested and it was concluded that the enone and the pyrone groups present in 2 may play an important biological role.

Interactions of anticholinesterases with Achatina fulica acetylcholine responses and electrogenic sodium pump.

The dose-dependent effects of the anticholinesterases, neostigmine and mycotoxin territrem-B, were determined on: (i) Cl(-)-responses of voltage clamped Achatina fulica neurons to microperfused acetylcholine; (ii) the 4 K(+)-induced outward currents evoked by an electrogenic sodium pump in the same neuron; and (iii) acetylcholinesterase activity of Achatina fulica ganglionic homogenates. Both compounds at low doses potentiated the peak acetylcholine responses. However, they had different effects at higher (> 1 microM) doses in that neostigmine now antagonized acetylcholine responses, while territrem-B still produced a maximal potentiation. At all doses neostigmine produced a dose-dependent inhibition of acetylcholinesterase activity. The cholinolytic effect of high doses of neostigmine was associated with the inhibition of 4 K(+)-induced current in the same neuron, while territrem-B neither altered the K(+)-induced current nor antagonized acetylcholine responses. The cholinolytic effect of neostigmine was completely antagonized by the inhibition of electrogenic sodium pump by ouabain or by perfusion with K(+)-free solution. These results suggest that neostigmine at high concentrations inhibits the electrogenic sodium pump and that the cholinolytic effect of high doses of neostigmine is secondary to this action. Territrem-B, on the other hand, had no effect on the electrogenic sodium pump and had no effect on the neuronal membrane properties other than to inhibit acetylcholinesterase. Thus, territrem-B may be a useful tool for studying the interaction between acetylcholinesterase and acetylcholine receptors.

NMR assignments of territrems A, B, and C and the structure of MB2, the major metabolite of territrem B by rat liver microsomal fraction.

The 1H- and 13C-nmr assignments of territrems A, B, and C [1-3] were made by using nOe and 2D nmr techniques. Following the same methods, the structure of MB2 [4], the major product of territrem B incubated with rat liver microsomal fraction, was determined as a hydroxylation product at the pro S methyl group of C-4 of territrem B.

Cleavage of territrems by alkaline hydrogen peroxide: Isolation and characterization of the aromatic moiety of territrems.

The chemical reaction of cleavaging territrem B to give 3,4,5-trimethoxy benzoic acid by alkaline hydrogen peroxide was investigated. The method was applied for confirmation of the chemical structure of the aromatic moiety of territrem A, A', B, and B'. The physicochemical properties of the aromatic cleavage product of territrem Aindicated the structure as 3,4-methylendioxy, 5-methoxy benzoic acid (or 4-methoxy, 6-carboxy, 1, 3-benzodioxole). The experiment also gave the evidences that territrem A and A', on the other hand territrem B and B' have the identical aromatic moieties on their structures.

Isolation, chemical structure, acute toxicity, and some physicochemical properties of territrem B' from Aspergillus terreus.

We have isolated a metabolite of territrem, designated territrem B', from the chloroform extract of a rice culture of Aspergillus terreus 23-1 by using the same isolation procedure as that for territrems A, B, and C. The present isolation procedure gave about 10 mg of territrem B' from 4 kg of rice culture per batch. Analysis of the high-resolution mass spectrum showed that the molecular composition of territrem B' is C29H34O10 (found, 542.2167; required, 542.200). Some results of physicochemical and acute tests are presented in this paper. Single-crystal X-ray diffractometry of territrem B' indicated that the three-dimensional structure of territrem B' has not changed significantly from that of territrem B except for the insertion of one oxygen atom into territrem B to make an additional pyron ring in the E ring. The tremorgenic activity of territrem B' is greatly reduced as tested by intraperitoneal injection in mice.

Transaminase B from Escherichia coli: quaternary structure, amino-terminal sequence, substrate specificity, and absence of a separate valine-alpha-ketoglutarate activity.

Transaminase B (branched-chain amino acid aminotransferase, EC 2.6.1.42), the ilvE gene product, was purified to apparent homogeneity from an Escherichia coli K-12 strain which carries the ilvE gene both on the host chromosome and on a plasmid. The oligomeric structure of the enzyme, as determined by analytical ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was confirmed to be that of a hexamer with a molecular weight of about 182,000 and apparently identical subunits. Cross-linking with dimethylsuberimidate yielded trimers, dimers, and monomers, but essentially no species of higher molecular weight. These results are consistent with a double-trimer arrangement of the subunits in native enzyme. The amino-terminal sequence was found to be: Gly Thr Lys Lys Ala Asp Tyr Ile (Trp) Phe Asn Gly (Thr) (Met) Val. Purified transaminase B catalyzed transamination between alpha-ketoglutarate and l-isoleucine, l-leucine, l-valine, and, to a lesser extent, l-phenylalanine and l-tyrosine, the latter reacting very sluggishly. The enzyme was free of aspartate transaminase and of transaminase C. The apparent K(m) values for the branched-chain alpha-ketoacids were smaller than those for the corresponding amino acids. The lowest K(m) was recorded for dl-alpha-keto-beta-methyl-n-valerate, and the highest was recorded for l-valine. The ratio of the valine- and isoleucine-alpha-ketoglutarate activities did not change significantly during purification, and both activities were quantitatively removed from crude extract by antibody raised against purified transaminase B. These observations argue against the existence of a separate valine-alpha-ketoglutarate transaminase. Anti-E. coli transaminase B antibody cross-reacted with crude extract from Salmonella typhimurium, but not with extract obtained from Pseudomonas aeruginosa.