No abstract

SUMMARYGlutamine synthetase (GSase), the enzyme that catalyses the conversion of glutamate and ammonia to glutamine, is present at high levels in vertebrate brain tissue and is thought to protect the brain from elevated ammonia concentrations. We tested the hypothesis that high brain GSase activity is critical in preventing accumulation of brain ammonia and glutamate during ammonia loading in the ammonia-intolerant rainbow trout. Trout pre-injected with saline or the GSase inhibitor methionine sulfoximine (MSOX, 6mgkg -1 ), were exposed to 0, 670 or 1000moll -1 NH 4 Cl in the water for 24 and 96h. Brain ammonia levels were 3-to 6-fold higher in ammonia-exposed fish relative to control fish and MSOX treatment did not alter this. Brain GSase activity was unaffected by ammonia exposure, while MSOX inhibited GSase activity by ~75%. Brain glutamate levels were lower and glutamine levels were higher in fish exposed to ammonia relative to controls. While MSOX treatment had little impact on brain glutamate, glutamine levels were significantly reduced by 96h. With ammonia treatment, significant changes in the concentration of multiple other brain amino acids occurred and these changes were mostly reversed or eliminated with MSOX. Overall the changes in amino acid levels suggest that multiple enzymatic pathways can supply glutamate for the production of glutamine via GSase during ammonia exposure and that alternative transaminase pathways can be recruited for ammonia detoxification. Plasma cortisol levels increased 7-to 15-fold at 24h in response to ammonia and MSOX did not exacerbate this stress response. These findings indicate that rainbow trout possess a relatively large reserve capacity for ammonia detoxification and for preventing glutamate accumulation during hyperammonaemic conditions.

The novel cytochrome P450 ⁄ redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated lowspin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH ⁄ NADH, with NADPH used more efficiently (K m[NADPH] = 0.9 ± 0.5 lM and K m[NADH] = 399.1 ± 52.1 lM). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multiangle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.Abbreviations CO, carbon monoxide; CPR, cytochrome P450 reductase; CYP101A1, Pseudomonas putida camphor hydroxylase P450cam or CYP102A1; CYP116B1, cytochrome P450 (116B1) from Cupriavidus metallidurans CH34; ddH 2 O, distilled, deionized water; EPTC, S-ethyl dipropylthiocarbamate; Fe-S, iron-sulfur; HS, high-spin; ImC10, imidazolyl decanoic acid; ImC11, imidazolyl undecanoic acid; ImC12, imidazolyl dodecanoic acid; IPTG, isopropyl thio-b-D-galactoside; LC, liquid chromatography; MALLS, multi-angle laser light scattering; NHE, normal hydrogen electrode; NO, nitric oxide; P450, cytochrome P450 or CYP; P450 BM3, cytochrome P450 BM3 from Bacillus megaterium or CYP102A1; PDO, phthalate dioxygenase; PDOR, Burkholderia cepacia phthalate dioxygenase reductase; SEC, size-exclusion chromatography; vernolate, S-propyl dipropylthiocarbamate.

Here we report the widespread natural occurrence of a known antibiotic and antineoplastic compound, hydroxyurea in animals from many taxonomic groups.Hydroxyurea occurs in all the organisms we have examined including invertebrates (molluscs and crustaceans), fishes from several major groups, amphibians and mammals. The species with highest concentrations was an elasmobranch (sharks, skates and rays), the little skate Leucoraja erinacea with levels up to 250 μM, high enough to have antiviral, antimicrobial and antineoplastic effects based on in vitro studies. Embryos of L. erinacea showed increasing levels of hydroxyurea with development, indicating the capacity for hydroxyurea synthesis. Certain tissues of other organisms (e.g. skin of the frog (64 μM), intestine of lobster (138 μM) gills of the surf clam (100 μM)) had levels high enough to have antiviral effects based on in vitro studies. Hydroxyurea is widely used clinically in the treatment of certain human cancers, sickle cell anemia, psoriasis, myeloproliferative diseases, and has been investigated as a potential treatment of HIV infection and its presence at high levels in tissues of elasmobranchs and other organisms suggests a novel mechanism for fighting disease that may explain the disease resistance of some groups. In light of the known production of nitric oxide from exogenously applied hydroxyurea, endogenous hydoxyurea may play a hitherto unknown role in nitric oxide dynamics.

To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of (15)N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R(d)), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R(d) values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R(d) values ranged from 0.9 μmol 100 g(-1) h(-1) (lysine) to 22.1 μmol 100 g(-1) h(-1) (threonine) with most values falling between 2 and 6 μmol 100 g(-1) h(-1). There was a significant correlation between R(d) and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.