A novel, endogenous inhibitor of 7-ethoxyresorufin-O-deethylase activity isolated from liver cytosolic fractions of bream (Abramis brama L.).

The cytosolic supernatant of bream (Abramis brama L.) liver homogenates inhibits the 7-ethoxyresorufin-O-deethylase (EROD) activity of pike (Esox lucius) microsomal fractions. The inhibitor shows no activity against 7-ethoxycoumarin-O-deethylase and benzo(a)pyrene hydroxylase indicating a high isoenzyme specificity. The inhibiting component is a heat-sensitive substance (56 degrees C for 5') which is not self regenerating after subsequent cooling. It can be isolated from the cytosolic fraction using two combined steps of ion exchange chromatography. The purification factor is 500-fold with a recovery rate of 70%. SDS-PAGE of the purified fractions indicate that electrophoretic purity was not achieved. However, a prominent band at about 97 kDa was present in all fractions in a close intensity activity relationship. The molecular weight of the native form of the purified protein was determined to be 175 +/- 35 kDa using gel filtration on a Sephacryl S 300 HR column. So far the inhibitor can be characterized as a protein. It shows strong tendencies to aggregate due to lipophilic interactions. These interactions can be repressed by the addition of 1% sodium cholate. The inhibitor has an optimum activity at 25 degrees C and pH 8.0. The inhibitor does not correspond to any of the known cytosolic, endogenous inhibitors of EROD activities in fish, including proteases, cytosolic phosphatases, kinases and resorufin reductase (e.g. DT-diaphorase), although a non-dicoumarol (10 microM)-inhibited menadione oxidoreductase activity of up to 46.7 +/- 0.4 nmol/min per mg inhibitory protein was measured. Kinetic studies using Michaelis-Menten kinetics with purified inhibitor fractions prove a non-competitive mode of inhibition. As this kind of inhibitor is not described yet it is named CERODIP (cytosolic, EROD-inhibiting protein).

The toxicity of soil samples containing TNT and other ammunition derived compounds in the enchytraeid and collembola-biotest.

The effect of ammunition-like compounds and armament waste on the mortality and reproduction of terrestrial invertebrates was assayed by using two biotests: the enchytraeid-biotest with Enchytraeus crypticus and the collembola-biotest with Folsomia Candida. Toxicity was investigated by using standard soil (Lufa 2.2) spiked with 2,4,6-trinitrotoluene (TNT), hexahydro-l,3,5-trinitro-l,3,5-triazine (hexogen, RDX), octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine (octogen, HMX) and 2,4,6-triaminotoluene (TAT). Ecotoxicity was investigated with ammunition-contaminated soil material from the former ammunition plant "Tanne" at Clausthal-Zellerfeld (CTNTla) and the Brandplatz (incineration site) in Torgau-Elsnig (TETNT1a), Germany. TNT increased mortality and reduced reproduction of both test organisms corresponding to the concentrations used, whereas hexogen, octogen and TAT had no effect in the tested concentrations (1000-2000 mg/kg). From the two soil materials, TETNT1a was much more toxic than CTNT1a. The LC50(7d) in the enchytraeid-biotest was 570 mg TNT/kg and the EC50(28d) 369 mg TNT/kg soil material (dw). In the collembola-biotest the LC50(7d) was 185 mg TNT/kg and the EC50(28d) 110 mg TNT/kg soil matter (dw).

Cytochrome P450 and dependent activities in unexposed and PAH-exposed terrestrial annelids.

The cytochrome P450 system of the oligochaetes Eisenia f. fetida (tiger worm) and Enchytraeus crypticus (pot worm) was analysed using ethoxy-, pentoxy- and benzoxyresorufin as substrates for monooxygenase activity. Whole body microsomes of the earthworm E.f. fetida displayed PentROD activity in the range from 0.26 to 1.05 pmol mg protein-1 min-1 and BenzROD activity in the range from 0.14 to 0.30 pmol mg protein-1 min-1. Exposure of the animals for up to four weeks to 100 mg fluoranthene or benzo[a]pyrene kg-1 soil (dry weight) did not induce significant changes in the activity of these monooxygenases. In E. crypticus EROD activity was in the range from 2.10 to 6.18 pmol mg protein-1 min-1 and PentROD activity in the range from 1.75 to 4.78 pmol mg protein-1 min-1. Short-term exposure to BaP by feeding reduced the EROD activity significantly by 45%, but did not effect PentROD activity. After long-term (8 weeks) exposure to BaP in the agar-agar medium EROD activity was not changed but PentROD had decreased to zero. In both species cytochrome P420 and NADPH-cytochrome C reductase activity were present. In E.f. fetida microsomes are associated with the giant haemoglobin. Both can be separated by gel filtration on a Sepharose B2 column or by hydrophobic interaction chromatography after solubilisation with cholate. NADPH-cytochrome C reductase elutes together with haemoglobin. Cytochrome P420 is eluted with Emulgen 911 and can be further purified by ion exchange chromatography using HA-Ultrogel. By SDS-PAGE of the purified microsomal proteins three protein bands are visualised in the range of cytochrome P450 displaying an apparent molecular mass of 54, 56 and 58 kDa. Only the 54-kDa protein interacts weakly with perch (Perca fluviatilis) CYP1A antibodies, while two proteins with an apparent molecular mass of 65 and 71 kDa give a strong antibody signal.

ACh and 5-HT induced changes in the concentration of cytosolic inositol trisphosphate (InsP3) and inositol bisphosphate (InsP2) in the ABRM of Mytilus edulis L.

1. For determination of the phosphoinositides and inositol phosphates present in anterior byssus retractor muscle (ABRM) of Mytilus edulis fiber bundles of this muscle were incubated with [3H]-inositol. Close-to-equilibrium labelling was achieved after 14-17 hr of incubation. 2. The phosphoinositides formed during incubation were identified as phosphatidylinositolphosphates by thin layer chromatography and as glycerophosphoryl esters by anion-exchange chromatography after deacylation. Besides PtdIns, PtdInsP and PtdInsP2 two labelled products are formed, which could not be identified. 3. Inositol phosphates were separated by anion-exchange chromatography. InsP, InsP2 and InsP3 are present, while InsP4 seemed to be absent. 4. Incubation of pre-labelled fibers with ACh induces the accumulation of InsP3 and InsP2 immediately. While 5-Ht accomplishes the accumulation after a lag time of 25 sec. The concentration of cytosolic InsP does not change.

Phosphorylation of molluscan paramyosin.

1. In Mytilus edulis two proteins of the contractile apparatus can be phosphorylated by cyclic AMP dependent protein kinases: a 295,000 d protein of unknown function, and paramyosin. 2. Paramyosin isolated from thick filaments by the selective extraction method contains the 106,000 d monomer only, whereas paramyosin extracted from ethanol ether dried powder contains equal amounts of the 108,000 d, and the 106,000 d monomers, and traces of the 104,000 d monomer. 3. Paramyosin isolated from ethanol ether dried powder incorporates up to four times the amount of 32P than paramyosin isolated by the selective extraction method. 4. Cytoplasmatic protein kinases show a higher affinity towards paramyosin as a phosphoryl acceptor than protein kinases associated with paramyosin. 5. Paramyosin of 5-HT treated catch muscles is phosphorylated 2 to 4 times better than paramyosin of ACh treated or untreated catch muscles.

5-HT induced accumulation of 3',5'-AMP and the phosphorylation of paramyosin in the ABRM of Mytilus edulis.

In Mytilus edulis 5-Ht induced relaxation of a muscle in catch is preceded by an increase in 3',5'-AMP content. In vitro two proteins of the contractile apparatus are phosphorylated by 3',5'-AMP dependent protein kinases. The 295000 dalton protein cannot be identified, the other one is paramyosin. Phosphorylated paramyosin inhibits actomyosin ATPase of smooth mollusc muscles at low and high Ca++ concentrations.