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

Abstract

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°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°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 μM)-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).