BACULOVIRUS EXPRESSION OF HUMAN P450 2E1 AND CYTOCHROME B(5) - SPECTRAL AND CATALYTIC PROPERTIES AND EFFECT OF B(5) ON THE STOICHIOMETRY OFP450 2E1-CATALYZED REACTIONS

The baculovirus (BV)-insect cell expression system has been successful ly used to express high levels of mammalian proteins. Here we report t he baculovirus-mediated expression of human P450 2E1 and cytochrome bg in Sf9 insect cells. The BglI-EcoRI fragment of the human P450 2E1 cD NA (Umeno ct al., Biochemistry 27, 1988) was used for the expression o f P450 2E1. Human cytochrome b(5) cDNA was obtained by the polymerase chain reaction using human liver cDNA as template. Infection of Sf9 in sect cells with a recombinant 2E1-BV resulted in the expression of a p rotein which comigrated with human liver P450 2E1 on SDS gels and cros s-reacted with a polyclonal antibody against rat liver P450 2E1. Inclu sion of hemin in the cell growth medium greatly enhanced the spectral activity of expressed 2E1. Expression levels of 1.5 nmol/mg cell lysat e were obtained after 7 days of infection. However, maximal turnover n umbers (min(-1)) were observed after 48 h of infection. The lambda(max ) of the CO:reduced CO difference spectrum of human P450 2E1 was found to be 451.1 nm. In the presence of exogenous hemin, BV-expressed huma n bs showed a typical reduced - oxidized difference spectrum with lamb da(max) and lambda(min) of 425 and 409 nm, respectively. With saturati ng levels of purified rat liver NADPH:P450 oxidoreductase and rat live r cytochrome b(5) included in the incubations, expressed human P450 2E 1 showed high catalytic activity for the metabolism of p-nitrophenol ( PNP), ethoxycoumarin, N-nitrosodiethylamine, and N-nitrosodimethylamin e (NDMA). The presence of b(5); in the incubations increased the activ ities several-fold. The K-m and k(cat) values for the N-demethylation of NDMA to HCHO in the presence of rat b(5) by expressed 2E1 were 36.0 mu M and 8.3 min(-1), respectively. In the absence of extra added b(5 ), the K-m was increased sixfold and the k(cat) decreased fourfold. In the presence of extra added b(5) expressed 2E1 showed a K-m for PNP m etabolism of 86 mu M and a k(cat) of 7.8 min(-1). Simultaneous infecti on of Sf9 insect cells with both 2E1 and human b(5) recombinant BV res ulted in a membrane fraction (2E1:b6) containing both proteins at a ra tio of b(5) to P450 of approximately 1.8:1. The K-m and k(cat) values for NDMA demethylase activity by the 2E1:b(5) membrane fraction were s imilar to those with exogenously added b(5). Stoichiometric analysis o f the rates of NADPH oxidation and product and H2O2 formation showed t hat the sum of the HCHO (product) and H2O2 formed could not account fo r the amount of NADPH utilized which is consistent with a pathway lead ing to H2O formation. The presence of substrate did not significantly affect the rates of either NADPH oxidation or H2O2 formation. Extra ad ded b(5) significantly decreased the rate of NADPH utilization and H2O 2 formation by approximately 63 and 86%, respectively, with or without substrate in the incubations. The results show that exogenous b(5) de creased H2O2 and H2O formation by an amount which could account for th e extra reducing equivalents needed for the increased rate of product formation, suggesting a mechanism for the b(5)-dependent stimulation. (C) 1995 Academic Press, Inc.