Abstract
The detergent 1-O-n-octyl-beta-D-glucopyranoside (octylglucoside) was found to replace the phospholipid requirement in the demethylation of benzphetamine by cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase purified from phenobarbital-treated rabbit liver. At low enzyme concentration (0.1 microM) in the absence of glycerol and phosphate, the maximum rate of benzphetamine-specific NADPH oxidation was approximately 35% of that observed in the presence of dilauroylglyceryl-3-phosphoryl choline. At higher enzyme concentration (2.5 microM) and in the presence of 0.15 M phosphate, 20% glycerol, octylglucoside was as effective as phospholipid in stimulating the production of formaldehyde from benzphetamine. The detergent concentration required for maximal enzymatic activity was 2.5-4.0 g/liter, depending on the cytochrome preparation used. At higher octylglucoside concentrations (5-7 g/liter), activity decreased to zero, although neither enzyme appeared to be irreversibly denatured at these detergent concentrations. Sedimentation equilibrium experiments with P-450LM2 alone or in the presence of equimolar reductase showed that increasing octylglucoside levels promoted disaggregation of the cytochrome. Pentamers and hexamers predominated at detergent concentrations where maximal activity was observed, while higher levels of detergent where activity was absent produced cytochrome dimers and, ultimately, monomers. The reductase was monomeric at detergent levels between at least 3 and 7 g/liter. Moreover, both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450LM2 and its reductase was not formed at octylglucoside concentrations where high activity was evident. These results are consistent with a model of P-450/reductase interaction in which functional aggregates of three to six cytochrome polypeptides move laterally in the microsomal membrane and interact with the reductase by random collision.
Highlights
Inhibitionof ethylmorphine N-demethylation wheremaximalactivity was observed, while higher by mersalyl in rat liver microsomes led Franklin and Estalevels of detergent where cytochrome dimers and, ualtcitmivaitteylymw, oansomabesresn. tThperored-ucesbderovoekral(4P)-4to50prmooploesceutlehsa t a single reductase molecule services withinone multicomponentcomplex, ductase wasmonomeric at detergentlevels betweenat least 3 and 7 g/liter. Both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450m2 and its reductase was not formed a t octylglucoside concentrations and that electron transfer does not occur between different complexes
Assays-The concentration of P-450LM2 was estimated by the CO difference spectral method [19].P-450Lw2oxidase activity was determined with d-benzphetamine as substrate by measuring the rate of NADPH oxidation or the production of HCHO
The following data demonstrate that the nonionic detergent octylglucoside can replace the phospholipid requirement for benzphetamine N-demethylation as measured both by NADPH oxidation in the standard assay system (Fig. 1) and by HCHO production under conditions of protein concentration, buffer, and temperature which were used for subsequent hydrodynamic and detergent-binding measurements (Fig. 2)
Summary
TThperored-ucesbderovoekral(4P)-4to50prmooploesceutlehsa t a single reductase molecule services withinone multicomponentcomplex, ductase wasmonomeric at detergentlevels betweenat least 3 and 7 g/liter Both gel filtration and sedimentation equilibrium experiments demonstrated that a stable complex between P-450m2 and its reductase was not formed a t octylglucoside concentrations and that electron transfer does not occur between different complexes. A similar conclusion was reached by Peterson et al [5] based on a study of the temperature dependenceof the reduction of microsomal P-450 by NADPH, and by Stier and where high activity was evident These resaurltescon- Sackmann [6] from a determination of the reduction kinetics sistent with a model of P-450/reductase interaction in of a membrane-soluble, spin-labeled fatty acid radical. Which functional aggregates of three to sicxytochrome On the other hand, experimentsof Duppel and Ullrich [7], polypeptides move laterally in the microsomal mem- Yang ( 8 ) , Taniguchi et al [9], and Ingelman-Sundberg and brane and interactwith the reductase by random col- Johansson [10]have been interpreted asbeing consistent with lision
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