Dynamic mobility of genetically expressed fusion protein between cytochrome P4501A1 and NADPH-cytochrome P450 reductase in yeast microsomes. |
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Authors: | M Yamada Y Ohta T Sakaki Y Yabusaki H Ohkawa S Kawato |
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Affiliation: | Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Japan. |
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Abstract: | A fusion protein of rat liver CYP1A1 with NADPH-cytochrome P450 reductase was expressed genetically in yeast microsomal membranes. This flavo-cytochrome is active in 6-hydroxylation of zoxazolamine. Rotational diffusion of the fusion protein was examined by observing the flash-induced absorption anisotropy r(t) of the P450.CO complex. Theoretical analysis of r(t) was performed based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 2 ms to a time-independent value r(3). Forty percent of the fusion protein rotated with a rotational relaxation time phi of 1.35 ms. Treatment with high salt increased the mobile population of the fusion protein to 62% with phi = 0.96 ms. The mobile population of the fusion protein is close to that of CYP1A1 coexpressed with the P450 reductase and greater than that of CYP1A1 alone [Iwase et al. (1991) Biochemistry 30, 8347-8351]. The large mobile population of the fusion protein provides evidence that CYP1A1 is mobilized by forming associations with P450 reductase in microsomal membranes. |
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