Dynamic mobility of genetically expressed fusion protein between cytochrome P4501A1 and NADPH-cytochrome P450 reductase in yeast microsomes.
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.
Yamada
M
M
Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Japan.
Ohta
Y
Y
Sakaki
T
T
Yabusaki
Y
Y
Ohkawa
H
H
Kawato
S
S
D000818:Animals; D002417:Cattle; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis / Q000235:genetics*; D004058:Diffusion; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D009251:NADPH-Ferrihemoprotein Reductase; D011189:Potassium Chloride / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012399:Rotation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D011189:Potassium Chloride; D019363:Cytochrome P-450 CYP1A1; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase