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.
Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Japan.
M
M
Yamada
Y
Y
Ohta
T
T
Sakaki
Y
Y
Yabusaki
H
H
Ohkawa
S
S
Kawato
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