Rotation and membrane topology of genetically expressed methylcholanthrene-inducible cytochrome P-450IA1 lacking the N-terminal hydrophobic segment in yeast microsomes.
A modified rat liver cytochrome P-450IA1, lacking amino acids 2-30, a proposed membrane anchor for cytochrome P-450, was expressed genetically in yeast microsomal membranes. This truncated cytochrome is practically active in the deethylation of 7-ethoxycoumarin. A full-length cytochrome P-450IA1 was also expressed in yeast microsomes. Rotational diffusion of P-450IA1 was examined by observing the flash-induced absorption anisotropy r(t) of the P-450.CO complex. The anisotropy decayed to a time-independent value within a 2-ms time range. Since the decay curve has the characteristics of a slow rotation of membrane-embedded cytochrome, the theoretical analysis of r(t) was performed based on a "rotation-about-membrane normal" model. 41% of the shortened P-450IA1 was rotating with the rotational relaxation time phi of 1020 microseconds, whereas 27% of the full-length P-450IA1 was mobile with phi = 1101 microseconds. The high salt treatment did not remove the shortened cytochrome from the membrane and also did not drastically weaken the interactions of the cytochrome with the membrane, as judged from the slow rotation characteristics (phi = 830 microseconds). These results demonstrate that the N-terminal shortened P-450IA1 is incorporated properly into the yeast microsomal membrane and that the N-terminal hydrophobic segment is not solely responsible for attachment to the membrane, providing evidence that additional segments of P-450IA1 are involved in the membrane binding.
Ohta
Y
Y
Institute of Physics, College of Arts and Sciences, University of Tokyo, Japan.
Sakaki
T
T
Yabusaki
Y
Y
Ohkawa
H
H
Kawato
S
S
D000818:Animals; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004058:Diffusion; D004790:Enzyme Induction; D005454:Fluorescence Polarization; D015870:Gene Expression; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D008433:Mathematics; D008748:Methylcholanthrene / Q000494:pharmacology*; D008861:Microsomes / Q000201:enzymology*; D008962:Models, Theoretical; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D013997:Time Factors
D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System