Membrane topology of cytochrome P450 2B4 in Langmuir-Blodgett monolayers.
Using Langmuir-Blodgett monolayers of both phosphatidylethanolamines and phosphatidylcholines as membrane mimics, we have examined the topology of cytochrome P450 2B4 anchoring. The interaction of wild-type P450 2B4 with phosphatidylethanolamine monolayers can be characterized as a biphasic reaction, with the initial fast phase explained by the specific insertion of membrane-spanning segments of the protein into the monolayer. Injection of cytochrome b5 (b5) beneath dipalmitoyl-phosphatidylcholine monolayers also resulted in biphasic kinetics. Regardless of the nature of the lipid employed, neither a truncated cytochrome P450 2B4 (P450 2B4 Delta2-27) lacking the amino-terminal hydrophobic residues widely believed to be the major transmembrane segment nor a soluble b5 fragment (Deltab5) lacking its carboxy terminus anchor exhibit the fast-phase behavior characteristic of specific insertion. To further characterize the membrane topology of P450 2B4, its insertion area in DPPE monolayers was measured and analyzed with use of the Gibbs equation for adsorption at an interface. The mean molecular insertion area derived from isotherms of P450 2B4 in a DPPE monolayer at a pressure of 19 mN/m, 680 +/- 95 A2 is large enough to accommodate two to four transmembrane helices. The large insertion area and the fact that the truncated cytochrome retains as much as 30% of its membrane localization when expressed in Escherichia coli (Pernecky, S. J., Larson, J. R., Philpot, R. M., and Coon, M. J. (1993) Proc. Natl. Acad. Sci. USA 90, 2651-2655) suggest that this cytochrome is not deeply embedded but that other regions, in addition to the amino-terminal 26 residues, may be involved in the interaction of cytochrome P450 with the membrane.
Shank-Retzlaff
M L
ML
Beckman Institute of Advanced Science and Technology and Department of Chemistry, University of Illinois, 405 North Mathews Avenue, Urbana, Illinois, 61801, USA.
Raner
G M
GM
Coon
M J
MJ
Sligar
S G
SG
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry; D008560:Membrane Fluidity; D008567:Membranes, Artificial; D008862:Microsomes, Liver; D009211:Myoglobin / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011312:Pressure; D011817:Rabbits; D051381:Rats; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000378:metabolism
D008567:Membranes, Artificial; D009211:Myoglobin; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D015060:1,2-Dipalmitoylphosphatidylcholine; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase