Lipids

, Volume 43, Issue 12, pp 1127–1132

Studies of Membrane Topology of Mitochondrial Cholesterol Hydroxylases CYPs 27A1 and 11A1

  • Irina A. Pikuleva
  • Natalia Mast
  • Wei-Li Liao
  • Illarion V. Turko
Original Article

DOI: 10.1007/s11745-008-3234-x

Cite this article as:
Pikuleva, I.A., Mast, N., Liao, WL. et al. Lipids (2008) 43: 1127. doi:10.1007/s11745-008-3234-x

Abstract

Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in metabolism of cholesterol. CYP27A1 hydroxylates cholesterol at position 27 and, thus, initiates cholesterol removal from many extrahepatic tissues. CYP11A1 is a steroidogenic P450 that converts cholesterol to pregnenolone, the first step in the biosynthesis of all steroid hormones. We utilized a new approach to study membrane topology of CYPs 27A1 and 11A1. This approach involves heterologous expression of membrane-bound P450 in E. coli, isolation of the P450-containing E. coli membranes, treatment of the membranes with protease, removal of the digested soluble portion and extraction of the membrane-associated peptides, which are then identified by mass spectrometry. By using this approach, we found four membrane-interacting peptides in CYP27A1, and two peptides in CYP11A1. Peptides in CYP27A1 represent a contiguous portion of the polypeptide chain (residues 210–251) corresponding to the putative F-G loop and adjacent portions of the F and G helices. Peptides in CYP11A1 are from the putative F-G loop (residues 218–225) and the C-terminal portion of the G helix (residues 238–250). This data is consistent with those obtained previously by us and others and provide new information about the membrane topology of CYPs 27A1 and 11A1.

Keywords

CYP27A1 CYP11A1 Membrane topology Mass spectrometry analysis 

Abbreviations

CYP27A1

Cytochrome P450 27A1

CYP11A1

Cytochrome P450 11A1

MS

Mass spectrometry

TFA

Trifluoroacetic acid

MALDI

MS matrix assisted laser desorption ionization mass spectrometry

TOF

Time-of-flight

Copyright information

© AOCS 2008

Authors and Affiliations

  • Irina A. Pikuleva
    • 1
    • 2
  • Natalia Mast
    • 1
    • 2
  • Wei-Li Liao
    • 3
  • Illarion V. Turko
    • 3
  1. 1.Department of Ophthalmology and Visual SciencesCase Western Reserve University and University Hospitals Case Medical CenterClevelandUSA
  2. 2.Department of Pharmacology and ToxicologyUniversity of Texas Medical BranchGalvestonUSA
  3. 3.Center for Advanced Research in BiotechnologyNational Institute of Standards and Technology and University of Maryland Biotechnology InstituteRockvilleUSA

Personalised recommendations