Membrane topology of guinea pig cytochrome P450 17 alpha revealed by a combination of chemical modifications and mass spectrometry.
Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH-cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17alpha was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17alpha was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 17alpha and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17alpha's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N- and C-terminal domains and the putative F-G loop were concluded to be in or near the membrane-binding domains of P450 17alpha.
Izumi
Shunsuke
S
Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima 739-8526, Japan.
Kaneko
Hiroshi
H
Yamazaki
Takeshi
T
Hirata
Toshifumi
T
Kominami
Shiro
S
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D005998:Glycine / Q000378:metabolism; D006168:Guinea Pigs; D007425:Intracellular Membranes / Q000737:chemistry* / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin
D003902:Detergents; D008563:Membrane Lipids; D008565:Membrane Proteins; D010446:Peptide Fragments; D011510:Proteolipids; C020485:proteoliposomes; D013254:Steroid 17-alpha-Hydroxylase; D014357:Trypsin; D005998:Glycine