Amino-terminal topology of thromboxane synthase in the endoplasmic reticulum.
The membrane topology of the NH2-terminal portion of human thromboxane synthase (TXS), a member of the cytochrome P450 superfamily, has been investigated. By sequence alignment, the first 6 residues of the mature TXS polypeptide are likely to form a distinctive "tail" structure not found in many other mammalian cytochromes P450 in the endoplasmic reticulum membrane. Peptides with either the ultimate 10 or 15 residues of the NH2 terminus of TXS were synthesized and used to produce site-directed antibodies. The resulting peptide antibodies were highly specific and recognized human TXS, as shown by binding assays and Western blot analysis. Binding of the peptide antibodies to recombinant TXS in transfected COS-1 and to endogenous TXS in THP-1 cells was analyzed by immunocytochemistry. Selective permeabilization of the plasma membrane to immunoglobulin was achieved with streptolysin O; general permeabilization, including the endoplasmic reticulum membrane, was accomplished with Triton X-100. Permeabilization of the plasma membrane was sufficient to produce binding of both peptide antibodies to their epitopes, indicating that the epitopes for both of the peptide antibodies were exposed on the cytoplasmic side of the endoplasmic reticulum membrane. The results with the peptide antibodies provide direct experimental evidence supporting the topological model for membrane-bound cytochrome P450 proposed by Nelson and Strobel (Nelson, D. R., and Strobel, H. W. (1988) J. Biol. Chem. 263, 6038-6050), in which the NH2 terminus is oriented toward the cytoplasmic side of the endoplasmic reticulum membrane.
Ruan
K H
KH
Vascular Biology Research Center, University of Texas Health Sciences Center, Houston 77030.
Wang
L H
LH
Wu
K K
KK
Kulmacz
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013930:Thromboxane-A Synthase / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D000906:Antibodies; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013930:Thromboxane-A Synthase