Molecular identification of adrenal inner zone antigen as a heme-binding protein.
The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25-Dx and membrane-associated progesterone receptor. IZA was purified as a glutathione S-transferase-fused or His(6)-fused protein, and its molecular properties were studied. The UV-visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high-spin type. Analysis of the heme indicated that it is of the b type. Site-directed mutagenesis studies were performed to identify the amino-acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99-K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21-hydroxylation reaction was investigated in COS-7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21-hydroxylase activity.
Min
Li
L
Department of Molecular Physiological Chemistry, Graduate School of Medicine, Osaka University, Japan.
Strushkevich
Natallia V
NV
Harnastai
Ivan N
IN
Iwamoto
Hiroko
H
Gilep
Andrei A
AA
Takemori
Hiroshi
H
Usanov
Sergey A
SA
Nonaka
Yasuki
Y
Hori
Hiroshi
H
Vinson
Gavin P
GP
Okamoto
Mitsuhiro
M
D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000941:Antigens / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003080:Cold Temperature; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011980:Receptors, Progesterone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D015383:Zona Fasciculata / Q000166:cytology / Q000378:metabolism; D015385:Zona Reticularis / Q000166:cytology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D008565:Membrane Proteins; C486424:Pgrmc1 protein, rat; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D006639:Histidine; D008156:Luciferases; D005982:Glutathione Transferase