Molecular identification of adrenal inner zone antigen as a heme‐binding protein

• Published in: The FEBS journal Vol. 272; no. 22; pp. 5832 - 5843
• Main Authors: Min, Li, Strushkevich, Natallia V., Harnastai, Ivan N., Iwamoto, Hiroko, Gilep, Andrei A., Takemori, Hiroshi, Usanov, Sergey A., Nonaka, Yasuki, Hori, Hiroshi, Vinson, Gavin P., Okamoto, Mitsuhiro
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-11-2005; Blackwell Publishing Ltd
• Subjects: Adrenal Cortex - cytology; Adrenal Cortex - metabolism; Adrenal glands; adrenal inner zone antigen; Amino Acid Sequence; Animals; Antibodies, Monoclonal - metabolism; Antigens; Antigens - metabolism; Carrier Proteins - analysis; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cercopithecus aethiops; Cold Temperature; COS Cells; Electron Spin Resonance Spectroscopy; Enzymes; Escherichia coli - genetics; Genes, Reporter; Glutathione Transferase - metabolism; HeLa Cells; Hemeproteins - analysis; Hemeproteins - chemistry; Hemeproteins - metabolism; heme‐binding protein; Histidine - chemistry; Humans; Luciferases - metabolism; Membrane Proteins; membrane‐associated progesterone receptor; Microscopy, Fluorescence; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptides; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Rats; Receptors, Progesterone - chemistry; Receptors, Progesterone - genetics; Receptors, Progesterone - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Rodents; Sequence Homology, Amino Acid; Spectrophotometry, Ultraviolet; steroidogenesis; Zona Fasciculata - cytology; Zona Fasciculata - metabolism; Zona Reticularis - cytology; Zona Reticularis - metabolism; zonae fasciculata and reticularis
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2005.04977.x

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 His6‐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.