Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adrenal Cortex / cytology
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Adrenal Cortex / metabolism*
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Amino Acid Sequence
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Animals
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Antibodies, Monoclonal / metabolism
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Antigens / metabolism*
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COS Cells
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Carrier Proteins / analysis
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Chlorocebus aethiops
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Cold Temperature
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Electron Spin Resonance Spectroscopy
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Escherichia coli / genetics
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Genes, Reporter
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Glutathione Transferase / metabolism
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HeLa Cells
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Heme-Binding Proteins
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Hemeproteins / analysis
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Hemeproteins / chemistry
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Hemeproteins / metabolism*
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Histidine / chemistry
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Humans
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Luciferases / metabolism
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Membrane Proteins
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Microscopy, Fluorescence
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Protein Binding
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Rats
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Receptors, Progesterone / chemistry
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Receptors, Progesterone / genetics
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Receptors, Progesterone / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Sequence Homology, Amino Acid
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Spectrophotometry, Ultraviolet
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Zona Fasciculata / cytology
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Zona Fasciculata / metabolism
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Zona Reticularis / cytology
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Zona Reticularis / metabolism
Substances
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Antibodies, Monoclonal
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Antigens
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Carrier Proteins
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Heme-Binding Proteins
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Hemeproteins
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Membrane Proteins
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Pgrmc1 protein, rat
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Receptors, Progesterone
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Recombinant Fusion Proteins
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Histidine
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Luciferases
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Glutathione Transferase