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

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 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.

Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

“…There is additional evidence for regulation of rat PGRMC1 (also known as Vema, IZA, 25-Dx, and sometimes mPR) by progesterone and involvement of PGRMC1 in cellular, non-genomic progesterone responses (37)(38)(39)(40)(41)(42). However, progesterone binding by rat PGRMC1, was later shown to be nonspecific and weak by Min et al (5). Instead, they found that rat PGRMC1 bound heme and activated the cytochrome P 450 , CYP21, a key enzyme of progesterone biosynthesis, similar to the biological function proposed by Hughes et al (8).…”

“…In addition, since the ferrous heme does not bind well to Dap1p, the heme would likely dissociate during redox catalysis (35). Finally, attempts to reduce the rat Dap1p homologue, IZA, by P 450 reductases and cytochrome b 5 have been unsuccessful in vitro (9).…”

Progesterone Receptor Membrane Component 1 Modulates Human Cytochrome P450 Activities in an Isoform-Dependent Manner

“…PGRMC1 is widespread in eukaryotes from yeast to human. Despite its name, it is controversial whether progesterone binds to PGRMC1 (Min et al, 2005;Peluso et al, 2008b), and its function has not been fully understood. PGRMC1 is expressed in many tissues including liver, kidney, brain, breast, and adrenals (Cahill, 2007;Lösel et al, 2008).…”

Suppression of the ecdysteroid‐triggered growth arrest by a novel Drosophila membrane steroid binding protein

“…In general, dimers can be separated under reducing condition. But the vertebrate PGRMC1 has been reported to form the homodimer even in reducing condition, suggesting the homodimer was linked covalently [9,18]. Dm_MSBP also may thus form a homodimer, and this dimerization may be involved in the function of Dm_MSBP at the beginning of metamorphosis.…”