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

Li Min; 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

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.

MONOCLONAL antibodies; ANTIGENS; HEME; HEMOGLOBINS; CARRIER proteins; PROGESTERONE receptors

FEBS Journal, 2005, Vol 272, Issue 22, p5832

1742-464X

Academic Journal

10.1111/j.1742-4658.2005.04977.x