Identification of the adrenocorticotropin and ginkgolide B-regulated 90-kilodalton protein (p90) in adrenocortical cells as a serotransferrin precursor protein homolog (adrenotransferrin).

Abstract

Two-dimensional electrophoresis (2D-PAGE) of metabolically labeled adrenocortical proteins, identified a series of spots at a molecular size of 90 kDa [isoelectric point (pI) 6.8-7.1; p90] that was induced by ACTH, but whose intensity was reduced in cells obtained from animals treated with an extract of Ginkgo biloba (EGb 761) and its purified component ginkgolide B (GKB). We have now identified p90. GKB (2 mg/kg x d, i.p.) was administered to rats for 8 d. Adrenocortical cells were prepared and stimulated with ACTH for 3 h. Cells obtained from saline-treated rats responded to ACTH by producing high amounts of corticosterone, an effect that was inhibited in cells obtained from GKB-treated animals. Samples were fractionated by 2D-PAGE and matrix-assisted laser desorption ionization mass spectrometry analysis of the p90 spots isolated from the gels revealed sequences sharing identity with the serotransferrin precursor protein. Further PCR screening of a rat adrenal cDNA library identified a sequence with a high degree of homology (79%) to serotransferrin precursor protein, and a lesser degree to rat transferrin (54%) and human melanotransferrin (32.8%). p90, in 2D-PAGE immunoblots, was also recognized by a monoclonal antibody raised against human 97-kDa melanotransferrin. Iron binding assays with rat adrenal cortex extracts further identified a 90-kDa melanotransferrin immunoreactive protein binding iron, suggesting that the identified protein, which we name "adrenotransferrin," may have iron-binding activity. This is the first report describing the presence of a serotransferrin precursor protein homolog belonging to the transferrin family and sharing epitopes with melanotransferrin in the adrenal, its induction by ACTH, and sensitivity to GKB.