Identification of the adrenocorticotropin and ginkgolide B-regulated 90-kilodalton protein (p90) in adrenocortical cells as a serotransferrin precursor protein homolog (adrenotransferrin).
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.
Amri
Hakima
H
Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20057, USA.
Li
Wenping
W
Drieu
Katy
K
Papadopoulos
Vassilios
V
D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003345:Corticosterone / Q000096:biosynthesis; D004224:Diterpenes / Q000494:pharmacology*; D046934:Ginkgolides; D007501:Iron / Q000378:metabolism; D033862:Iron-Binding Proteins / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification*; D007783:Lactones / Q000494:pharmacology*; D008297:Male; D058950:Melanoma-Specific Antigens; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000276:immunology; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014168:Transferrin / Q000031:analogs & derivatives*
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D004224:Diterpenes; D046934:Ginkgolides; D033862:Iron-Binding Proteins; D007783:Lactones; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D011498:Protein Precursors; D014168:Transferrin; C483681:adrenotransferrin, rat; D000324:Adrenocorticotropic Hormone; C045856:ginkgolide B; D007501:Iron; D003345:Corticosterone