title: Characterization of corticotropin-releasing hormone binding protein in human plasma by chemical cross-linking and its binding during pregnancy.
abstract: A human plasma CRH-binding protein (CRH-BP) was identified and characterized by chemical cross-linking of 125I-Tyr-hCRH to human plasma using disuccinimidyl suberate. The apparent mol wt of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 43,000. The mol wt was slightly lower in the nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the mol wt of 125I-Tyr-CRH, the BP appeared to have a mol wt of approximately 38,000. Binding was specific since the appearance of the 43,000 dalton band was not affected by unlabeled ACTH, vasopressin, serum albumin, or gamma-globulin, but was inhibited by unlabeled hCRH dose dependently. Pretreatment of plasma with 0.1 mol/L HCl, 0.01 mol/L NaOH, 10 mmol/L dithiothreitol, or trypsin before cross-linking abolished its ability to bind 125I-Tyr-hCRH. Rat, rabbit, or goat plasma or human cerebrospinal fluid did not bind 125I-Tyr-CRH. It is unlikely that CRH-BP is a CRH receptor, because the estimated mol wt of the CRH-BP is smaller than the reported size of CRH receptors, and the CRH-BP did not bind to ovine CRH. The binding of 125I-Tyr-CRH to CRH-BP decreased in the third trimester of pregnancy, when plasma CRH levels were markedly elevated. However, after dissociating endogenous CRH from the CRH-BP, the binding was almost the same as in nonpregnant subjects. In addition, CRH-BP inhibited CRH-induced ACTH secretion from cultured rat anterior pituitary cells. We conclude that most of the increased plasma CRH found in pregnant women is bound to CRH-BP, and so is inactive, therefore plasma ACTH levels do not increase to above the normal range.
authors.affiliation: Department of Medicine, Tokyo Women's Medical College, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Suda
authors.forename: M
authors.initials: M
authors.lastname: Iwashita
authors.forename: F
authors.initials: F
authors.lastname: Tozawa
authors.forename: T
authors.initials: T
authors.lastname: Ushiyama
authors.forename: N
authors.initials: N
authors.lastname: Tomori
authors.forename: T
authors.initials: T
authors.lastname: Sumitomo
authors.forename: Y
authors.initials: Y
authors.lastname: Nakagami
authors.forename: H
authors.initials: H
authors.lastname: Demura
authors.forename: K
authors.initials: K
authors.lastname: Shizume
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000328:Adult; D000818:Animals; D001667:Binding, Competitive; D001798:Blood Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000097:blood* / Q000502:physiology; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000097:blood*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008297:Male; D010903:Pituitary Gland, Anterior / Q000378:metabolism; D011247:Pregnancy / Q000097:blood*; D011485:Protein Binding; D011863:Radioimmunoassay; D051381:Rats
chemical_list: D001798:Blood Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D000324:Adrenocorticotropic Hormone; D003346:Corticotropin-Releasing Hormone