Identification of a protein in adrenal particulates that binds adrenocorticotropin specifically and with high...

This paper is concerned with the identification and isolation in cross-linked form of a protein of bovine adrenal cortical particulates that binds the ACTH probe 125I-[Phe2,Nle4,DTBct25]ACTH-(1-25) amide specifically, reversibly, and with high affinity. This protein may well represent the long sought, adenylate cyclase-linked, low affinity ACTH receptor or a portion thereof. Evaluation of the binding data by Scatchard analysis afforded a linear plot corresponding to a dissociation constant of 2.7 X 10(-9) M with a single class of binding sites. Competitive binding studies using nonradioactive ACTH analogs served to establish the specificity of the binding. ACTH-(1-24), was the most active competitor, followed by [Gln5]ACTH-(1-20) amide, [Gln5,Phe9] ACTH-(1-24), and ACTH-(11-20) amide, the weakest binder of the group. These findings correlate well with the ability of the peptides to stimulate cAMP formation in bovine adrenal cortical cells, i.e. ACTH-(1-24) greater than [Gln5]ACTH-1-20) amide greater than [Gln5,Phe9]ACTH-(1-24). ACTH-(11-20) amide is biologically inactive but inhibits ACTH-(1-24)-stimulated adenylate cyclase with a 50% inhibition ratio of 400:1. Nonspecific binding was suppressed by inclusion in the incubates of the protease inhibitors pepstatin, bacitracin, and benzamidine. The binding protein was cross-linked to the radioactive probe with disuccinimidyl suberate with a high cross-linking yield. The cross-linked material was solubilized with sodium dodecyl sulfate (SDS), and the 100,000 X g supernatant was subjected to SDS-polyacrylamide gel electrophoresis, followed by a autoradiography. The gel showed the presence of a band corresponding to an apparent mol wt of 43,000 (assuming a molecule of ligand bound). This band was absent when cross-linking was performed in the presence of unlabeled ACTH-(1-24). Similar results were obtained when cross-linking was performed with dithiobis (succinimidyl)propionate or ethyleneglycolbis (succinimidyl)succinate. The soluble cross-linked material bound to a column of succinoylavidin Sepharose and could be eluted with guanidinium chloride at pH 1.5. SDS-polyacrylamide gel electrophoresis and autoradiography of the affinity-purified material afforded the same pattern as the unpurified material; however, considerably more radioactivity was present in the high mol wt region of the gels.