Studies on the Role of Basic Amino Acid Residues of ACTH Peptide in Steroidogenesis by Isolated Adrenal Cells
1972 Volume 71 Issue 6 Pages 1029-1041
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1972 Volume 71 Issue 6 Pages 1029-1041
The biological significance of the basic amino acid residues between positions 15 and 18 of the N-terminal region of ACTH peptide was investigated by assaying the steroidogenic activities of various synthetic ACTH peptides, mainly in isolated cells of rat adrenals.
1. The tetradecapeptide Gly1-(1-14) ACTH exhibited about 1/3, 000 of the activity of the octadecapeptide (1-18) ACTH on a molar basis. The concentration-response curves of the two peptides were parallel and there was no difference in the maximum steroidogenic levels or the slopes of the curve.
2. Contrary to results in vivo, (1-18) ACTH and the carboxyl terminal-blocked amide analog, (1-18) ACTH-NH2 were equally potent when assayed in isolated adrenal cells.
3. Change from L-phenylalanine to the D-isomer at position 7 of the octadecapeptide, β-Ala1, Orn15-(1-18) ACTH-NH2 resulted in marked decrease in activity. The log-concentration response curve of β-Ala1, D-Phe7, Orn15-(1-18) ACTH-NH2 was shifted to a higher concentration range and its activity was about 3% of that of the parent L-peptide. Moreover, on simultaneous addition of both peptides, no competitive in-hibition was seen under the conditions examined.
4. The octapeptide (11-18) ACTH-NH2 potentiated ACTH-induced steroidogenesis both in vivo and in isolated adrenal cells. Kinetic experiments revealed two types of interaction, non-specific and specific, between ACTH and adrenal cells: the former involves the positive charges on the peptide and the latter is due to two portions distinguished as “active” and “binding” sites. (11-18) ACTH-NH2 decreased the amount of ACTH adsorbed on non-specific sites of adrenal cells and resulted in enhanced corticosterone production.
These results indicate that the active site of ACTH peptide is located within the first 14 amino acid residues of the N-terminal region of ACTH and that elongation with the sequence Lys-Lys-Arg-Arg at positions 15 to 18 increases the affinity of ACTH peptide for adrenal cells. The correct configuration of amino acid residues in the N-terminal region is also important for the affinity of ACTH peptide for adrenal cells.
