Preparation and characterization of [N alpha-(4-azido-2-nitrophenyl)Ala1,Tyr36]-parathyroid hormone related peptide (1-36)amide: a high-affinity, partial agonist having high cross-linking efficiency with its receptor on ROS 17/2.8 cells.
The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide [[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-1 and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the alpha-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent KdS of 1 X 10(-9) M and 0.6 X 10(-9) M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)
Jüppner
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Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Abou-Samra
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Uneno
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Keutmann
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HT
Potts
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JT
Segre
G V
GV
D000345:Affinity Labels / Q000138:chemical synthesis; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D007700:Kinetics; D010281:Parathyroid Hormone / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D011506:Proteins / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D018016:Receptors, Parathyroid Hormone
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; C066362:parathyroid hormone-related protein (1-36)amide, N(alpha)-(4-azido-2-nitrophenyl)-Ala(1)-Tyr(36)-