The synthesis and biological activity of four novel fluorescent vasopressin analogs

W. H. Lutz, J. M. Londowski, R. Kumar

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

We synthesized and tested the biological properties of four fluorescent vasopresin analogs: [1-(2-mercapto)propionic acid]-8-lysine-N6-5-dimethylamino-naphthalene-1-sulfonyl vasopressin (D-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxyfluorescein vasopressin (F-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-2-N-methylanthranilamide vasopressin (MA-MLVP), and [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxytetramethylrhodamine vasopressin (R-MLVP). All fluorescent analogs were prepared by coupling the appropriate fluorochrome to the 6-amino group of the lysine residue in [1-(2-mercapto)propionic acid]-8-lysine vasopressin (MLVP) which was synthesized by the Merrifield solid-phase method. The structures of high performance liquid chromatography-purified MLVP and the fluorescent analogs were confirmed by fast atom bombardment mass spectrometry. F-MLVP, MA-MLVP, and R-MLVP effectively competed for 8-arginine vasopressin (AVP)-binding sites in canine renal plasma membranes and on the surface of porcine kidney cells (LLC-PK1, ATCC CL101). Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 32, 8.8, and 26 nM, respectively, were calculated from the results of competition binding assays conducted with membranes. D-MLVP did not bind to plasma membranes. Dissociation constants for F-MLVP, MA-MLVP, and B-MLVP of 390, 38, and 160 nM, respectively, were calculated from the results of competition binding assays conducted with cells. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10-6 M increased adenylate cyclase activity in canine renal plasma membranes to values 2.4, 2.9 and 2.6 times that of basal activity, respectively. A maximally active concentration of AVP (1 μM) increased adenylate cyclase activity in canine renal plasma membranes to a value 2.7 times that of basal activity. D-MLVP did not stimulate adenylate cyclase activity. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10-6 M increased the cAMP content of porcine kidney cells from a basal level of 43 to 267, 160, and 469 pmol/mg of cell protein, respectively. Specific binding of these fluorescent analogs to receptors on the surface of LLC-PK1 cells was observed by fluorescence microscopy. These observations indicate that F-MLVP, MA-MLVP, and R-MLVP are biologically active fluorescent vasopressin analogs which are well-suited to the study of renal vasopressin receptors by fluorescence microscopy.

Original languageEnglish (US)
Pages (from-to)4657-4663
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number8
StatePublished - 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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