Synthesis of a tritiated human growth hormone releasing peptide.

Tritium-labeled growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 was synthesized by tritium-halogen exchange on the precursor His-5,7-Br2-D-Trp-Ala-Trp-D-Phe-Lys-NH2. The radiolabeled peptide had a specific activity of 29 Ci/mmol and a radiochemical purity of 95%. The tritium label was shown by 3H NMR to be located mostly at the expected 5,7-positions of the indole nucleus in the D-Trp residue. The dibromopeptide was prepared by solid-phase peptide synthesis, employing racemic 5,7-Br2-Trp as a building block and separation of the resulting epimeric mixture by HPLC. 5,7-Br2-Trp was prepared by a five-step sequence beginning with 2,4-dibromoaniline. The use of anisole as an additive in the HF resin/peptide cleavage was rejected because anisole was found to undergo electrophilic substitution of the dibromoindole nucleus; a modified HF deprotection/cleavage procedure was developed and used instead.

A radiometric assay for HIV-1 protease.

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

Binding of a growth hormone releasing hexapeptide to specific hypothalamic and pituitary binding sites.

The drug SK&F 110679 (His-D-Trp-Ala-Trp-D-Phe-LysNH2), is an enkephalin-derived hexapeptide, which specifically releases growth hormone in a wide variety of species in vivo and in vitro. Previous binding studies, using ligands which are specific for mu and delta opioid binding sites, demonstrated an inverse relationship between the opioid binding potency and the potency in releasing growth hormone of a series of peptides related to SK&F 110679. In an attempt to understand its mode of action better, a binding assay for the peptide was established using a ligand which had been tritium labelled at the D-Trp2 residue. Membrane fragments from both the hypothalamus and anterior pituitary tissue were found to contain sites to which [3H]SK&F 110679 reversibly and saturably bound. The binding curves for [3H]SK&F 110679 to membrane fragments of both hypothalamus and anterior pituitary were resolved into two binding components with the computer program LIGAND. The Kd's obtained were in the 10(-8) M and 10(-5) M range. The relationship of these binding sites to the growth hormone-releasing activity of the peptide was explored by examining the relationship between the binding and potency in releasing growth hormone of a series of peptides related to SK&F 110679. For sites in both the hypothalamus and pituitary, a significant correlation between binding and the release of growth hormone was obtained. Thus, these binding sites appeared to be involved in the release of growth hormone by SK&F 110679-related peptides.(ABSTRACT TRUNCATED AT 250 WORDS)