Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.     

Subcellular localization of thyrotropin-releasing hormone (TRH)- and Neuropeptide Y (NPY)-like immunoreactivity in the neurointermediate lobe of the frog pituitary

The presence of thyrotropin-releasing hormone (TRH) and neuropeptide Y (NPY) has been demonstrated in the neural lobe and in the intermediate lobe of the frog pituitary by immunocytochemistry on ultrathin sections of neurointermediate lobes obtained by cryoultramicrotomy. In the neural lobe, separate populations of TRH- or NPY-immunoreactive nerve fibers were observed. Both neuropeptides were contained in dense-core secretory vesicles about 200 nm in diameter. In intermediate lobe cells, TRH- and NPY-like immunoreactivities were observed in the cytoplasmic matrix and more sparsely in secretory granules. Occasionally, immunoreactive TRH could be visualized at the plasma membrane level. In the nucleus, both peptides were detected in the euchromatin, in the vicinity of the heterochromatin and in the nucleolus. Conversely, gonadotropin-releasing hormone-like immunoreactivity could not be detected. These results provide immunocytological evidence for the presence of endogenous TRH and NPY in frog melanotrophs indicating that these peptides may participate in the regulation of intermediate lobe secretion.     

Apparent Co-Sequestration of Immunoreactive Corticotropin, α-Melanot opin, and γ-Lipotropin in Hypothalamic Granules

Abstract: In the present study, the question of whether immunoreactive α-melanotropin (α-MSH₁), corticotropin (ACTH₁), and β-melanotropin (β-MSH₁) are co-sequestered in hypothalamic granules of adult male rats was addressed. When a 900 ×g supernatant fluid prepared from a hypothalamic homogenate was fractionated on continuous sucrose density gradients under non-equilibrium Conditions, two populations of particles containing α-MSH₁, ACTH₁, or β-MSH₁ Were observed. However, when fractionated under equilibrium conditions, the two populations of particles containing α-MSH₁ ACTH₁, or β-MSH₁ were recovered as a single band. This sedimentation characteristic indicates that the particles containing a given peptide differ in size but are similar in density. In their sedimentation, the small particles containing α-MSH₁, ACTH₁, and β-MSH₁ are indistinguishable from granules containing α-MSH₁, whereas the large particles containing α-MSH₁ (ACTH₁, and β-MSH₁ are indistinguishable from synaptosomes containing α-MSH₁, β-MSH₁ had an apparent molecular weight (M.W.) of about 5,000, which is similar to that of γ-lipotropin. ACTH₁ was comprised of three species of molecules: big (M.W. ≥ 10,000), 5.7K (M.W. ≌ 5,700), and 4.5K (M.W. ≌ 4,500). Big ACTH was the predominant and 5.7K ACTH the minor component of ACTH₁ present in granules as well as in synaptosomes. These results are suggestive that α-MSH, ACTH and its precursors, and γ-lipotropin are co-sequestered in hypothalamic granules.     

Regulated Secretion of Pro-Opiomelanocortin Converting Enzyme and an Aminopeptidase B-Like Enzyme from Dispersed Bovine Intermediate Lobe Pituitary Cells

Coordinate secretion of two prohormone/proneuropeptide processing enzymes [pro-opiomelanocortin converting enzyme (PCE) and an aminopeptidase B-like enzyme (APBE)] and alpha-melanotropin (alpha-MSH) from bovine intermediate lobe pituitary cells was studied. Stimulation of secretion with 8-bromo-cyclic AMP produced significant increases in levels of immunoreactive alpha-MSH, PCE, and APBE. Treatment of cells with the dopaminergic agonist 2-bromo-alpha-ergocryptine resulted in significant decreases in secretion of alpha-MSH, PCE, and APBE. In neither case were there significant changes in levels of cytosolic lactic dehydrogenase or lysosomal beta-glucuronidase in the medium. The secreted PCE activity was shown to process frog and mouse pro-opiomelanocortin primarily to 23,000-Mr corticotropin (ACTH), 13,000-Mr ACTH, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin, products comparable to those synthesized by the mouse and frog intermediate lobe in situ. The secreted enzymatic activity had a pH optimum between 4.0 and 5.0, was strongly inhibited by pepstatin A, and had an inhibitor profile similar to the purified bovine intermediate lobe PCE. The secreted APBE activity cleaved Argo-[Met]-enkephalin to [Met]-enkephalin and had a pH optimum and inhibitor profile similar to that previously reported for an activity from purified secretory vesicle fractions of bovine intermediate and neural lobes. The coordinate regulated secretion of alpha-MSH and enzyme activities (PCE and APBE) strongly indicates their colocalization in the same secretory vesicle compartment within the cell. The characteristics of the two enzymes secreted in the medium paralleled those seen in the tissue and further support their role in pro-opiomelanocortin processing in vivo.     

Big form of gamma-melanotropin-like immunoreactivity in normal human plasma

Using a radioimmunoassay for gamma 3-melanotropin (gamma 3-MSH), gamma-melanotropin-like immunoreactivity (gamma-MSH-LI) was detected in plasma extracts of normal subjects before and after metyrapone administration. Plasma gamma-MSH-LI from four normal men rose significantly after a single oral administration of metyrapone (30 mg/kg of body weight). Gel chromatographic study of plasma extract after metyrapone administration showed a single peak of gamma-MSH-LI near the elution position of mouse 16K fragment, however smaller forms of gamma-MSH-LI were not detected.     

Pro-opiocortin processing in the pituitary: A model for neuropeptide biosynthesis

The biosynthesis of α-MSH, β-endorphin and ACTH in the pituitary is reviewed. These neuropeptides are synthesized from a common pro-protein, pro-opiomelanocortin. The pro-protein is cleaved intragranularly, at pairs of basic residues in the molecule to yield the respective peptide products. An unique, thiol protease (pro-opiocortin converting enzyme) and a carboxypeptidase B-like enzyme, both localized within pituitary secretory granules and having a pH optimum of 5–6, appear to be involved in the proteolytic processing of pro-opiomelanocortin.