60 YEARS OF POMC: Purification and biological characterisation of melanotrophins and corticotrophins.

The remarkable conservation of the primary structures and anatomical location of dogfish α-melanocyte-stimulating hormone (MSH), corticotrophin-like intermediate lobe peptide (CLIP) and adrenocorticotrophic hormone (ACTH) compared with mammals reinforced the tissue-specific processing hypothesis of ACTH peptides in the pituitary gland. The cloning of dogfish pro-opiomelanocortin (POMC) led to the identification of δ-MSH and simultaneously revealed the high conservation of the γ-MSH sequence during evolution. These studies have also shown that ß-MSH is much less conserved during evolution and in some species is not even processed from ß-LPH. Human pro-γ-MSH potentiates the corticosteroidogenic activity of ACTH and peptides generated from its N-terminal, in particular big-γ-MSH, appear to have adrenal mitogenic activity. Human big-γ-MSH (from the zona intermedia) may also cause the adrenache. The review finishes with a cautionary note with regard to the misdiagnosis of the ectopic ACTH syndrome in which partial processing of ACTH can result in large concentrations of α-MSH and CLIP, which can interfere in the performance of two-site immunoassays, and the problem of the correct disulphide bridge arrangement in synthetic N-POMC peptides is also discussed.

Characterization of triacetyl-α-melanocyte-stimulating hormone in carp and goldfish.

A triacetyl form of α-melanocyte-stimulating hormone (MSH) was found in carp (Cyprinus carpio) and goldfish (Carassius auratus), by selective detection of mass profile for cell secretory granules using direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) analysis during the investigation of fish pituitaries. The structure of triacetyl-α-MSH in carp and goldfish was further analyzed using a collision-induced dissociation with electrospray ionization mass spectrometry, and determined to be N,O-diacetyl Ser as the N-terminal residue and O-acetyl Tyr at position 2. These modifications for α-MSH in carp and goldfish are structurally different from that of medaka hormone, in which [N,O-diacetyl Ser(1), O-acetyl Ser(3)]-α-MSH has been identified. The profiles of four α-MSH variants, des-, mono-, di- and tri-acetyl forms in goldfish and medaka pituitaries were also examined by direct tissue MALDI-TOF MS analysis, and the percentages as a total of α-MSH molecules were compared for fish reared in a white or black tank for 5 days. Among structural variants, diacetyl-α-MSH was the predominant form in goldfish and N-desacetyl-α-MSH in medaka, respectively. In both species, the relative level of the predominant form in the pituitary of white-adapted fish tended to be lower than that of black-adapted fish. In goldfish, no significant difference was observed in the relative content of triacetyl-α-MSH in both backgrounds, whereas the lowest content of triacetyl-α-MSH was found in black-adapted medaka. These preliminary data indicate that it is difficult to elucidate the relations between the physiological roles and acetylated pattern of α-MSH molecule, depending on species.

Isolation and characterization of melanotropins from lamprey pituitary glands.

Three peptides containing the melanotropin-core amino-acid sequence, YXMXHFRWG, were isolated from the pituitary glands of a modern representative of the most primitive vertebrates, the sea lamprey, Petromyzon marinus. MSH-A, a nonadecapeptide (NPELYQMNHFRWGQPPTHF), is free at both ends. MSH-B, an eicosapeptide (VQESADGYRMQHFRWGQPLP), is free at the N-terminus and amidated at the C-terminus. They differ strikingly from gnathostome MSHs in structure. The third peptide, with an apparent molecular weight of 15 kDa, was tentatively designated lamprey ACTH, based on a structural feature: the N-terminal 22-residue-MSH (SVSSPKYAMGHFRWGSPDKATI) is followed by four consecutive basic amino acids (RKRR) and a ACTH-like sequence (PVRPNTSDSPEIPDYAF--). MSH-B is 10 and 100 times more potent than alpha-MSH and MSH-A, respectively, in a frog skin assay in vitro, whereas the lamprey ACTH showed no melanotropic activity. Lamprey ACTH did, however, show corticotropic activity on the lamprey pronephric and mesonephric tissue.

Isolation and characterization of [Pro2]somatostatin-14 and melanotropins from Russian sturgeon, Acipenser gueldenstaedti Brandt.

A new form of somatostatin (SRIH), along with melanotropins (MSHs), was isolated from pituitaries of the Russian sturgeon Acipenser gueldenstaedti Brandt by gel filtration, ion exchange, and reversed-phase HPLC following acid-acetone extraction. The sturgeon SRIH consists of 14 amino acid residues and differs from mammalian SRIH-14 by the substitution Pro for Gly at position 2. Synthetic [Pro2]SRIH-14 was as potent as mammalian SRIH-14 in inhibiting release of growth hormone into medium from the organ-cultured pituitary of rainbow trout. Sturgeon alpha-MSH has the same amino acid sequence as those found in mammals. Sturgeon beta-MSH is composed of 17 amino acid residues, and its amino acid sequence is identical to the N-terminal 15 residues of salmon beta-MSH I and to the C-terminal 2 residues of mammalian beta-MSH.

Immunocytochemical localization and biochemical characterization of melanotropin-like peptides in the gonads of a protochordate.

The compound gonads of the protochordate ascidian Styela plicata were investigated by immunocytochemistry, HPLC, and radioimmunoassay to verify the presence of melanotropin-like peptides, alpha-MSH-like immunoreactivity is localized in the follicular cells and in the perinuclear cytoplasm of different types of ovaric follicles, as well as in the spermatogonia and spermatocytes of testicular lobules. The ascidian immunoreactive peptides occurring in the gonads consist of alpha-MSH and ACTH(1-13)-NH2 and their amounts are higher in summer than in winter.

Isolation and structural characterization of a novel peptide related to gamma-melanocyte stimulating hormone from the brain of the leech Theromyzon tessulatum.

This paper reports the purification of a novel pro-opiomelanocortin derivative peptide (a gamma-melanocyte stimulating hormone-like (gamma-MSH-like) molecule) from the brain of the leech Theromyzon tessulatum. After reverse-phase HPLC purification, the sequence of the gamma-MSH-like peptide (YVMGHFRWDKFamide) was established by a combination of automated Edman degradation, electrospray mass spectrometry measurement, enzymatic treatment and co-elution experiments in reverse-phase HPLC with synthetic peptides.

Isolation and structural characterization of peptides related to alpha- and gamma-melanocyte-stimulating hormone (MSH) from the frog brain.

Peptides that are derived from the processing of proopiomelanocortin were isolated in pure form from the brain of the frog Rana ridibunda. The primary structure of the most abundant of those peptides was established as: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. This amino acid sequence is identical to that of mammalian and frog pituitary alpha-melanocyte-stimulating hormone (MSH) and the peptide co-eluted with synthetic desacetyl alpha-MSH, indicating that it is COOH-terminally alpha-amidated. A second component, which exhibited a shorter retention time, co-eluted with the glycine-extended form of desacetyl alpha-MSH [ACTH(1-14)]. The primary structure of the third peptide isolated in pure form from the brain extract was established as: Lys-Tyr-Val-Met-Ser-His-Phe-Arg-Trp-Asn-Lys-Phe-NH2. This sequence corresponds to Lys-gamma 1-MSH as predicted from the nucleotide sequence of frog proopiomelanocortin. The presence of substantial amounts of desacetyl alpha-MSH and Lys-gamma 1-MSH in the frog brain supports the concept that, in amphibia, melanotropins may act as neurotransmitters and/or neuromodulators as well as hormonal peptides.

Detection and partial characterization of proopiomelanocortin-related end-products from the pars intermedia of the toad, Bombina orientalis.

Steady-state analyses were performed on the proopiomelanocortin (POMC)-related end-products present in acid extracts of the pars intermedia of the anuran amphibian, Bombina orientalis. Sephadex G-75 gel filtration chromatography indicated that immunoreactive alpha-MSH-sized material and N-acetylated beta-endorphin-related material are the major POMC-related products present in this tissue. The alpha-MSH-sized immunoreactivity was further fractionated by reversed phase HPLC. The major peak of immunoreactivity isolated by this procedure eluted with the same retention time as synthetic ACTH(1-13)amide. Cation exchange chromatography supported the conclusion that the major storage form of alpha-MSH in the pars intermedia of Bombina is ACTH(1-13)amide. Analysis of Bombina pars intermedia in culture indicated that mono-acetylated and di-acetylated alpha-MSH were the major forms of alpha-MSH secreted into the medium. The major peak of N-acetylated beta-endorphin-related material was further analyzed by cation exchange chromatography and Sephadex G-25 gel filtration column chromatography. The major storage form of beta-endorphin in this tissue is N-acetylated, has a net positive charge at pH 2.75 of +1, and has an apparent molecular weight of 1.2K. The beta-endorphin present in the pars intermedia of this tissue does not undergo further N-acetylation at the time of secretion. These results indicate that in the pars intermedia of the archaeobatrachian, Bombina orientalis, the N-acetylation of alpha-MSH is a cosecretory processing event, whereas N-acetylation of beta-endorphin is a post-translational processing event. These results are compared to other archaeobatrachian and neobatrachian pituitary POMC systems that have been analyzed.

Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.

Gamma 2-MSH immunoreactivity in the human heart.

In patients undergoing aorto-coronary by-pass surgery, we found a 26% arterial-venous difference of immunoreactive gamma 2-melanocytostimulating hormone (MSH), a proopiomelanocortin (POMC) derived peptide known to possess profound hemodynamic effects. These results prompted an investigation of the presence of gamma 2-MSH in the human heart. Using a two-step extraction procedure, regions of human hearts were examined by sensitive and specific radioimmunoassays to determine their gamma 2-MSH content. Mean (+/- SEM) concentrations of 0.14 +/- 0.023 pmol/g and 0.12 +/- 0.017 were found in right atrium and right ventricle, respectively. High performance liquid chromatography indicated that 80-90% of the total immunoreactivity eluted in a single sharp peak in a position identical to that of synthetic gamma 2-MSH.

High-performance displacement chromatography of melanotropins and their derivatives.

Mixtures containing derivatives of the biological active peptide alpha-melanocyte stimulating hormone (alpha-MSH) or beta-melanocyte stimulating hormone (beta-MSH) were purified by using reversed-phase chromatography in the displacement mode. On a 250 x 4.6 mm octadecyl silica column and with instrumentation used in analytical high-performance liquid chromatography about 30 mg of the peptide mixture were separated by using an aqueous solution of benzyldimethyldodecylamonium bromide as the displacer in a single chromatographic run. The results demonstrate the advantages of displacement over elution in preparative chromatography of peptides on the scale of tens of milligrams that is typical for physiological tissue extracts and in solid-phase peptide synthesis.

Isolation of alpha-melanotropin from the pars intermedia of the larval amphibian, Ambystoma tigrinum.

The effect of background adaptation on the steady-state levels of alpha-melanotropin in the pars intermedia of the larval amphibian. Ambystoma tigrinum, was investigated. Acid extracts of pars intermedia obtained from light-adapted and dark-adapted animals were analyzed by radioimmunoassay following Sephadex gel filtration chromatography, reverse-phase HPLC, and Sulfopropyl Sephadex cation-ion-exchange chromatography. For both background adaptation conditions similar results were obtained. The major form of alpha-melanotropin present in the pars intermedia has the following properties: (1) an apparent molecular mass of 1.5 kDa; (2) a net charge at pH 3.5 of +4; and (3) a retention time following reverse-phase HPLC similar to that of synthetic ACTH(1-13)amide. In dark-adapted animals a minor form of alpha-melanotropin which has a net charge of +3 at pH 3.5 was also detected. The latter form represented approximately 10% of the total alpha-melanotropin immunoreactivity in the pars intermedia of dark-adapted animals. These results strongly suggest that the predominant form of alpha-melanotropin in the pars intermedia of larval A. tigrinum is a nonacetylated ACTH(1-13)amide-like polypeptide.

Isolation, characterization, and corticotropic activity of rabbit adrenocorticotropin.

Rabbit (r) ACTH was extracted from 600 pituitaries, and 2 forms of immunoreactive ACTH were identified with the least polar form accounting for approximately 90% of the total. Peptide mapping and sequence analysis indicated that three tryptic peptides had retention times identical to those obtained from human (h) ACTH. The least polar tryptic fragment from rACTH had a shorter retention time than the corresponding one from hACTH. Sequence analysis indicated that rACTH differed from hACTH at three different loci, namely, an Asn in place of Asp in position 29; a Val in place of Leu in position 37, and a Val in place of Phe in position 39. Biological activity of the ACTH was compared with synthetic hACTH in 2 bioassays with adrenals from 10-day-old pups, the first using dispersed rabbit adrenal cells and the second using monolayer adrenal cells in culture. The biological potencies of the two ACTH preparations were identical with respect to corticosterone (B) release in the short term bioassay, with an ED50 value of 1.67 X 10(-10) M. The ED50 value for cortisol (F) release for rACTH and hACTH were 1.1 X 10(-10) M and 1.67 X 10(-10) M, respectively, which were not statistically different. The biological potency of rACTH in the monolayer adrenal cell system for both F and B was significantly greater than the hACTH, and the ED50 values were 4.4 X 10(-10) M and 8.9 X 10(-10) M, respectively. There was a progressive decrease in the B/F ratios with increasing concentrations of ACTH in both the bioassay systems suggesting that ACTH stimulated the 17 alpha-hydroxylase activity even when the exposure of cells to ACTH was as short as 2 h.

Proportions of mono- and diacetylated forms of alpha MSH in individual neurointermediate lobe extracts of Cyprinus carpio L.

The proportions of the mono- and diacetylated forms of alpha MSH in individual carp neurointermediate lobe (NIL) extracts, as assessed by HPLC and RIA, fluctuate within a narrow range (mono/mono- + diacetyl alpha MSH = 3-14%). These results obtained on individuals of different sex and age show that the diacetylated form predominates in the NIL of all individuals where it represents in the mean 90% of the acetylated forms. The relatively low fluctuation of the proportions of the acetylated forms suggests that the possibility for a physiological modulation of the diacetylation step may be limited in this species of fish.

Theoretical interpretation of extraction (in brain) of peptides including concentration variations.

The transport properties of several peptides across blood-brain barrier (BBB) have been investigated theoretically in terms of simple diffusion and facilitated diffusion processes. Comparison of the calculated results from the simple diffusion and the experimental data reveals the presence of the facilitated diffusion of these substances which we have conceived of as a carrier-mediated process. The values of the partition coefficients f for these peptides were in the range 7 X 10(-4) less than or equal to f less than or equal to 200 X 10(-4). The calculated f values gave permeabilities, Ps, in lipids between 10(-7) less than or equal to Ps less than or equal to 14 X 10(-7) cm/s. These values were then used to estimate the extraction for peptides from simple diffusion alone which vary from 0.3 to 3.5% compared with the experimental extraction (0.4-12%) indicating the inadequacy of the simple diffusion alone to explain the experimental data. As for the carrier-mediated facilitated diffusion process we have used the activated-complex theory. The extraction in this case depends on the maximal rate of transport (Tmax)f and the reciprocal of the affinity constant Kt for the transport of peptides through BBB. We have deduced that (Tmax)f approximately 0.46 X 10(-3) pmol/g X s and Kt approximately 0.35 nM for Met-enkephalin (Met-ENK), Leu-enkephalin (Leu-ENK), glutathione, carnosine, alpha-MSH and MIF and (Tmax)f approximately 10 X 10(-3) pmol/g X s and Kt approximately 7 nM for AVP, beta LT, beta E and alpha E to explain the observed results. We have also obtained the quantitative variation of extraction with concentration of peptides in the brain-capillary and have established that the extraction decreases with increasing concentration of peptides, tending to a small constant value at high concentrations. It has been inferred that carrier-mediated facilitated diffusion is important for the transport of peptides across BBB.

Chemical and biological characterization of salmon melanocyte-stimulating hormones.

Ten peptides related to melanocyto-stimulating hormone (MSH) have been identified in an acid acetone extract of the chum salmon pituitary. All these peptides are related to the alpha-MSH and beta-MSH families, but no peptide related to gamma-MSH has been found. This result is in accordance with the finding that the gamma-MSH segment is deleted from the N-terminal peptide of salmon pro-opiocortin (NPP I). Based on the structures of newly isolated peptides, the maturation process of MSH is discussed. The major components of salmon MSH were tested for biological activities. In the lipolytic assay with rabbit fat cells, alpha-MSH I and alpha-MSH II were equipotent, but beta-MSH I and NPP I exhibited very low or no activity. On the other hand, the des-acetyl-alpha-MSH I was found to be four times as potent as alpha-MSH I in this assay. The steroidogenic activities of alpha-MSH I and N-des-acetyl-alpha-MSH I were approximately 0.05% of the potency of ovine ACTH. All other peptides exhibited less than 0.01% potency. Salmon alpha-MSHs were found to be somewhat more potent melanophore-stimulating agents than the beta-MSHs.

A corticotropin (ACTH) related peptide with cytotoxic activity.

A commercially available preparation of corticotropin was found to have potent cytotoxic activity for several established cell lines. Neither synthetic corticotropin nor alpha melanocyte stimulating hormone demonstrated this cytotoxic activity. Gel filtration allowed separation of a 2100 dalton cytotoxic peptide from the actual corticotropin present in the commercially prepared material. The structural relatedness of the cytotoxic peptide to corticotropin was demonstrated by neutralization with antisera to alpha melanocyte stimulating hormone. These studies indicate the existence of a newly identified ACTH related peptide with cytotoxic activity.