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.

The biosynthesis, intracellular transport, and packaging of melanocyte-stimulating peptides in the amphibian pars intermedia.

Experiments in which glycine-(3)H has been introduced into excised neurointermediate lobes of Xenopus laevis incubated in a modified Krebs-Ringer bicarbonate medium have shown that approximately 50% of the incorporated radioactivity is present in small peptides which have an electrophoretic mobility characteristic of the melanocyte-stimulating (MSH) peptides shown to be elaborated within the tissue. Based on these results and the demonstration that a discrete approximately 7 min pulse of the label can be introduced into the tissue, electron microscope radioautography has been employed to follow the subcellular events concerned with the synthesis, intracellular transport, and packaging of the labeled secretory product. Together, these studies indicate that the newly synthesized material arises in peptide form, rather than as part of a larger prohormone molecule, on the ribosomes of the rough endoplasmic reticulum within the parenchymal cells of the intermediate portion of the lobe. A proportion is then incorporated into and remains for an extended period within the intracisternal granules which are a feature of the rough endoplasmic reticulum within these cells in vitro Most ( approximately 60%) of the labeled secretory product, however, is transferred to the Golgi complex within 30 min and, within a further 10 min, becomes packaged into small ( approximately 200 mmicro) electron-opaque secretory granules. It is probable that under the conditions employed these granules represent the final intracellular location of secretory product before it is released

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.

Biosynthesis, processing, and control of release of melanotropic peptides in the neurointermediate lobe of Xenopus laevis.

The neurointermediate lobes of dark-adapted toads Xenopus laevis were incubated for 30 min in [3H]arginine and then "chased" for various time periods. By use of this pulse-chase paradigm there were detected 10 trichloroacetic acid (TCA)-precipitable peptides separated on acid-urea polyacrylamide gels and one TCA-soluble peptide separated by high-voltage electrophoresis (pH 4.9) with melanotropic activity. Each of these peptides had a different degree of melanocyte stimulating hormone (MSH) activity as revealed by the Anolis skin bioassay. Three of these TCA-precipitable peptides comigrated with ACTH, beta-lipotrophin, and alpha-MSH on acid-urea gels. Evidence suggesting a precursor-product mode of biosynthesis of the melanotropic peptides is presented. 7 of the 10 TCA-precipitable peptides and the one TCA-soluble peptide with melanotropic activity were released into the medium. The half-time of release of the TCA-precipitable peptides was about 2 h, whereas the half-time of TCA-soluble peptide release was about 30 min. The release of these peptides was inhibited by 5 X 10(-5) M dopamine. Dopamine inhibition of release did not appear to affect the biosynthesis of the melanotropic peptides, but did appear to enhance the degradation of the newly synthesized TCA-soluble peptide in the tissue. White adaptation of the toads greatly decreased the biosynthesis of all of the TCA-precipitable melanotropic peptides.

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.

alpha-Melanocyte-stimulating hormone-like peptides in the intermediate lobe of the rat pituitary gland: characterization of content and release in vitro.

Reverse phase high performance liquid chromatography (HPLC) followed by RIA of the chromatographic fractions was used to separate and quantify, respectively, the alpha MSH-like peptides stored in the intermediate lobe (IL) of the rat pituitary gland and released from IL cells in vitro. Immunoreactive material eluting with the same HPLC retention time as N,O-diacetyl alpha MSH accounted for approximately 80% of the total immunoreactive alpha MSH (IR-alpha MSH) in either the neurointermediate lobe or dispersed IL cells. The remainder of the IR-alpha MSH coeluted with either synthetic desacetyl alpha MSH or alpha MSH. Furthermore, the predominant alpha MSH-like compound released in vitro from dispersed IL cells eluted from the HPLC column with the same retention time as synthetic N,O-diacetyl alpha MSH. Treatment of dispersed IL cells with drugs known to enhance (l-isoproterenol or A 23187) or to inhibit (apomorphine or lisuride) the release of IR-alpha MSH revealed that N,O-diacetyl alpha MSH was the primary form released. Finally, an evaluation of the stability of the alpha MSH-like peptides indicated that N,O-diacetyl alpha MSH was readily converted to alpha MSH in the presence of 0.1 N hydrochloric acid.

Dynamics and characterization of plasma immunoreactive beta-melanocyte stimulating hormone in hemodialysis patients: its relationship to ACTH.

We studied the plasma immunoreactive beta-MSH ("beta-MSH") in hemodialysis patients to determine its basal level, plasma disappearance rate, gel filtration and immunological characteristics. All patients had increased plasma "beta-MSH" (90--440 pg/ml; normal less than 90 pg/ml). Plasma ACTH and cortisol values were within the normal range. Cortisol infusion over 2 h induced almost no plasma "beta-MSH" variation as compared to controls where "beta-MSH" decreased rapidly (apparent half-life 90 min.); more prolonged administration of corticosteroids (dexamethasone 0.5 mg every 6 h for two days) caused a slight (20%) but significant (P less than 0.001) decrease of "beta-MSH." On Sephadex G-50 endogenous "beta-MSH" eluted in a molecular weight range of 6,000--10,000. In our radioimmunoassay dilution curves of endogenous "beta-MSH" paralleled that of synthetic human beta-MSH, but not that of purified human beta-LPH. In conclusion, hemodialysis patients show a clear dissociation between elevated "beta-MSH" and normal ACTH plasma levels. "beta-MSH" probably has a decreased plasma disappearance rate and seems related to a substance different from human beta-MSH.

On the isolation of human pituitary hormones.

Six human pituitary hormones can be obtained from a single batch of human pituitary glands by the procedure described. Starting from an acetone powder of the glands, the hormones are segregated into three fractions; one containing GH and PRL, a second containing the glycoprotein hormones, and the third fraction containing smaller peptides including ACTH and MSH. Clinical grade GH is extracted from the residue at alkaline pH, followed by a series of ammonium sulfate and pH fractionations. Further chromatography on G-100 yields a highly purified GH preparation. FSH is purified by chromatography on carboxymethyl cellulose at pH 5.4 followed by gel filtration on G-100 and fractionation on sulfopropyl Sephadex C-50 at pH 5.4. LH and TSH are separated by DEAE-cellulose chromatography at pH 9.5. The GH and glycoprotein hormones are recovered in a highly purified form and good yields. The GH is obtained in an active form which is easily soluble and well suited for clinical use. A large part of the PRL is also saved during the extraction procedure.

Peptides related to the NH2-terminal end of proopiocortin in man.

Peptides related to the NH2-terminus of proopiocortin in man were studied with three different RIAs directed toward gamma 3MSH, human 16K, and mouse 16K. The culture medium derived from a human corticotropic adenoma (SCH medium), which had previously been used as a human reference standard, generated competitive binding curves parallel to that of purified human 16K in all three RIA systems. Gel exclusion chromatography performed with pituitary-derived materials (adenoma extract and medium, and plasma from patients with Nelson's syndrome) showed that the overall immunoreactive gamma 3MSH eluted as one major peak at the position of human 16K. Its molecular weight estimated under denaturing conditions was 11,000. Gel exclusion chromatography performed with nonpituitary-derived materials (tumor extract and plasma from patient with the ectopic ACTH syndrome) showed that a major peak eluted at the position of human 16K, and a smaller molecular weight peptide eluted in a position intermediary between that of human 16K and synthetic gamma 3MSH. These data show that immunoreactive gamma 3MSH is indeed identical to human 16K in pituitary-derived materials. A different processing of the proopiocortin molecule is likely to occur in nonpituitary tumors and will result in the release of a smaller molecular weight peptide. The exact nature of this peptide is not known. It is speculated that it may serve as a nonpituitary tumor marker.

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.

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.

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.

The frog pars intermedia contains only the non-acetylated form of alpha-MSH: acetylation to generate alpha-MSH occurs during the release process.

Pulse-chase experiments revealed that the frog pars intermedia synthesizes the desacetyl form of alpha-MSH. Its structure was shown to be similar, if not identical, to the mammalian structure. During release two additional peptides derived from desacetyl alpha-MSH appeared, one being alpha-MSH. We conclude that the N-acetylation of newly synthesized MSH is associated with release of the hormone. Radioimmunoassays and bioassays showed that the non-acetylated peptide is the only tissue form of MSH and confirmed that acetylation is linked to release.

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.

Regional heterogeneity in the processing of pro-opiomelanocortin in rat brain.

The occurrence of alpha-N-acetylated forms of beta-endorphin (beta END) and alpha-melanotropin (alpha MSH) in rat pituitary and brain regions was investigated using high-performance liquid chromatography and radioimmunoassay. Acetylated derivatives of both peptides predominated in neurointermediate pituitary but represented minor components in anterior pituitary, in agreement with previous reports. Regional differences in the degree of acetylation were also observed in brain. Arcuate nucleus, the major source of beta END and alpha MSH in brain, contained small amounts of acetylated forms but most of the immunoreactivity chromatographed with the unmodified beta END 1-31 and des-acetyl-alpha MSH. Amygdala and periaquaductal grey contained the non-acetylated forms only, whereas in nucleus accumbens the major immunoreactive species were the acetylated derivatives of beta END 1-31 and beta END 1-27 along with the mono- and di-acetyl forms of alpha MSH. The selective distribution of acetylated beta END and alpha MSH suggests heterogeneity in the processing of POMC in brain and may indicate a role for alpha-N-acetylation in regulating the biological activities of these peptides in specific brain regions.

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.