Melanocyte-stimulating Activity Research Articles

Corticotrophin-like peptides in the rat pituitary.

SUMMARY The pars intermedia of the rat pituitary contains a peptide resembling the 18–39 portion of adrenocorticotrophic hormone (ACTH), which has been termed 'corticotrophin-like intermediate lobe peptide' (CLIP). It can be detected by its cross-reaction with an antiserum directed against the CO2H-terminal portion of the ACTH molecule; it has an amino acid composition identical to the 18–39 portion of human ACTH, except for one less glycine and an extra valine residue, and it is rapidly released from neurointermediate lobes maintained in organ culture. The pars intermedia also contains a peptide with an amino acid composition and biological potency identical to that of melanocyte-stimulating hormone (α-MSH) isolated from other mammals, and which accounts for the bulk of melanocyte-stimulating activity in the pituitary. Rat ACTH resembles human ACTH in amino acid composition, except for an extra valine and one less glycine residue. On the basis of these data it is proposed that ACTH is the precursor of α-MSH and CLIP, which are both present in the cells of the pars intermedia.

Isolation of a tridecapeptide from Natriuretic fractions of bovine posterior pituitary.

Abstract. Crude Van Dyke protein prepared from frozen bovine posterior pituitary glands was treated with formic acid to release small bound and adsorbed peptides. Gel filtration, continuous free‐flow electrophoresis and descending paper electrophoresis were used to separate the small peptides into successively purer fractions which were assayc sdfor pressor, utero tonic, natriuretic and melanocyte stimulating activities under double blind conditions. The highest peak of natriuretic activity was associated with only a small pressor and no uterotonic activities, and the same fractions contained a ninhydrin‐positive spot on paper electrophoresis, the mobility of which at pH 5.6 was similar to that of alpha‐melanocyte stimulating hormone (MSH) and ACTH‐(i‐13‐amide)‐tri‐decapeptide (1–13 ACTH‐amide) but differed from vasopressin and oxytocin. Material from this spot had an amino acid composition which corresponded with the first 13 residues of ACTH. Quantitative bioassay of the final fraction in the frog demonstrated a melanotropic activity which, however, was lower than would have been expected for pure alpha‐MSH. The natriuretic activity of the isolated fraction was far higher than the potency of separately administered peptides which might be present: arginine‐vasopressin, alpha‐MSH or 1–13 ACTH. It is suggested that the isolated fraction represents a mixture of alpha‐MSH and 1–13 ACTH, and that alpha‐MSH may be an acetylated end‐product of ACTH cleavage in the pituitary.

Biologic and immunologic characterization and physical separation of ACTH and ACTH fragments in the ectopic ACTH syndrome.

Extracts of tumors from 32 patients with the ectopic ACTH syndrome were subjected to simultaneous bioassay and radioimmunoassays for ACTH. Radioimmunoassays were performed using three antisera, one of which reacts with the extreme N-terminal 1-13 amino acid sequence of ACTH, the second with the N-terminal 1-23 sequence of the ACTH molecule, and the third with the C-terminal 25-39 amino acid sequence of ACTH. There was, in general, good correlation between bioactivity and N-terminal ACTH immunoreactivity. However, there were large excesses of both extreme N-terminal and C-terminal immunoreactive materials in most tumor extracts, which were not found in extracts of three human pituitaries. Three tumor extracts were subjected to molecular sieve chromatography on Sephadex G-50 fine resin. The bioactive ACTH eluted in the same fractions as pituitary ACTH (mol wt approximately 4,500 daltons) and reacted equally in all three ACTH radioimmunoassay systems. The bioactive tumor ACTH was neutralized by incubation with the C-terminal antiserum, indicating it has an intact C-terminal sequence of amino acids. The next several fractions from the Sephadex column contained a material, mol wt approximately 3,100, which was biologically inactive and had C-terminal immunoreactivity but no N-terminal or extreme N-terminal immunoreactivity. Incubation with the N-terminal 1-23 ACTH antiserum did not adsorb these C-terminal fragments, indicating they lacked an intact sequence of amino acids in this region. A smaller ACTH fragment (mol wt approximately 1,800 daltons) eluted in still later fractions and reacted with the extreme N-terminal antiserum but not with the N-terminal or C-terminal antisera. It had no steroidogenic activity, but appeared to have significant melanocyte-stimulating activity. It is concluded that, in addition to an ACTH similar, if not identical, to pituitary ACTH, tumors of patients with the ectopic ACTH syndrome contain both N-terminal and C-terminal ACTH fragments.

Electrophysiological correlates of melanocyte-stimulating hormone activity in the frog.

Electrophysiological correlates of melanocyte-stimulating hormone activity in the frog.

Electroencephalographic measures of melanocyte-stimulating hormone activity.

Electroencephalographic measures of melanocyte-stimulating hormone activity.

Melanocyte stimulating activities of the [formula omitted]-nitrophenyl sulfenyl derivatives of adrenocorticotropin and [5-glutamine] -α-melanotropin

The o -nitrophenyl sulfenyl derivative of adrenocorticotropin in which the single tryptophan residue of the molecule is modified, is found to be fully active in stimulating amphibian melanophores. The melanophore stimulating activity of [5-glutamine] -α-melanotropin, a synthetic analog of α-melanotropin, is also found to be undiminished by chemical modification of the tryptophan residue.

ACTH and protein synthesis in a transplantable ACTH-secreting tumor. Evidence for the absence of MSH.

The synthesis of ACTH and protein was studied in vitro in a transplantable mammotropic pituitary tumor (MtT.F4) in female Fisher rats. In tumor-bearing rats the thymus was involuted and the adrenals were 15 times normal weight. Based on radioimmunoassay, the ACTH concentration in MtT.F4 was at least 10-fold less than the concentration of the hormone in the normal pituitary. Crude ACTH was isolated from MtT.F4 by extraction with HC1, heat coagulation, and oxycellulose adsorption. The hormone was purified by gel filtration and carboxymethylcellulose chromatography. In vitro incubations were performed with MtT.F4 and with normal bovine anterior pituitary. The incorporation of radioactive phenylalanine into both ACTH and protein was about 3-fold greater in the tumor than in the normal pituitary. However, the relative rate of amino acid incorporation into ACTH, compared to that into protein, was no greater in the tumor than in the normal pituitary. These data suggested that ACTH was secreted 30–300 times more rapidly from the tumor than from the normal pituitary. When extracts from MtT.F4 were assayed for MSH content by the frog-skin assay, all the activity could be accounted for by the intrinsic melanocytestimulating activity of the ACTH present in the tumor. MtT.F4 was incubated with radioactive phenylalanine, and extracts were analyzed chromatographically for MSH. No evidence was found of radioactivity or optical density where either α- or β-MSH is normally found. It was concluded that MtT.F4 in female Fisher rats produces ACTH but not α- or β-MSH. (Endocrinology 86: 1394, 1970)

Melanocyte-stimulating activity in serum and the aqueous flare response in rabbits.

Melanocyte-stimulating activity in serum and the aqueous flare response in rabbits.

Melanocyte-stimulating hormone: activity in thermal polymers of alpha-amino acids.

Thermal polymers of arginine, glutamic acid, glycine, histidine, phenylalanine, and tryptophan have melanocyte-stimulating activity. The fact that similar polymers lack such activity indicates that the effect is related to the specific amino acid residues. The active polymers are discussed as a model of an evolutionary precursor of contemporary melanocyte-stimulating hormone.

Biological proprtie of ovine beta-lipotropic hormone.

Ovine β-lipotropic hormone LPH) was assayed for hormonal activities associated with anterior and intermediate lobe pituitary hormones and was found to have melanocyte-stimulating, lipolytic and adrenal-stimulating activities. As a lipolytic agent, β-LPH is most active in rabbit adipose tissue; it is only weakly active in rat and appears to be inactive in mouse adipose tissue. Studies with fragments of the β-LPH molecule produced during the reaction of β-LPH with cyanogen bromide have shown that the center for lipolytic and melanocyte-stimulating activities resides in region 41–58, which contains the sequence of β-MSH. Some conclusions about the effect of structure on activity have been drawn. Results of a study revealing the presence of β-LPH in lamb serum are also presented. (Endocrinology 82: 898, 1968)

Hypothalamic and cerebral cortical inhibition of melanocyte-stimulating hormone secretion in the frog, Rana pipiens.

Aqueous extracts of hypothalamic and cerebral cortical tissues of light and dark adapted frogs were assayed in vitro for their ability to inhibit the secretion of melanocyte-stimulating hormone (MSH) from the pars intermedia of the dark adapted frog. The extracts also were assayed for melanocyte-stimulating activity. The pars intermedia secreted large quantities of MSH in vitro. The extracts of the hypothalamus and of cerebral cortex of both light and dark adapted frogs contained a factor(s) that inhibited the secretion of MSH from the pars intermedia. Extracts of the hypothalamus, but not extracts of the cerebral cortex, also contained a melanocytestimulating substance. There was no significant difference between extracts of the hypo hilamus and cerebral cortex of light and dark adapted frogs in their ability to inhibit MSH secretion. The possible chemical nature of the factor(s) was discussed. The ability of the cerebral cortical extracts to inhibit MSH secretion was attributed to the catecholamine cont...

Melanocyte-stimulating activity in a transplantable mouse pituitary tumor.

Levels of melanocyte-stimulating activity were determined in the circulation and in the tumors of mice bearing a transplantable pituitary tumor which produced darkening of the coat color. An average concentration of 5.1 (±0.6) X104 U/mg dry weight was found for 8 tumors. The level of activity in the circulation (C.L.A.) was followed as a function of time following transplantation of the tumor and was correlated with the change in the reflectance of the coat and the growth of the tumor. An average C.L.A. of approximately 6 U per ml per mg tumor wet weight was found. Changes in coat color became apparent while the tumor was still quite small and the C.L.A. was low (less than 100 U/ml); however, maximal darkening occurred at a time when the C.L.A. was approximately 1000 U/ml or more. The half-lives of the endogenous circulating activity in the mouse and of exogenous α-MSH in the rat were both found to be approximately 1.7 min. A secretion rate of approximately 1.5 X104 U per day per mg tumor wet weight was calculated, indicating that the tumor activity turned over an average of 1.65 times/day. In spite of secretion rates as high as 3.7 X107 U/day, less than 75 U of activity was found in the 24-hr urine. Preliminary separation experiments suggest that the tumor contains and secretes α-MSH. (Endocrinology79: 97, 1966)

Sodium hydroxide treatment of l-histidyl- l-phenylalanyl- l-arginyl- l-tryptophylglycine and its potentiated melanocyte-stimulating activity in vitro

Treatment of l-histidyl- l-phenylalanyl- l-arginyl- l-tryptophylglycine, a common fragment of α- and β-MSH and ACTH, with 0.1 N sodium hydroxide at 100° for 10 min brought about a 40-fold increase in the MSH activity on frog-skin melanocytes in vitro. Chemical and enzymatic examinations of this treated peptide showed that one quarter of the arginine was converted to ornithine and four amino acid residues: histidine, phenylalanine, arginine and tryptophan, were racemized. As far as the MSH activity in vitro is concerned, potentiation is the only phenomenon which was observed. Neither retarded nor prolonged effects of its action could be detected in this product. When the relative potencies of the synthetic stereoisomers of the pentapeptide were measured against the darkening value produced by a constant amount of the all- l pentapeptide, l-histidyl- d-phenyalanyl- l-arginyl- l-tryptophylglycine and l-histidyl- l-phenylalanyl- l-arginyl- d-tryptophylglycine exhibited some 160 and 20 times higher activity than that of the all- l pentapeptide respectively. Based on these observations, the relationship between structure and the potentiated activity of the alkali-treated all- l pentapeptide is discussed. A possible explanation for the phenomenon of appearance or disappearance of the potentiated activity in alkali-treated ACTH and MSH's is proposed.

The effect of ionizing radiation of melanocyte-stimulating and steroidogenic activities of corticotropin

Purified porcine corticotropin (ACTH) was irradiated with 2.0-MeV electrons in reduced Argon atmosphere. Residual melanocyte-stimulating and steroidogenic activities of the irradiated samples were measured. Irradiated samples of corticotropin had greater melanocyte-stimulating activities than non-irradiated samples. At 400·106 rad melanocyte-stimulating activity was 340% of the at o rad. Steroidogenic activity diminished with increasing doses of radiation, and a molecular weight estimate of 4.0·103 was obtained. The ratio of melanocyte-stimulating activity to steroidogenic activity rose from 1 in non-irradiated samples to 97 in samples irradiated at 600-106 rad.Irradiation of purified α-melanocyte-stimulating hormone in Ar atmosphere caused diminution of melanocyte-stimulating activity.Irradiation of corticotropin in air caused loss of both steroidogenic and melanocyte-stimulating activities at about the same initial rate. This rate was considerably more rapid than that for loss of steroidogenic activity of corticotropin irradiated in Argon.

Studies on peptides. VI. Synthesis of three stereoisomeric pentapeptides of histidylphenyl-alanylarginyltryptophylglycine possessing D-phenylalanyl-D-arginyl moiety within the sequence and their melanocyte-stimulating activities in vitro.

The syntheses of three pentapeptide isomers, L-histidyl-D-phenylalanyl-D-arginyl-L-tryptophylglycine (II), D-histidyl-D-phenylalanyl-D-arginyl-L-tryptophylglycine (III) and L-histidyl-D-phenylalanyl-D-arginyl-D-tryptophylglycine (IV) were described. Nα-Ben-zyloxycarbonyl-D-phenylalanyl-NG-nitro-D-arginine served as a key intermediate for the synthesis of these isomers. Melanocyte-stimulating activities of these compounds were examined using the frog-skin from Rana pipiens. It was found that II possessed the activity identical to that of L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophylglycine (I). The peptides (III) and (IV) were both inactive.

INCREASED PERMEABILITY OF THE BLOOD-AQUEOUS HUMOR BARRIER IN THE RABBIT'S EYE PROVOKED BY MELANOCYTE STIMULATING PEPTIDES.

The relative protein content in the aqueous humor of an intact eye can be determined by a new photoelectric method. The systemic administration of certain hypophysial polypeptides causes a transitory increase in permeability of the blood-aqueous humor barrier in the rabbit's eye. This has been denoted the “aqueous flare response” (AFR). The following peptides have been tested for their ability to cause the AFR: synthetic α-MSH, porcine α- and β-MSH, porcine corticotropin, porcine peptide I, synthetic Nα-Ac-β1–10-corticotropin and β1–10-corticotropin. The AFR provoking effect of these preparations is correlated with their melanocyte stimulating activity but not with their corticotropic activity. A number of factors influencing the response have been investigated. It is shown that an AFR is more easily evoked 1) in fasted than in normally fed rabbits, 2) in pigmented than in albino rabbits, and 3) in adult than in young animals. (Endocrinology 74: 255, 1964)

LIPOTROPIN, A NEW ACTIVE PEPTIDE FROM PITUITARY GLANDS.

A BIOLOGICALLY active peptide which is chemically and physiologically distinct from known pituitary hormones has recently been isolated and characterized1. Its purity has been established by various criteria such as column chromatography and gel filtration, zone electrophoresis and disk electrophoresis, countercurrent distribution and sedimentation. This peptide, which was obtained in the course of the isolation of adrenocorticotropin (ACTH) by a revised method2, is more acidic than the latter hormone, and consists of 59 amino-acids. Its molecular weight was determined by sedimentation equilibrium investigations and found to be 6,900. It is structurally different from ACTH, having lysine rather than phenylalanine at the COOH-terminus, and glutamic acid rather than the serine at the NH2-terminus; furthermore, it has a different amino-acid composition. It has a very low adrenocorticotropic activity, and resembles ACTH in melanocyte-stimulating activity; however, it is not potentiated by treatment with boiling NaOH, as is ACTH. Although the lipolytic potencies in the rabbit as determined in vitro were comparable for both peptides, they differed markedly in the rat.

Adrenocorticotropins: XXIX. The action of sodium hydroxide on adrenocorticotropin

Heating of a dilute adrenocorticotropin (ACTH) solution in 0.1 M NaOH has been found to cause a loss of both adrenal-stimulating and lipolytic activities. However, the same treatment not only potentiated but also prolonged the intrinsic melanocyte-stimulating potency of ACTH. Extensive physical and chemical studies of the NaOH-treated ACTH have been carried out. It was found that a limited cleavage of the second bond in the hormone occurred and that a number of amino acid residues within the molecule have been racemized. Possible explanations for the potentiation of melanocyte-stimulating activity in ACTH have been discussed.

Some aspects of the relationship between chemical structure and melanocyte-stimulating properties of several peptides related to adrenocorticotropin

The melanocyte-stimulating activity of several synthetic peptides whose amino acid sequences correspond to partial structures of ACTH has been determined. The duration of the response of frog melanocytes to these pepetides in vivo was studied, and two. ACTH and a synthetic nonadecapeptide, were found to have longer-lasting effects than native melanotropins. Heating with NaOH solution prolonged the melanocyte-expanding response to α-MSH and larger peptides. This treatment also incresed the potency of ACTH and of two synthetic peptides, a decapeptide and a nonadecapeptide. Evidence to suggest that the N-terminal serine may be implicated in the potentiating action of NaOH is presented.

Occurrence of three electrophoretic components with melanocyte-stimulating activity in extracts of single pituitary glands from ungulates.

An in vitro method in which the skin of a single test animal (Anolis carolinensis) is used, and which can be used to determine the melanocyte-stimulating activity of a large number of samples, is described. The electrophoretic behavior of the melanocyte-stimulating components present in the pituitary glands of different species of Ungulata was studied. It was found that single beef, pig and sheep pituitaries contain three melanocyte-stimulating components, which behaved in electrophoresis like β-glutamyl-MSH, β-seryl-MSH and a-MSH respectively. These MSH components were present in each single pituitary gland in a ratio which appeared to be more or less constant within a species and which was characteristic for that species.