Ichiro Kawazoe

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.

Coexistence of immunoreactivity for melanin-concentrating hormone and α-melanocyte-stimulating hormone in the hypothalamus of the rat

Coexistence of immunoreactivity for melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) within rat hypothalamic neurons has been examined by the unlabeled antibody enzyme method. Neurons exhibiting both MCH- and alpha-MSH-like immunoreactivities were found in the dorsolateral hypothalamus, whereas no MCH-like immunoreactive perikarya were seen in the arcuate nucleus, where some neurons were stained with alpha-MSH antiserum. There seem to be two distinct alpha-MSH-like immunoreactive neurons in the rat hypothalamus, one exhibiting coexistence with MCH-like immunoreactivity and the other not showing any cross-reaction with MCH antiserum.

Melanin-concentrating hormone-like immunoreactive material in the rat hypothalamus; characterization and subcellular localization

SummaryMelanin-concentrating hormone (MCH) is a neurosecretory peptide that induces melanin concentration within teleost melanophores. Here, we characterized MCH-like substance in the rat brain by both an in vitro fish-scale melanophore bioassay and a radioimmunoassay with a salmon MCH antiserum that is directed toward the carboxy-terminus and requires the cyclic configuration for recognition. Furthermore, subcellular localization of the MCH in the rat brain was examined by immunocytochemistry using electron microscopy. We confirmed that MCH-immunoreactivity and MCH-bioactivity were present together in the same effluent fractions of the rat hypothalamic extracts by reverse-phase high-performance liquid chromatography (HPLC). At electron microscopic level, MCH-immunoreactivity was located specifically in secretory granules in MCH-positive cell bodies confined to the hypothalamus with their neuronal processes projecting widely in the rat brain. Although full characterization of substance must await its isolation, our results strongly support the notion that rat MCH-like substance may be homologous but not identical to salmon MCH, and simultaneously may serve some neurotransmitter and/or neuromodulator role in the brain of the rat.

Fish melanin-concentrating hormone disperses melanin in amphibian melanophores

Melanin-concentrating hormone (MCH), which was isolated from salmon pituitary and caused melanin concentration in fish scale melanophores, has been tested on cultured chromatophores of an amphibian, the bullfrog tadpole. MCH induced dispersion of melanin in cultured melanophores of the tadpole. The duration of the dispersing effect of MCH was relatively short compared with that of alpha-melanocyte-stimulating hormone (alpha-MSH). MCH also induced the concentration of cultured iridophores of bullfrog tadpole.

Characterization of melanin concentrating hormone in teleost hypothalamus

Melanin concentrating hormone (MCH) is a heptadecapeptide isolated from chum salmon (Oncorhynchus keta) pituitaries. The peptide has been isolated from whole brain extract at a low yield of 1.2 micrograms/1300 brains. MCH activity in the hypothalamus was characterised by in vitro scale bioassay and radioimmunoassay. Specificity of these assay systems was examined with neurotransmitters such as epinephrine, norepinephrine, and dopamine, hypothalamic hormones such as somatostatin, isotocin, Arg-vasotocin, oxytocin, and Arg-vasopressin, and salmon prolactin and its chymotryptic peptide or salmon PRL176-187. Among them only salmon PRL176-187 exhibited weak activities in both assays. The neurotransmitters were 10(4) to 10(5) times less potent than MCH in the bioassay. MCH concentrations in a pituitary and a hypothalamus were estimated as 5300 +/- 750 ng (ca. 106 micrograms/g) and 48 +/- 9.5 ng (ca. 1.6 micrograms/g), respectively, by radioimmunoassay. Lysyl endopeptidase digestion of the hypothalamic extract resulted in a significant increase of biological activity as well as of immunoreactivity. Gel filtration of the hypothalamic extract and subsequent enzymatic digestion revealed that the fractions at higher molecular weight were contributory to the increase in the activities.

Purification and characterization of recombinant tuna growth hormone produced in Escherichia coli

Abstract Cloned cDNA of tuna growth hormone (tGH) was expressed in Escherichia coli cells and recombinant tGH (r-tGH) was accumulated as insoluble inclusion bodies. The r-tGH extracted from inclusion bodies was renaturated through treatment with guanidine·HCl and urea, and then purified to apparent homogeneity by reversed-phase liquid chromatography. About 30 mg of r-tGH was obtained from 100 mg of purified inclusion bodies. The circular dichroism spectrum of r-tGH was closely similar to that of native tGH prepared from tuna pituitary glands. The amino terminal sequence Ala-Gln-Pro-Ile-Thr- of r-tGH was also the same as that of tGH, except for the first Ala, which is not contained in native tGH. The amino terminus Met in r-tGH was thought to be removed by N-terminal methionine aminopeptidase. The growth of juvenile rainbow trout (Salmo gairdneri) was remarkably accelerated when the r-tGH was administrated by four intraperitoneal injections at intervals of 7 d: 1.5 fold in length and 1.8 fold in body weight/28 d.

A sensitive bioassay for melanotropic hormones using isolated medaka melanophores

Melanophore-stimulating hormones (MSHs) from chum salmon cause pigment dispersion in isolated melanophores of medaka, a teleost. The in vitro medaka melanophore bioassay that responded to light with pigment dispersion and to the dark with pigment aggregation was utilized for measuring the activity of melanotropic hormones. alpha-MSH I was the most potent melanophore-dispersing agent tested. The minimal dose for the induction of pigment dispersion was 10(-15) M alpha-MSH I, 10(-13) M N-des-acetyl(Ac)-alpha-MSH, and 10(-11) M beta-MSH I, respectively. The melanosome-dispersing activity of beta-MSH I was enhanced about 40% by salmon N-acetyl-endorphin I (N-Ac-EP). The results suggest that N-Ac-EP may act as an enhancer for the activity of certain MSHs. The present bioassay provides a unique method for determining the biological activity of melanotropic peptides.