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Molecular Brain Research, 15 (1992) 1-7 © 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00
BRESM 70452
Research Reports
Isolation and structural characterization of peptides related to a- and y-melanocyte-stimulating hormone (MSH) from the frog brain Denis Tranchand Bunel a, j. Michael Conlon b, Nicolas Chartrel a, Marie-Christine Tonon a and Hubert Vaudry a a European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University of Rouen, Mont-Saint-Aignan (France) and b Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178 (USA) (Accepted 17 March 1992)
Key words: Neuropeptide; a-Melanocyte-stimulating hormone; y-Melanocyte-stimulating hormone; Proopiomelanocortin; Amphibian
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 a-melanocyte-stimulating hormone (MSH) and the peptide co-eluted with synthetic desacetyl a-MSH, indicating that it is COOH-terminally a-amidated. A second component, which exhibited a shorter retention time, co-eluted with the glycine-extended form of desacetyl a-MSH [ACTH(I-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-y~-MSH as predicted from the nucleotide sequence of frog proopiomelanocortin. The presence of substantial amounts of desacetyl a-MSH and Lys-yrMSH in the frog brain supports the concept that, in amphibia, melanotropins may act as neurotransmitters and/or neuromodulators as well as hormonal peptides.
INTRODUCTION The multifunctional precursor protein proopiomelanocortin (POMC) is synthesized in corticotrope cells of the distal lobe of the pituitary and melanotrope cells of the pars intermedia 6'22. Processing of POMC yields a number of bioactive peptides including adrenocorticotropin (ACTH), lipotropins (/3- and y-LPH), melanotropins (a-, /3- and y-MSH) and /3-endorphin 2z'23. Immunocytochemical studies have shown the presence of peptides derived from POMC in discrete populations of neurons in the brain of mammals 1°,16, and sub-mammalian vertebrates 3,7,36,37. In particular, aMSH-immunoreactive neurons have been detected in the basal hypothalamus of various species, namely in the infundibular nucleus in human 8, in the arcuate nucleus in rat 16'28 and in the ventral infundibular nucleus in frog 3'7'36. Similarly, y-MSH-like immunoreac-
tivity has been measured in extracts of bovine hypothalamia2, 33. Several lines of evidence indicate that melanotropins are functionally important neuropeptides. (i) Central administration of a-MSH induces a number of behavioral effects, such as enhancement of attention and memory 9, grooming behavior 14, agression 2°'z4 and reduction of body temperature2k (ii) Immunoelectron microscopic studies revealed that a-MSH is sequestered in dense core synaptic vesicles which are in a favorable position to be discharged upon nerve depolarization 2s. (iii) The release of a-MSH from rat hypothalamic neurons is stimulated by high potassium and veratridine in a calcium-dependent manner 17,35. Biochemical studies combining HPLC analysis and radioimmunological identification have shown the occurrence of peptides closely related to authentic aMSH in the central nervous system of both
Correspondence: H. Vaudry, European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University of Rouen, 76134 Mont-Saint-Aignan, France~ Fax: (33) 35-146349.
vertebrates3.~s,37 3~ and insects:'. However, the primary structure of a melanotropin in brain tissue has never been determined. In the present study, we have isolated two forms of a - M S H and Lys-y~-MSH in the brain of the European green frog, Rana ridibunda.
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Molecular Brain Research, 15 (1992) 1-7 © 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00
BRESM 70452
Research Reports
Isolation and structural characterization of peptides related to a- and y-melanocyte-stimulating hormone (MSH) from the frog brain Denis Tranchand Bunel a, j. Michael Conlon b, Nicolas Chartrel a, Marie-Christine Tonon a and Hubert Vaudry a a European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University of Rouen, Mont-Saint-Aignan (France) and b Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178 (USA) (Accepted 17 March 1992)
Key words: Neuropeptide; a-Melanocyte-stimulating hormone; y-Melanocyte-stimulating hormone; Proopiomelanocortin; Amphibian
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 a-melanocyte-stimulating hormone (MSH) and the peptide co-eluted with synthetic desacetyl a-MSH, indicating that it is COOH-terminally a-amidated. A second component, which exhibited a shorter retention time, co-eluted with the glycine-extended form of desacetyl a-MSH [ACTH(I-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-y~-MSH as predicted from the nucleotide sequence of frog proopiomelanocortin. The presence of substantial amounts of desacetyl a-MSH and Lys-yrMSH in the frog brain supports the concept that, in amphibia, melanotropins may act as neurotransmitters and/or neuromodulators as well as hormonal peptides.
INTRODUCTION The multifunctional precursor protein proopiomelanocortin (POMC) is synthesized in corticotrope cells of the distal lobe of the pituitary and melanotrope cells of the pars intermedia 6'22. Processing of POMC yields a number of bioactive peptides including adrenocorticotropin (ACTH), lipotropins (/3- and y-LPH), melanotropins (a-, /3- and y-MSH) and /3-endorphin 2z'23. Immunocytochemical studies have shown the presence of peptides derived from POMC in discrete populations of neurons in the brain of mammals 1°,16, and sub-mammalian vertebrates 3,7,36,37. In particular, aMSH-immunoreactive neurons have been detected in the basal hypothalamus of various species, namely in the infundibular nucleus in human 8, in the arcuate nucleus in rat 16'28 and in the ventral infundibular nucleus in frog 3'7'36. Similarly, y-MSH-like immunoreac-
tivity has been measured in extracts of bovine hypothalamia2, 33. Several lines of evidence indicate that melanotropins are functionally important neuropeptides. (i) Central administration of a-MSH induces a number of behavioral effects, such as enhancement of attention and memory 9, grooming behavior 14, agression 2°'z4 and reduction of body temperature2k (ii) Immunoelectron microscopic studies revealed that a-MSH is sequestered in dense core synaptic vesicles which are in a favorable position to be discharged upon nerve depolarization 2s. (iii) The release of a-MSH from rat hypothalamic neurons is stimulated by high potassium and veratridine in a calcium-dependent manner 17,35. Biochemical studies combining HPLC analysis and radioimmunological identification have shown the occurrence of peptides closely related to authentic aMSH in the central nervous system of both
Correspondence: H. Vaudry, European Institute for Peptide Research, Laboratory of Molecular Endocrinology, CNRS URA 650, UA INSERM, University of Rouen, 76134 Mont-Saint-Aignan, France~ Fax: (33) 35-146349.
vertebrates3.~s,37 3~ and insects:'. However, the primary structure of a melanotropin in brain tissue has never been determined. In the present study, we have isolated two forms of a - M S H and Lys-y~-MSH in the brain of the European green frog, Rana ridibunda.
NPY
I
NKB
E
cO
m
m U.l Z
MATERIALS AND METHODS
I, / k'\ nO rn
