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Primary structure of a novel mast cell degranulating peptide from the skin of the African frog, Hylambates maculatus, exhibiting an unusual post-translational modification
Abstracts from the 11th InternationalSymposium on Regulatory Peptides
CHARACTERISATION OF ADRENOMEDULL1N RECEPTORS AND THE PRESENCE OF ADRENOMEDULLIN mRNA AND IMMUNOREACT1VITY IN THE RAT SPINAL CORD. David M. Smith, Mohammad A. Ghatei, Ali A. Owji, James V. Gardiner, Paul D. Upton, and Stephen R. Bloom. Division of Endocrinology and Metabolic Medicine, Depaaament of Medicine, Royal Postgraduate Medical School, Hanunersmith Hospital, London, UK. Adrenomedullin is a 52 eanino acid peptide first isolated from human phaeochromocytomas in 1993 by its ability to activate platelet adenylyl cyclase. It is a potent and long lasting hypotensive agent with abundant specific binding sites explessed ill rat lung and hea~. Adrenomedullin and its binding sites have also been demonstrated in the CNS. We now show abm~dant (B,,~ = 723 + 71 fmol/mg protein), lfigh affinity (K o = 0.45 + 0.06 nM) binding sites for ~2q-adrenomedullin in rat spinal cord membranes. The structm'ally related peptides, CGRP, amylin and calcitonin did not bind at this site (1C~0> 10 ~M). Chemical cross-linking of the binding siteJ2Sl-adrenomedullin complex revealed a major band of relative molecular weight (Mr) = 84,000 + 1000 and a minor band of Mr = 122,000 ~: 9000. Enzymic deglycosylation of this site showed a considerable caJ'bohydrate content. Adrenomedullin binding sites have been associated with stimulation of the cAMP second messenger system, but no increase in adenylyl cyclase activity could be detected in spinal cord homogenates or membranes. To examine a possible paracrine role for adrenomedullin in flae spinal cord we detemfined the presence of adrenomednllin mRNA and immunoreactivity. Northern analysis revealed the presence of a single band con'esponding to that found in lung but one third of the intensity as measured by densitometry. Adrenomedullin-lR (eluting in the same fraction as rat adrenomedullin standard by reversed phase FPLC) was also detected although the concentration (40.5 ± 6.0 fmol/g wet weight tissue) was vel3' low compared to the message. Thus the presence of abundant binding sites and mRNA anticipates an as yet undefined function for this peptide in the spinal cord.
Primary structure of a novel mast cell degranulating peptide from the skin of the African frog, Hylambates maculatus, exhibiting an unusual post-translational modification A. F. Smyth, M. Cross, A. Johnsen*, D. W. Halton and C. Shaw Comparative Neuroendocrinology Research Group, Schools of Clinical Medicine and Biology & Biochemistry, The Queen's University of Belfast, Northern Ireland and *Department of Clinical Chemistry, Rigshospitalet, Copenhagen, Denmark. The skin of the African frog, Hylambates maculatus, is rich in biologically-active peptides including the tachykinins, hylambatin and (Glu 2, ProS)-kassinin, (Asn2, LeuS)-caemlein and the chemotactic peptide, XO-4. Using a rat peritoneal mast cell degranulation assay, we have isolated a novel active peptide from this species. The primary structure of this novel active peptide was established as: FKEGLLLTVTGVINAVTNA. This peptide displays limited structural homology to the previously isolated chemotactic peptide, XO-4. However, in cycle 3 of the sequencer, two PTH-amino acids were detected - a minor peak coincident with PTH-GIu and and a major peak eluting immediately after PTH-Val. This phenomenon has been previously encountered in an antimicrobial peptide from another species of frog. The identity of the major PTH-amino acid peak was established as Glu-OEt. The presence of this glutamic acid ethyl ester may be artefactual, caused by the ethanolic extraction medium, or may represent an actual post-translational modification. In each case, the glutamic acid residue is next to a basic residue in the structure. The effect of this modification on bioactivity is the subject of current investigation.
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CHARACTERISATION OF ADRENOMEDULL1N RECEPTORS AND THE PRESENCE OF ADRENOMEDULLIN mRNA AND IMMUNOREACT1VITY IN THE RAT SPINAL CORD. David M. Smith, Mohammad A. Ghatei, Ali A. Owji, James V. Gardiner, Paul D. Upton, and Stephen R. Bloom. Division of Endocrinology and Metabolic Medicine, Depaaament of Medicine, Royal Postgraduate Medical School, Hanunersmith Hospital, London, UK. Adrenomedullin is a 52 eanino acid peptide first isolated from human phaeochromocytomas in 1993 by its ability to activate platelet adenylyl cyclase. It is a potent and long lasting hypotensive agent with abundant specific binding sites explessed ill rat lung and hea~. Adrenomedullin and its binding sites have also been demonstrated in the CNS. We now show abm~dant (B,,~ = 723 + 71 fmol/mg protein), lfigh affinity (K o = 0.45 + 0.06 nM) binding sites for ~2q-adrenomedullin in rat spinal cord membranes. The structm'ally related peptides, CGRP, amylin and calcitonin did not bind at this site (1C~0> 10 ~M). Chemical cross-linking of the binding siteJ2Sl-adrenomedullin complex revealed a major band of relative molecular weight (Mr) = 84,000 + 1000 and a minor band of Mr = 122,000 ~: 9000. Enzymic deglycosylation of this site showed a considerable caJ'bohydrate content. Adrenomedullin binding sites have been associated with stimulation of the cAMP second messenger system, but no increase in adenylyl cyclase activity could be detected in spinal cord homogenates or membranes. To examine a possible paracrine role for adrenomedullin in flae spinal cord we detemfined the presence of adrenomednllin mRNA and immunoreactivity. Northern analysis revealed the presence of a single band con'esponding to that found in lung but one third of the intensity as measured by densitometry. Adrenomedullin-lR (eluting in the same fraction as rat adrenomedullin standard by reversed phase FPLC) was also detected although the concentration (40.5 ± 6.0 fmol/g wet weight tissue) was vel3' low compared to the message. Thus the presence of abundant binding sites and mRNA anticipates an as yet undefined function for this peptide in the spinal cord.
Primary structure of a novel mast cell degranulating peptide from the skin of the African frog, Hylambates maculatus, exhibiting an unusual post-translational modification A. F. Smyth, M. Cross, A. Johnsen*, D. W. Halton and C. Shaw Comparative Neuroendocrinology Research Group, Schools of Clinical Medicine and Biology & Biochemistry, The Queen's University of Belfast, Northern Ireland and *Department of Clinical Chemistry, Rigshospitalet, Copenhagen, Denmark. The skin of the African frog, Hylambates maculatus, is rich in biologically-active peptides including the tachykinins, hylambatin and (Glu 2, ProS)-kassinin, (Asn2, LeuS)-caemlein and the chemotactic peptide, XO-4. Using a rat peritoneal mast cell degranulation assay, we have isolated a novel active peptide from this species. The primary structure of this novel active peptide was established as: FKEGLLLTVTGVINAVTNA. This peptide displays limited structural homology to the previously isolated chemotactic peptide, XO-4. However, in cycle 3 of the sequencer, two PTH-amino acids were detected - a minor peak coincident with PTH-GIu and and a major peak eluting immediately after PTH-Val. This phenomenon has been previously encountered in an antimicrobial peptide from another species of frog. The identity of the major PTH-amino acid peak was established as Glu-OEt. The presence of this glutamic acid ethyl ester may be artefactual, caused by the ethanolic extraction medium, or may represent an actual post-translational modification. In each case, the glutamic acid residue is next to a basic residue in the structure. The effect of this modification on bioactivity is the subject of current investigation.
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