Peptides,Vol. 11, pp. 613-617. ©PergamonPressplc, 1990.Printedin the U.S.A.
0196-9781/90$3.00 + .00
BRIEF COMMUNICATION
Isolation and Microsequence Analysis of a Novel Form of Neuromedin U From Guinea Pig Small Intestine R. MURPHY, *l C. A. TURNER,* J. B. FURNESS,* L. P A R K E R t AND A. G I R A U D t
*Department of Anatomy and Histology and Centre for Neuroscience, School of Medicine Flinders University, Bedford Park, S.A. 5042, Australia and "PDepartment of Medicine, University of Melbourne, Western Hospital Footscray, Victoria 3011, Australia Received 11 December 1989
MURPHY, R., C. A. TURNER, J. B. FURNESS, L. PARKERAND A. GIRAUD.Isolationand microsequenceanalysisof a novel form of neuromedin Ufrom guineapig small intestine. PEPTIDES 11(3) 613--617, 1990.--A multidimensionalchromatographic regimen has been used to isolate and purify a peptide showingimmunoreactivityfor neuromedinU from guinea pig small intestine. Microsequence Edman N-terminalanalysisand C-terminalanalysisby enzymaticdigestionshowed this peptide to be a nonapeptide with the followingsequence:H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH 2. The C-terminaloctapeptideof this sequenceis the same as porcine NMU-8, and the C-terminalheptapeptide is identicalto rat NMU(17-23). NeuromedinU
Neuropeptides
Isolation
S e q u e n c e Guineapig
NEUROMEDIN U-8 and neuromedin U-25 are two related neuropeptides that have been isolated and sequenced from porcine spinal cord (9). The octapeptide constitutes the C-terminal region of NMU-25, flanked by a dibasic sequence (Arg-Arg) and is thus likely to be derived from the larger peptide. Both peptides show uterine contractile activity and pressor effects (9,13), and NMU-8 enhances water and electrolyte secretion in the porcine intestine, but does not affect gut muscle contractility (3). The distribution of neuromedin U-like immunoreactivity (NMU-LI) has been mapped in rat brain and intestine (1, 2, 5, 8), and in the guinea pig small intestine (1,7), where it has been shown to be localized extensively in neurons and in some endocrine cells. The molecular form of NMU present in rat tissues has been shown to differ from the porcine peptides by chromatographic analyses (1,4), and has recently been isolated and sequenced from rat small intestine (10). The rat peptide is 23 amino acids long and, whilst the C-terminal heptapeptide is identical to the porcine peptide, the remainder of the molecule shares only limited sequence homology with the porcine peptide and the dibasic cleavage point is not present. NMU-LI has also been chromatographically characterized in
Intestine
extracts of human and guinea pig brain and intestine (1,4). NMU-LI from guinea pig brain contains two major components that have similar chromatographic behavior to the porcine peptides, but NMU-LI extracted from the intestine of this species is much more heterogeneous. The major intestinal component elutes in a similar position to porcine NMU-8, and a minor component coelutes with porcine NMU-25, but there are also other immunoreactive components which do not coelute with any known forms of neuromedin U. The present study was undertaken to isolate the major component of NMU-LI present in guinea pig small intestine and to determine its amino acid sequence. METHOD
General Carboxypeptidase Y was purchased from Boehringer (Australia), and dimethylaminonaphthalenesulphonyl chloride (dansyl chloride) was purchased from Sigma Chemical Co. (U.S.A.). All other chemicals were purchased from Ajax Chemicals (Australia)
1Requestsfor reprintsshouldbe addressedto Dr. Roger Murphy,Departmentof Anatomy,Hinders MedicalCentre, BedfordPark, S.A. 5042, Australia.
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614
MURPHY, TURNER, FURNESS, PARKER AND GIRAUD
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0196-9781/90$3.00 + .00
BRIEF COMMUNICATION
Isolation and Microsequence Analysis of a Novel Form of Neuromedin U From Guinea Pig Small Intestine R. MURPHY, *l C. A. TURNER,* J. B. FURNESS,* L. P A R K E R t AND A. G I R A U D t
*Department of Anatomy and Histology and Centre for Neuroscience, School of Medicine Flinders University, Bedford Park, S.A. 5042, Australia and "PDepartment of Medicine, University of Melbourne, Western Hospital Footscray, Victoria 3011, Australia Received 11 December 1989
MURPHY, R., C. A. TURNER, J. B. FURNESS, L. PARKERAND A. GIRAUD.Isolationand microsequenceanalysisof a novel form of neuromedin Ufrom guineapig small intestine. PEPTIDES 11(3) 613--617, 1990.--A multidimensionalchromatographic regimen has been used to isolate and purify a peptide showingimmunoreactivityfor neuromedinU from guinea pig small intestine. Microsequence Edman N-terminalanalysisand C-terminalanalysisby enzymaticdigestionshowed this peptide to be a nonapeptide with the followingsequence:H-Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH 2. The C-terminaloctapeptideof this sequenceis the same as porcine NMU-8, and the C-terminalheptapeptide is identicalto rat NMU(17-23). NeuromedinU
Neuropeptides
Isolation
S e q u e n c e Guineapig
NEUROMEDIN U-8 and neuromedin U-25 are two related neuropeptides that have been isolated and sequenced from porcine spinal cord (9). The octapeptide constitutes the C-terminal region of NMU-25, flanked by a dibasic sequence (Arg-Arg) and is thus likely to be derived from the larger peptide. Both peptides show uterine contractile activity and pressor effects (9,13), and NMU-8 enhances water and electrolyte secretion in the porcine intestine, but does not affect gut muscle contractility (3). The distribution of neuromedin U-like immunoreactivity (NMU-LI) has been mapped in rat brain and intestine (1, 2, 5, 8), and in the guinea pig small intestine (1,7), where it has been shown to be localized extensively in neurons and in some endocrine cells. The molecular form of NMU present in rat tissues has been shown to differ from the porcine peptides by chromatographic analyses (1,4), and has recently been isolated and sequenced from rat small intestine (10). The rat peptide is 23 amino acids long and, whilst the C-terminal heptapeptide is identical to the porcine peptide, the remainder of the molecule shares only limited sequence homology with the porcine peptide and the dibasic cleavage point is not present. NMU-LI has also been chromatographically characterized in
Intestine
extracts of human and guinea pig brain and intestine (1,4). NMU-LI from guinea pig brain contains two major components that have similar chromatographic behavior to the porcine peptides, but NMU-LI extracted from the intestine of this species is much more heterogeneous. The major intestinal component elutes in a similar position to porcine NMU-8, and a minor component coelutes with porcine NMU-25, but there are also other immunoreactive components which do not coelute with any known forms of neuromedin U. The present study was undertaken to isolate the major component of NMU-LI present in guinea pig small intestine and to determine its amino acid sequence. METHOD
General Carboxypeptidase Y was purchased from Boehringer (Australia), and dimethylaminonaphthalenesulphonyl chloride (dansyl chloride) was purchased from Sigma Chemical Co. (U.S.A.). All other chemicals were purchased from Ajax Chemicals (Australia)
1Requestsfor reprintsshouldbe addressedto Dr. Roger Murphy,Departmentof Anatomy,Hinders MedicalCentre, BedfordPark, S.A. 5042, Australia.
613
614
MURPHY, TURNER, FURNESS, PARKER AND GIRAUD
A
B - 60
F-"
Vo
