Regulatory Peptides, 41 (1992) 1-8
1
© 1992 Elsevier Science Publishers B.V. All fights reserved 0167-0115/92/$05.00
REGPEP 01203
The purification and sequence analysis of an avian neuromedin U Jan Domin l, Miguel A. Benito-Orfila, Kiran A. Nandha, Alastair Aitken 2 and Stephen R. Bloom Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London (UK) (Received 28 January 1992; revised version received and accepted 6 May 1992)
Key words: Neuromedin U; Chicken; Purification; Radioimmunoassay
Summary In this study we have purified an avian homologue of neuromedin U from the chicken. Each step of the purification process was followed by a specific radioimmunoassay developed using porcine neuromedin U. Microsequence analysis characterised the peptide to be 25 amino acid residues long with the following sequence: Y-K-V-DE-D-L-Q-G-A-G-G-I-Q-S-R-G-Y-F-F-F-R-P-R-N. Chicken neuromedin U has marked sequence similarity with the porcine peptide at its bioactive C-terminal region. Our findings demonstrate that the amino acid sequence of neuromedin U is markedly conserved in species which have diverged millions of years ago in evolutionary terms.
Introduction The bioactive neuropeptide neuromedin U was originally purified from extracts of porcine spinal cord in two molecular forms, neuromedin U8 and an N-terminally extended form termed neuromedin U25. In addition to its presence in the central nervous system, neuromedin U has also been localised in the neurons of the gastrointestinal tract, the pituitary and thyroid glands and urogenital tract [2,3]. In most species 1 Present address: Department of Growth Regulation, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK. 2 Present address: Laboratory of Protein Structure, National Institute For Medical Research, Mill Hill, London NW7 IAA, UK. Correspondence to: S.R. Bloom, 2nd Floor Francis Fraser Lab, Hammersmith Hospital, Du Cane Rd, Hammersmith, London W12 ONN, UK
however, only neuromedin U25-1ike molecular forms appear to be present. The guineapig is an exception since both neuromedin U8- and neuromedin U25-1ike molecular forms have been identified in tissues from this species [4]. Despite the similarity in their apparent molecular weights, chromatographic studies predicted a degree of inter-species molecular heterogeneity based on differential elution times. Such studies have prompted the purification and sequence analysis of neuromedin U peptides from several species including rat [ 5 ], rabbit [ 6 ], frog [ 7 ] and guinea-pig [ 8 ]. The aim of this investigation was to purify neuromedin U from an avian species. The chicken was chosen since its gastrointestinal tract was found to contain high concentrations of neuromedin U immunoreactivity. Analysis of this peptide would allow us to determine the degree to which its primary sequence is conserved between species as diverse as chicken and rat or pig. Such information would thereby give further insight into the degree of evolutionary pressure which has existed to conserve this sequence and consequently its biological role in these species.
Materials and Methods
Small intestines from 40 adult chickens were obtained from a local poultry farm and transported to the laboratory on ice. After washing the tissue with water, the mucosa was removed by tangentially scraping the lumenal surface with a stainless steel spatula. The remaining muscular layers (769 g wet wt.) were rinsed once more, diced and extracted with boiling 0.5 M acetic acid (3 volumes). This extract was decanted and the process repeated using flesh acid. Both supernatants were pooled and used for the purification process. The time between tissue collection and extraction did not exceed 4 h. At all stages of the purification, neuromedin U-like immunoreactivity was measured using a radioimmunoassay described previously [4]. The extract was next filtered through a muslin gauze under gravity and then under vacuum (Whatman No. 5). Aliquots (10 ml) were loaded onto activated Sep-Pak C18 cartridges (Waters Associates, Milford, MA, USA). The cartridges were then sequentially washed with water and 20~o acetonitrile (ACN), whereafter neuromedin U was eluted with 40 ~o aqueous ACN. The volume of the 40 ~o eluate was reduced by vacuum centrifugation (Speed Vac Concentrator, Uniscience, UK) and the pH of the solution adjusted to pH 6.0. The precipitate which had formed was removed by centrifugation (2000 g, 10 rain) and aliquots of the supernatant passed through anion exchange cartridges (QMA Sep-Pak, Waters Associates). Effluates were collected and lyophilised. The residue was resuspended in aqueous 0.05 ~ trifluoroacetic acid (TFA) and applied to a phenyl column (Radial Pal
1
© 1992 Elsevier Science Publishers B.V. All fights reserved 0167-0115/92/$05.00
REGPEP 01203
The purification and sequence analysis of an avian neuromedin U Jan Domin l, Miguel A. Benito-Orfila, Kiran A. Nandha, Alastair Aitken 2 and Stephen R. Bloom Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London (UK) (Received 28 January 1992; revised version received and accepted 6 May 1992)
Key words: Neuromedin U; Chicken; Purification; Radioimmunoassay
Summary In this study we have purified an avian homologue of neuromedin U from the chicken. Each step of the purification process was followed by a specific radioimmunoassay developed using porcine neuromedin U. Microsequence analysis characterised the peptide to be 25 amino acid residues long with the following sequence: Y-K-V-DE-D-L-Q-G-A-G-G-I-Q-S-R-G-Y-F-F-F-R-P-R-N. Chicken neuromedin U has marked sequence similarity with the porcine peptide at its bioactive C-terminal region. Our findings demonstrate that the amino acid sequence of neuromedin U is markedly conserved in species which have diverged millions of years ago in evolutionary terms.
Introduction The bioactive neuropeptide neuromedin U was originally purified from extracts of porcine spinal cord in two molecular forms, neuromedin U8 and an N-terminally extended form termed neuromedin U25. In addition to its presence in the central nervous system, neuromedin U has also been localised in the neurons of the gastrointestinal tract, the pituitary and thyroid glands and urogenital tract [2,3]. In most species 1 Present address: Department of Growth Regulation, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK. 2 Present address: Laboratory of Protein Structure, National Institute For Medical Research, Mill Hill, London NW7 IAA, UK. Correspondence to: S.R. Bloom, 2nd Floor Francis Fraser Lab, Hammersmith Hospital, Du Cane Rd, Hammersmith, London W12 ONN, UK
however, only neuromedin U25-1ike molecular forms appear to be present. The guineapig is an exception since both neuromedin U8- and neuromedin U25-1ike molecular forms have been identified in tissues from this species [4]. Despite the similarity in their apparent molecular weights, chromatographic studies predicted a degree of inter-species molecular heterogeneity based on differential elution times. Such studies have prompted the purification and sequence analysis of neuromedin U peptides from several species including rat [ 5 ], rabbit [ 6 ], frog [ 7 ] and guinea-pig [ 8 ]. The aim of this investigation was to purify neuromedin U from an avian species. The chicken was chosen since its gastrointestinal tract was found to contain high concentrations of neuromedin U immunoreactivity. Analysis of this peptide would allow us to determine the degree to which its primary sequence is conserved between species as diverse as chicken and rat or pig. Such information would thereby give further insight into the degree of evolutionary pressure which has existed to conserve this sequence and consequently its biological role in these species.
Materials and Methods
Small intestines from 40 adult chickens were obtained from a local poultry farm and transported to the laboratory on ice. After washing the tissue with water, the mucosa was removed by tangentially scraping the lumenal surface with a stainless steel spatula. The remaining muscular layers (769 g wet wt.) were rinsed once more, diced and extracted with boiling 0.5 M acetic acid (3 volumes). This extract was decanted and the process repeated using flesh acid. Both supernatants were pooled and used for the purification process. The time between tissue collection and extraction did not exceed 4 h. At all stages of the purification, neuromedin U-like immunoreactivity was measured using a radioimmunoassay described previously [4]. The extract was next filtered through a muslin gauze under gravity and then under vacuum (Whatman No. 5). Aliquots (10 ml) were loaded onto activated Sep-Pak C18 cartridges (Waters Associates, Milford, MA, USA). The cartridges were then sequentially washed with water and 20~o acetonitrile (ACN), whereafter neuromedin U was eluted with 40 ~o aqueous ACN. The volume of the 40 ~o eluate was reduced by vacuum centrifugation (Speed Vac Concentrator, Uniscience, UK) and the pH of the solution adjusted to pH 6.0. The precipitate which had formed was removed by centrifugation (2000 g, 10 rain) and aliquots of the supernatant passed through anion exchange cartridges (QMA Sep-Pak, Waters Associates). Effluates were collected and lyophilised. The residue was resuspended in aqueous 0.05 ~ trifluoroacetic acid (TFA) and applied to a phenyl column (Radial Pal
