Purification and characterisation of a new hypothalamic satiety peptide, cocaine and amphetamine regulated transcript (CART), produced in yeast.
Cocaine and amphetamine regulated transcript (CART) is a newly discovered hypothalamic peptide with a potent appetite suppressing activity following intracerebroventricular administration. When the mature rat CART sequence encoding CART(1-102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation broth reflecting processing at dibasic sequences. None of these corresponded to the naturally occurring CART(55-102). In order to obtain CART(55-102) the precursor Glu-Glu-Ile-Asp-CART(55-102) has been produced and CART(55-102) was generated by digestion of the precursor with dipeptidylaminopeptidase-1. All four generated CART peptides have been characterised by N-terminal amino acid sequencing and mass spectrometry. The CART peptides contain six cysteine residues and using the yeast expressed CART(62-102) the disulphide bond configuration was found to be I-III, II-V and IV-VI. When the four CART peptides were intracerebroventricularly injected in fasted mice (0.1 to 2.0 microg) they all produced a dose dependent inhibition of food intake.
Thim
L
L
Department of Protein Chemistry, Health Care Discovery, Novo Nordisk A/S, Bagsvaerd, Denmark. lth@novo.dk
Nielsen
P F
PF
Judge
M E
ME
Andersen
A S
AS
Diers
I
I
Egel-Mitani
M
M
Hastrup
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004220:Disulfides / Q000032:analysis; D005285:Fermentation; D007031:Hypothalamus / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein
D017931:DNA Primers; D004220:Disulfides; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein