PURIFICATION AND CHARACTERIZATION OF A NEW HYPOTHALAMIC SATIETY PEPTIDE, COCAINE AND AMPHETAMINE REGULATED TRANSCRIPT (CART), PRODUCED IN YEAST

Cocaine and amphetamine regulated transcript (CART) is a newly discove red hypothalamic peptide with a potent appetite suppressing activity f ollowing intracerebroventricular administration. When the mature rat C ART sequence encoding CART(1-102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation b roth reflecting processing at dibasic sequences. None of these corresp onded 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 dipe ptidylaminopeptidase-1. All four generated CART peptides have been cha racterised by N-terminal amino acid sequencing and mass spectrometry. The CART peptides contain sis cysteine residues and using the yeast ex pressed CART(62- 102) the disulphide bond configuration mas found to b e I-III, II-V and IV-VI. When the four CART peptides were intracerebro ventricularly injected in fasted mice (0.1 to 2.0 mu g) they all produ ced a dose dependent inhibition of food intake. (C) 1998 Federation of European Biochemical Societies.