Chemical and enzymatic treatment of endothelin.
Endothelin-1 (ET), the most potent vasoconstrictor yet discovered, is a peptide containing 21 amino acids with two intrachain disulfide bridges. With the aim of obtaining two-chain derivatives, Et was submitted to chemical and enzymatic treatments. Reaction of ET with CNBr in 70% HCOOH gave, in addition to the expected [Hse7 lactone]-7,8-seco-ET and unreacted material, a by-product whose molecular weight was 25 m.u. greater than that of ET. When the reaction mixture, after lyophilisation, was immediately quenched with NH3-saturated dry MeOH, two products could be recovered in a 5:1 ratio, both obtained by nucleophilic attack of the homoserine lactone: the expected [Hse7-NH2]-7,8-seco-ET and [Hse7]ET, resulting from competitive intramolecular reaction of the deprotonated alpha-amino group of the Asp8 residue. The Lys9-Glu10 bond turned out to be very resistant to enzymatic attack both by Lys-C-endopeptidase and trypsin. The 9,10-seco-ET derivative could be obtained by treatment with Lys-C-endopeptidase only by using a high enzyme/ET ratio and after a prolonged incubation time. Cleavage of the Lys9-Glu10 bond could not be achieved by treatment with trypsin, even with a high enzyme/substrate ratio. The main product was 13,14-seco-ET, deriving from the action of chymotripsin (present as an impurity in the trypsin preparation) on Tyr13. The structure of these peptides was confirmed by amino-acid sequence analysis and fast atom bombardment mass spectrometry (FAB-MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives.
Perego
R
R
Pharmacia, Nerviano, Milan, Italy.
Gozzini
L
L
Arlandini
E
E
Bolis
G
G
de Castiglione
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D016232:Endothelins / Q000737:chemistry* / Q000378:metabolism; D005612:Freeze Drying; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D016232:Endothelins; D010446:Peptide Fragments; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide