Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase".
Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D015260:Neprilysin; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits; D012844:Sincalide / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013758:Tetragastrin / Q000378:metabolism; D013997:Time Factors
D010446:Peptide Fragments; D013758:Tetragastrin; C025997:cholecystokinin (27-33); D010450:Endopeptidases; D015260:Neprilysin; D012844:Sincalide