Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides.
The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation.
Pauwels
S
S
Department of Nuclear Medicine, University of Louvain, Brussels, Belgium.
Najdovski
T
T
Dimaline
R
R
Lee
C M
CM
Deschodt-Lanckman
M
M
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D005755:Gastrins / Q000378:metabolism*; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D015260:Neprilysin / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D012844:Sincalide / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013431:Sulfates
D000596:Amino Acids; D005755:Gastrins; D010446:Peptide Fragments; D013431:Sulfates; D015260:Neprilysin; D012844:Sincalide