Tryptic cleavage of gastric lipases: location of the single disulfide bridge.
Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.
Aoubala
M
M
UPR 9025 "Lipolyse Enzymatique" du Centre National de la Recherche Scientifique, Marseille, France.
Bonicel
J
J
Bénicourt
C
C
Verger
R
R
De Caro
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D013270:Stomach / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism*
D004220:Disulfides; D010446:Peptide Fragments; D008049:Lipase; D014357:Trypsin