TRYPTIC CLEAVAGE OF GASTRIC LIPASES - LOCATION OF THE SINGLE DISULFIDE BRIDGE

Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin a nd the resulting peptides were characterized. Exposure of HGL to tryps in 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 (Ly s-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the mol ecule 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 inhibit ory mAbs immunoreacted selectively with the N-terminal fragment H2, wh ereas 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 R 4). 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.