An enzymatically active truncated form (-55 N-terminal residues) of rabbit gastric lipase. Correlation between the enzymatic activity and disulfide bond oxydo-reduction state.
Rabbit gastric lipase (RGL) was subjected to proteolysis with trypsin and led to cleavage occurring at three defined sites (Lys-4, Arg-55 and Arg-229). The tryptic hydrolysate contained four fragments: Gly-230-Lys-379 (T1), Gly-56-Arg-229 (T2), Ser-5-Arg-55 (T3), as well as a 45 kDa molecular form consisting of peptides T1 and T2 linked by a disulfide bridge. The tryptic hydrolysate of RGL as well as the 55 N-terminal amino acid deleted forms conserved 30% of the initial enzymatic activity in a tributyrin assay. Two out of the three cysteine residues which are present in all the known gastric lipases were found to be involved in a disulfide bridge. Unlike HGL, RGL appears to have a heterogenous pattern of cysteine residues. The 30% enzymatic activity of RGL persisting after trypsin treatment may be attributable to the 45 kDa molecular form (with the Cys-227-Cys-236 or Cys-227-Cys-244 disulfide bridge). Trypsin-treated HGL, which was completely inactivated, showed that a single location of the disulfide bridge existed between cysteine residues 236 and 244. It can be concluded that the existence of one disulfide bridge is necessary to maintain the lipase activity of the 45 kDa form of RGL.
De Caro
J
J
Laboratoire de Lipolyse Enzymatique, UPR 9025 de l'IFRC 1 du CNRS, BP 71, 31, Chemin Joseph-Aiguier, F-13402 Marseille Cedex 20, France. caro@ibsm.cnrs-mrs.fr
Verger
R
R
De Caro
A
A
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D008049:Lipase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008066:Lipolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D017421:Sequence Analysis; D013270:Stomach / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D004220:Disulfides; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D003553:Cystine; D008049:Lipase; D014357:Trypsin; D003545:Cysteine; D004229:Dithiothreitol