title: Proteolytic cleavage of stingray phospholipase A2: isolation and biochemical characterization of an active N-terminal form.
abstract: BACKGROUND
  Mammalian GIB-PLA2 are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. The aim of this study was to check some biochemical and structural properties of a marine stingray phospholipase A2 (SPLA2).
  
  
  RESULTS
  The effect of some proteolytic enzymes on SPLA2 was checked. Chymotrypsin and trypsin were able to hydrolyze SPLA2 in different ways. In both cases, only N-terminal fragments were accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic and chymotryptic attack generated 13 kDa and 12 kDa forms of SPLA2, respectively. Interestingly, the SPLA2 13 kDa form was inactive, whereas the SPLA2 12 kDa form conserved almost its full phospholipase activity. In the absence of bile slats both native and 12 kDa SPLA2 failed to catalyse the hydrolysis of PC emulsion. When bile salts were pre-incubated with the substrate, the native kinetic protein remained linear for more than 25 min, whereas the 12 kDa form activity was found to decrease rapidly. Furthermore, The SPLA2 activity was dependent on Ca²⁺; other cations (Mg²⁺, Mn²⁺, Cd²⁺ and Zn²⁺) reduced the enzymatic activity notably, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca²⁺.
  
  
  CONCLUSIONS
  Although marine and mammal pancreatic PLA2 share a high amino acid sequence homology, polyclonal antibodies directed against SPLA2 failed to recognize mammal PLA2 like the dromedary pancreatic one. Further investigations are needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of phospholipases.
authors.affiliation: Biochemistry Department, Science College, King Saud University, PO Box 22452, 11495 Riyadh, Saudi Arabia. abirghanouchi@yahoo.fr
authors.forename: Abir G Ben
authors.initials: AG
authors.lastname: Bacha
authors.forename: Hafedh
authors.initials: H
authors.lastname: Mejdoub
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D003429:Cross Reactions; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D054500:Group IB Phospholipases A2 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D012621:Seasons; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012860:Skates, Fish / Q000378:metabolism*; D013045:Species Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D001647:Bile Acids and Salts; D029941:Fish Proteins; D010446:Peptide Fragments; D054500:Group IB Phospholipases A2; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium