Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate.
Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0-9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10(-2) M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000+/-500 and 2800+/-60 micromol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240+/-17 and 432+/-62 U/mg, respectively) on MGDG.
Amara
Sawsan
S
CNRS-Aix-Marseille Université-Enzymologie Interfaciale et Physiologie de la Lipolyse-UPR 9025, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Lafont
Dominique
D
Fiorentino
Brice
B
Boullanger
Paul
P
Carrière
Frédéric
F
De Caro
Alain
A
D001647:Bile Acids and Salts / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D038983:Galactolipids / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008823:Micelles; D010179:Pancreas / Q000201:enzymology*; D013379:Substrate Specificity / Q000187:drug effects
C011439:1,2-diacylglycerol; D001647:Bile Acids and Salts; D004075:Diglycerides; D038983:Galactolipids; D008823:Micelles; D002265:Carboxylic Ester Hydrolases; C019262:galactolipase; D008049:Lipase; C075988:pancreatic lipase related protein 2; D002118:Calcium