Contribution of blotting techniques to the study of rapeseeds (Brassica napus L.) lipases.
A recent advance in the study of plant lipases involving immunological techniques is presented. In an attempt to characterize lipases of cotyledons from germinating rapeseed seedlings and to investigate an eventual cross-reactivity with animal lipases, we have prepared anti-porcine pancreatic lipase antibodies raised in rabbit. It is shown by enzyme-linked immunosorbent assay and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Preincubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. We demonstrate immunochemical cross-reactivity between rapeseed and porcine pancreatic lipase. Using the immunoblotting procedure, it is found that antibodies bind specifically to a single polypeptide with a molecular mass of about 55 kDa. Rapeseed lipase activity decreased after immunoprecipitation suggesting that antibodies were bound to some catalytic site residues. We conclude from the data obtained in this study that the two different lipase species present close similarities in amino acid sequence and antigen characteristics.
Belguith
H
H
Laboratoire de Biochimie des protéines, Faculté des Sciences de Tunis, Tunis, Tunisia.
Khodjet el Khil
H
H
Fattouch
S
S
Jridi
T
T
Ben Hamida
J
J
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western / Q000379:methods; D001937:Brassica / Q000201:enzymology*; D018548:Cotyledon / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D008049:Lipase / Q000032:analysis*; D011817:Rabbits; D012639:Seeds / Q000201:enzymology; D013347:Subcellular Fractions; D013379:Substrate Specificity; D013552:Swine; D014280:Triglycerides / Q000378:metabolism
D014280:Triglycerides; D008049:Lipase