Lipases of the euphorbiaceae family: purification of a lipase from Euphorbia characias latex and structure-function relationships with the B chain of ricin.
A lipase from the latex of Euphorbia characias was purified using a method involving extraction with apolar solvent and adsorption chromatography on silica gel. The lipase (specific activity, 1500 international units/mg of protein) was eluted from silica gel complexes with a lipid. The main protein fraction, which had a molecular mass of 38 kDa, was inactive when dissociated from the lipid fraction. When the lipid and protein fractions were reassociated, 72% of the lipolytic activity was recovered. This lipolytic activity was inhibited by diethyl p-nitrophenyl phosphate, which was shown to bind the lipase with a molar ratio of 0.75. High specific activities (1000 international units/mg) were measured for the lipase of E. characias on lipid extracts rich in galactosyl diacylglycerols. The apolipase was sequenced up to residue 23. The B chain of ricin has a strong homology (43.5%) with that sequence and cross-reacted with antibodies raised against the purified lipase from E. characias. The activity of the B chain of ricin was comparable (54 international units/mg) to that of the apolipase of E. characias (100 international units/mg) mixed with the same lipid cofactor complex. The primary structure (residues 68-72) of the B chain of ricin contains the lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly. Its reactivity with diethyl p-nitrophenyl phosphate indicates the presence of an activated serine that, in addition to its well-documented lectin activity for galactosides, suggests that the B chain of ricin may be a galactosyl diacylglycerol lipase, closely analogous to the lipase from E. characias.
Centre de Biochimie et de Biologie Moléculaire, Centre National de la Recherche Scientifique, Marseille, France.
A
A
Moulin
M
M
Teissère
C
C
Bernard
G
G
Piéroni
D000595:Amino Acid Sequence; D007840:Latex / Q000737:chemistry; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D010944:Plants / Q000201:enzymology*; D012276:Ricin / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007840:Latex; D037121:Plant Lectins; D010940:Plant Proteins; D012276:Ricin; D008049:Lipase