Identification and characterization of plant glycerophosphodiester phosphodiesterase.
GPX-PDE (glycerophosphodiester phosphodiesterase; EC 3.1.4.46) is a relatively poorly characterized enzyme that catalyses the hydrolysis of various glycerophosphodiesters (glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, glycerophosphoserine and bis-glycerophosphoglycerol), releasing sn-glycerol 3-phosphate and the corresponding alcohol. In a previous study, we demonstrated the existence of a novel GPX-PDE in the cell walls and vacuoles of plant cells. Since no GPX-PDE had been identified in any plant organism, the purification of GPX-PDE from carrot cell walls was attempted. After extraction of cell wall proteins from carrot cell suspension cultures with CaCl2, GPX-PDE was purified up to 2700-fold using, successively, ammonium sulphate precipitation, gel filtration and concanavalin A-Sepharose. Internal sequence analysis of a 55 kDa protein identified in the extract following 2700-fold purification revealed strong similarity to the primary sequence of GLPQ, a bacterial GPX-PDE. To confirm the identity of plant GPX-PDE, an Arabidopsis thaliana cDNA similar to that encoding the bacterial GPX-PDE was cloned and overexpressed in a bacterial expression system, and was used to raise antibodies against the putative Arabidopsis thaliana GPX-PDE. Immunochemical assays performed on carrot cell wall proteins extracted by CaCl2 treatment showed a strong correlation between GPX-PDE activity and detection of the 55 kDa protein, validating the identity of the plant GPX-PDE. Finally, various properties of the purified enzyme were investigated. GPX-PDE is a multimeric enzyme, specific for glycerophosphodiesters, exhibiting a K(m) of 36 microM for glycerophosphocholine and active within a wide pH range (from 4 to 10). Since these properties are similar to those of GLPQ, the bacterial GPX-PDE, the similarities between plant and bacterial enzymes are also discussed.
Van Der Rest
Benoît
B
Laboratoire de Physiologie Cellulaire Végétale, UMR 5168, CEA, CNRS, INRA Université Joseph Fourier, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
Rolland
Norbert
N
Boisson
Anne-Marie
AM
Ferro
Myriam
M
Bligny
Richard
R
Douce
Roland
R
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D001419:Bacteria / Q000201:enzymology; D002473:Cell Wall / Q000201:enzymology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018552:Daucus carota / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D019143:Evolution, Molecular; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
D007106:Immune Sera; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase