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. 2004 May 1;379(Pt 3):601–607. doi: 10.1042/BJ20031489

Identification and characterization of plant glycerophosphodiester phosphodiesterase.

Benoît Van Der Rest 1, Norbert Rolland 1, Anne-Marie Boisson 1, Myriam Ferro 1, Richard Bligny 1, Roland Douce 1
PMCID: PMC1224124  PMID: 14750903

Abstract

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.

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Selected References

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