A new method for accurately measuring Delta(1)-pyrroline-5-carboxylate synthetase activity.
Proline is a key factor in plant adaptation to environmental stresses. The Delta(1)-pyrroline-5-carboxylate synthetase catalyzes the first committed step and the rate-limiting step for proline biosynthesis in both plants and mammals. This enzyme catalyzes the reduction of glutamate to pyrroline-5-carboxylate in two sequential steps including the phosphorylation and the reduction of its precursor. Several methods were established to assay P5CS activity but however none of them are fully reliable. Therefore, we developed a new simple and reliable assay which is based on the quantification of Pi. This assay allowed us to determine the optimal pH, the apparent K(m) and V(m) of P5CS with regard to ATP and glutamate.
Parre
Elodie
E
Université Pierre et Marie Curie-Paris6, CNRS, PCMP, Paris, France.
de Virville
Jacques
J
Cochet
Françoise
F
Leprince
Anne-Sophie
AS
Richard
Luc
L
Lefebvre-De Vos
Delphine
D
Ghars
Mohamed Ali
MA
Bordenave
Marianne
M
Zachowski
Alain
A
Savouré
Arnould
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D057075:Enzyme Assays / Q000379:methods*; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010936:Plant Extracts / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology; D012995:Solubility; D013379:Substrate Specificity
D010936:Plant Extracts; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor)