Purification, sequencing and characterization of phospholipase D from XXXX seeds.

@entity844 @entity1718 @entity39377 @entity1078 @entity4613 @entity2434 @entity70550 @entity3633 @entity15130 @entity141 @entity13344 @entity3689 @entity3634
UNASSIGNED: Phospholipase D (PLD; E.C. 3.1.4.4) is widespread in plants where it fulfills diverse functions in growth and in the response to stresses. The enzyme occurs in multiple forms that differ in their biochemical properties. In the present paper PLD from medicinally relevant @entity13344 seeds was purified by @entity141 -mediated hydrophobic interaction and anion exchange chromatography to electrophoretic homogeneity. Based on mass-spectrometric sequence analysis of tryptic protein fragments, oligonucleotide primers for cloning genomic DNA fragments that encoded the enzyme were designed and used to derive the complete @entity3689 sequence of this PLD. The sequence data, as well as the molecular properties (molecular mass of 92.0kDa, pI 5.39, maximum activity at pH 5.5-6.0 and @entity141 ion concentrations    60mM), allowed the assignment of this enzyme to the class of a-type PLDs. The apparent kinetic parameters Vmax and Km, determined for the hydrolysis of @entity2434 (PC) in an aqueous mixed-micellar system were 356  15 molmin(-1)mg(-1) and 1.84  0.17mM, respectively. @entity4613 analogs such as @entity70550 and @entity39377 displayed strong inhibition of the enzyme. @entity1078 had a strong activating effect at 2-10mM @entity15130 . PLD was inactivated at temperatures >45  C. The enzyme exhibited the highest activity toward PC followed by @entity3633 and @entity3634 . PCs with short-chain @entity1718 were better substrates than PCs with long @entity844 chains. Lyso-PC was not accepted as substrate.
