The membrane bound phosphatidic add phosphatase from avocado

Abstract

Phosphatidate Phosphatase (EC 3.1.3.4) is an important enzyme in plant lipid metabolism as it lies at a theoretical branchpoint between phospholipid and triacylglycerol biosyntheses. Since it's identification in 1955, it has received very little attention in plant lipid research. Previous studies reported in the literature have been limited to the use of crude plant cell extracts, making an accurate evaluation of the data difficult. The enzyme was characterised and purified to homogeneity from the microsomes of maturing avocado fruit, for the first time from any plant source. The novel procedure utilised detergent solubilisation in CHAPS, followed by anion exchange and Affi-Gel Blue chromatography, ammonium sulphate precipitation and Phenyl Superose chromatography. The enzyme had a subunit molecular mass, as determined by SDS-PAGE of 49kDa. Gel filtration studies revealed it was monomeric. Enzyme activity had a pH optimum of 6.0, was insensitive to N- ethylmaleimide and was stimulated by Mg(^2+). The homogenous enzyme was examined for the ability to hydrolyse sn-1,2- dioleoylglycerol-3-phosphate(PA), sn-1 -oleoylglycerol-3 -phosphate(LPA), sn-2- oleoylglycerol-3-phosphate, ceramide-1-phosphate and p-nitrophenylphosphate. All substrates were used, but the apparent V(_MAX) values for PA and LPA were considerably higher than the other substrates tested. The Michaelis-Menten kinetic model of enzyme catalysis was found to be inappropriate as the surface active enzyme was shown to be dependent on the bulk and surface concentration of the substrate in Triton X-100 mixed micelles. The surface dilution kinetic model was used to study PA and LPA hydrolysis. LPA was a better substrate and was also a potent competitive inhibitor of PA hydrolysis. Considering the specificities of the other enzymes in triacylglycerol biosynthesis, this premature dephosphorylation of LPA would prevent triacylglycerol formation. These findings possibly indicate that strict metabolic channelling is in operation with very low steady state concentrations of LPA with respect to PA, thus preventing any interaction with LPA in vivo.