Phosphatidate phosphatase from developing seeds and microspore-derived cultures of Brassica napus

Phosphatidate phosphatase (EC 3.1.3.4) was characterized in developing seeds (Brassica napus L. cv. Westar) and microspore-derived (MD) cultures of oilseed rape (B. napus L.). Differential centrifugation studies were conducted with homogenate prepared from developing seeds, MD embryos (B. napus L. cv. Reston) and an embryogenic MD culture (B. napus L. cv. Jet Neuf). Among the three tissue types, the level of microsomal phosphatidate phosphatase ranged from 11 to 17% of the total recovered enzyme activity whereas soluble phosphatidate phosphatase ranged from 25 to 61% of the total activity recovered. Microsomal phosphatidate phosphatase from developing seed displayed optimal activity in the range pH 6-7 whereas soluble phosphatidate phosphatase had a pH optimum of 5. The activity of phosphatidate phosphatase from microsomes of MD embryos exhibited a similar pH dependence. Activation energies for dephosphorylation of phosphatidate catalysed by phosphatidate phosphatase in microsomal and soluble fractions from developing seed were 15.6 and 9.4 kcal mol(-1), respectively. Assays with p-nitrophenyl phosphate as a substrate at pH 6.75 and 5 indicated that the overall character of phosphatase activity in the microsomal fraction was different from that of the enzyme in the soluble fraction. Tween 20 was used to solubilize phosphatidate phosphatase from microsomes of MD embryos (B. napus L. cv. Topas) with the most effective solubilization of enzyme occurring at a concentration of 0.4% (w/v) Tween 20 at a detergent to protein ratio of 1 : 1 (w/w). Solubilized microsomal phosphatidate phosphatase eluted within the sieving range of a Superose 6 column and displayed a minimum apparent M(r) of ca 40 000. The solubilized fraction catalysed the hydrolysis of a number of forms of phosphatidate as well as various other phosphorylated compounds.