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) cult ures of oilseed rape (B. napus L.). Differential centrifugation studie s were conducted with homogenate prepared from developing seeds, MD em bryos (B. napus L. cv. Reston) and an embryogenic MD culture (B. napus L. cv. Jet Neuf). Among the three tissue types, the level of microsom al phosphatidate phosphatase ranged from 11 to 17% of the total recove red enzyme activity whereas soluble phosphatidate phosphatase ranged f rom 25 to 61% of the total activity recovered. Microsomal phosphatidat e phosphatase from developing seed displayed optimal activity in the r ange 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 de phosphorylation of phosphatidate catalysed by phosphatidate phosphatas e in microsomal and soluble fractions from developing seed were 15.6 a nd 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 solub ilize phosphatidate phosphatase from microsomes of MD embryos (B. napu s L. cv. Topas) with the most effective solubilization of enzyme occur ring at a concentration of 0.4% (w/v) Tween 20 at a detergent to prote in ratio of 1 : 1 (w/w). Solubilized microsomal phosphatidate phosphat ase eluted within the sieving range of a Superose 6 column and display ed a minimum apparent M(r) of ca 40 000. The solubilized fraction cata lysed the hydrolysis of a number of forms of phosphatidate as well as various other phosphorylated compounds.