Characteristics of phosphatidate phosphatase from developing seeds and microspore-derived cultures of oilseed rape
Kocsis, Michael G.
University of Lethbridge. Faculty of Arts and Science
Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 1994
Phosphatidate phosphatase (PAP. EC 22.214.171.124) was charaterized from developing seeds and microspore-derived (MD) cultures of oilseed rape. In studies with homogenate from developing seeds (Brassica napus L. cv Westar) the time course for release of inorganic phosphate from phosphatidate was linear for at least 60 min and the enzyme was stable to at least three cycles of freezing and thawing. Differential centrifugation studies were conducted with homogenate prepared from developing seeds (B. napus L. cv Westar), MD embryos (B. napus L. cv Reston), and an embryogenic MD cell suspension culture (B. napus L. cv Jet Neuf). Among the three tissue types, the level of microsomal PAP ranged from 11% to 17% of the total recovered PAP activity whereas soluble PAP ranged from 25% to 61% of the total activity recovered. Microsomal PAP displayed optimal activity in the pH range of 6 to 7 whereas soluble PAP had a pH optimum of 5. Microsomal and soluble PAP exhibited temperature reaction optima of 40 degrees celsius and 50 degrees celsius, respectively, with activation energies of 15.6 kcal/mol and 9.4 kcal/mol. Assays with p-nitrophenyl phosphate as a substrate at pH 6.75 and pH 5 indicated that the overal character of phosphatase activity in the microsomal fraction was different from the enzyme in the soluble microsomal PAP from MD embryos of B. napus L. cv Topas. Tween 20 solubilized PAP effectively with concomitant maintenance of enzyme in the soluble fraction. A number of detergents were screened for their ability to solubilize microsomal PAP from MD embryos of B. napus L. cv Topas. Tween 20 solubilized PAP effectively with concomitant maintenance of enzyme activity. The most effective solubilization of enzyme occurred at a concentration of 0.4% (w/v) Tween 20 at a detergent to protein ratio of 1:1 (w/w). The pH optimum (pH 6-7) of solubilized PAP was similar to that of the particulate enzyme and the assay of the solubilized enzyme was free from interference by phospholipase action. Solubilized microsomal PAP had an apparent Mr of about 300,000 based on gel filtration chromatography on a column of Superose 6. Polyclonal antibodies raised in mice against a crude extract from microsomes of MD embryos inhibited microsomal PAP activity.
xii, 128 leaves : ill. ; 29 cm.
Oilseeds , Rapeseed , Phosphatidate phosphatase , Rape oil , Dissertations, Academic