Characterization of triacylglycerol biosynthetic enzymes from microspore-derived cultures of oilseed rape

Particulate and solubilized preparations of phosphatidate (PA) phosphatase (EC 3.1.3.4) and dia-cylglycerol acyltransferase (DGAT, EC 2.3.1.20) from microspore-derived (MD) cultures of Brassica napus L. cv Topas were characterized. The activity of solubilized PA phosphatase decreased by about 50% following storage for 24 h at 4 degrees celsius, whereas the activity of DGAT decreased by 30%. Bovine serum albumin increased the stability of both enzymes. Both preparations were enriched in the target enzyme and thus, may be useful in studies of regulation with limited influence by the other Kennedy pathway enzymes. Solubilized PA phosphatase was shown to dephosphoryolate a number of phosphate-containing compounds and showed a preference for dioleoyl-PA and dipalmitoyl-PA over other forms of PA tested. Microsomal PA phosphatase from MD embryos was partially dependent on Mg2+ and partially inhibited by the thioreactive agent, N-ethylmaleimide (NEM). The partial sensitivity to NEM suggest that MD embryos of B. napus may contain forms of PA phosphatase involved in glycerolipid synthesis and signal transduction. NEM-sensitive and NEM-insensitive PA phosphatase activity was found in microsomes of a cell suspension culture of B. napus L. cv Jet Neuf. PA phosphatase, solubilized from MD embryos, was partially purified using ammonium sulfate fractionation followed by anion exchange chromatography. PA phosphatase was resolved into two distinct peaks following anion-exchange chromatography. The peaks contained both NEM-sensitive and NEM-insensitive PA phosphatase activity. Following gel filtration, solubilized PA phosphatase displayed a minimum apparent Mr of about 40 000. Antibodies raised against partially purified preparations of PA phosphatase and DGAT from MD embryos of B. napus L. cv Topas were used in the development of immunochemical probes for these enzymes. Inhibitory anti-PA phosphatase antibodies were developed. Attempts were also made to identify a sub-class of antibodies which could interact with both denatured and native DGAT.

Authors

• Furukawa-Stoffer, Tara L.
• University of Lethbridge. Faculty of Arts and Science

• Enzymes
• Canola oil
• Oilseeds
• Fatty acids -- Synthesis
• Dissertations, Academic

Identifier

hdl:10133/63

OAI identifier oai:identifier

oai:opus.uleth.ca:10133/63