مرکز منطقه ای اطلاع رساني علوم و فناوري - The treatment of purified maize oil bodies with organic solvents and exogenous diacylglycerol allows the detection and solubilization of diacylglycerol acyltransferase

Latin Abstract :

In spite of its importance in the biosynthesis of reserve oils in plants, diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) has not been purified to homogeneity, and its study has remained incomplete. We found that the microsomal preparations from developing maize embryos contained substantial amounts of endogenous diacylglycerol (DAG). A solubilization procedure for extracting DAGAT from the microsomes (D. Little, R. Weselake, K. Pomeroy, S.T. Furukawa, J. Bagu, Biochem. J. 304 (1994)) was ineffective in eliminating the endogenous DAG, even after gel filtration. DAG removal through the preparation of acetone powders from the embryos led to the loss of DAGAT activity. Labelled triacylglycerol (TAG) was produced in the standard DAGAT assay when labelled DAG was supplied in benzene solution to the freeze-dried microsomes and the sample was dried and resuspended in an aqueous buffer. In contrast, no labelled TAG was produced when a similar sample supplied with non-labelled DAG was assayed with emulsified labelled DAG and acyl-CoA. Repeated washing of the microsomal freeze-dried fraction with benzene resulted in a complete loss of DAGAT activity in the standard assay, but the activity was restored by the addition of DAG plus phosphatidylcholine or Tween 20 in benzene. Although DAGAT has been reported to be confined mainly to the endoplasmic reticulum, we found that DAGAT activity was high in the purified oil bodies from both developing and mature maize embryos and was not removed by repeated washing with 6 M urea. The DAGAT activity was restored from delipidated oil bodies and from microsomes after the preparations had been resuspended in methanol/acetic acid/water (1:1:1, v/v). Although most of the proteins in the suspension were eluted as a single peak at the void volume after gel filtration chromatography, DAGAT activity was found in later fractions. SDS–PAGE of the peak activity fraction revealed no protein bands after silver staining, and the finding suggest that DAGAT protein is of low abundance and has a high kcat.