SOLUBILIZATION AND CHARACTERIZATION OF DIACYLGLYCEROL ACYLTRANSFERASEFROM MICROSPORE-DERIVED CULTURES OF OILSEED RAPE

articulate fractions prepared from microspore-derived (MD) embryos of oilseed rape (Brassica napus L. cv. Reston) and an embryogenic MD cell suspension culture of oilseed rape (B. napus L. cv. Jet Neuf) were us ed as a source of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) f or enzyme characterization and development of a solubilization procedu re. DGAT activity in the 1500-100000 g fraction from MD embryos was st imulated 4-5-fold by 3 to 4 mg of BSB/ml of reaction mixture. DGAT act ivity from MD embryos was stimulated 2-3-fold by fluoride salts and 1. 4-fold by NaCl, whereas iodide salts caused substantial inhibition of enzyme activity. The effect of the various 1:1 electrolytes on enzyme activity appeared to be related more to their differential effects on solution structure rather than ionic strength. DGAT was solubilized fr om membranes of MD embryos and the cell suspension culture by about 80 and 50% respectively, using 2 M NaCl in 1% (w/v) octanoyl-N-methylglu camide (MEGA-8) (pH 8.0 buffer) at a detergent to protein ratio of 2:1 . The specific activity of solubilized DGAT was about 2-fold greater t han that of the particulate enzyme. The mechanism of solubilization ap peared to be related to the lowering of the critical micellar concentr ation of MEGA-8 in the presence of NaCl. DGAT, solubilized from MD emb ryos, eluted with an M(r) of about 2x10(6) during gel-filtration chrom atography on a Superose 6 column equilibrated in buffer containing 0.1 % (w/v) MEGA-8. The solubilized enzyme exhibited optimal activity at p H 7. At concentrations above 2 mu M acyl-CoA, the specificity of solub ilized DGAT for oleoyl-CoA and palmitoyl-CoA was considerably greater than for stearoyl-CoA.