Diacylglycerol Acyltransferase in Maturing Oil Seeds of Maize and Other Species

Abstract

Diacylglycerol acyltransferase (EC 2.3.1.20) activity was detected in the microsomal fractions of maturing maize scutellum, soybean cotyledon, peanut cotyledon, and castor bean endosperm. The activity detected was high enough to account for the in vivo rate of triacylglycerol synthesis. The activity of the maize enzyme was characterized using diolein micelles prepared by sonication in Tween 20 as the substrate. The activity was highest at pH values of 6 to 7. The activity was proportional to the amount of enzyme added, and the reaction rate was linear for about 2 minutes. The enzyme was not inactivated by Tween 20, Zwitterion 3-08, Triton-X 100, and cholate, but was inactivated completely by sodium dodecyl sulfate. The enzyme was active on linoleoyl coenzyme A (CoA), palmitoyl CoA, and oleoyl CoA, although the activity was highest on linoleoyl CoA. Endogenous diacylglycerol was present in the microsomes, and the enzyme activity was only partially dependent on the addition of external diolein. Subcellular fractionation of the total scutellum extract in sucrose density gradients was performed. By comparing the migration of the enzyme between rate and equilibrium centrifugation, and between equilibrium centrifugation in the presence and absence of magnesium ions in the preparative media, the enzyme was shown to be associated with the rough endoplasmic reticulum. Some of the above findings on the maize enzyme were extended to the enzymes from castor bean, soybean, and peanuts.Diacylglycerol acyltransferase (EC 2.3.1.20) catalyzes the final step in the synthesis of triacylglycerols in oil seeds (18,22). It is also the only known enzyme unique to the long biosynthetic pathway of triacylglycerols, since the diacylglycerol produced could also be used to produce phospholipids or galactolipids. In spite of the importance of this enzyme in triacylglycerol biosynthesis in oil seeds, its properties have not been well studied.Diacylglycerol acyltransferase in the microsomal fraction of developing seeds has been assayed directly or together with other enzymes (6,10,20,21). In a recent, and so far the most detailed, study, the general properties of the enzyme in a safflower microsomal fraction were characterized (10). However, the detected level ofactivity was almost a magnitude lower than that required to catalyze the sequence of reaction from glycerol phosphate to triacylglycerol (21, 22). Using an enzyme assay which had been used successfully to study the enzyme in spinach leaves, Martin and Wilson (13) failed to detect enzyme activity in the cotyledon extract of developing soybean.An important but unknown aspect of diacylglycerol acyltransferase is its subcellular location. In general, the microsomal fractions were used to study the enzyme activity, and they 'Supported by National Science Foundation grant DMB 85-15556.presumably contained vesicles of the ER as well as membranes of other subcellular particles, including broken plastids. In a recent detailed analysis of the subcellular location ofthe enzyme in spinach...

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Endoplasmic Reticulum, Oleosins, and Oils in Seeds and Tapetum Cells

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