Possibly there are similarities among
Possibly there are similarities among euphorb plant species but extraction of the D609 pathway present in the microsomal fraction is needed for testing their preferences for substrates. Although R. communis, E. lathyris and E. lagascae belong to the same family, the DGAT activity of their microsomal preparations has not been compared. The objective of this work was to test in vitro the preferences for oleoyl-CoA and 1,2–diolein, which are the predominant native substrates in E. lathyris and to compare with the microsomes of R. communis and E. lagascae.
Materials and methods
Results and discussion Table 1 summarizes the results of Reaction 1, where [14C] oleoyl-CoA was used as acyl donor and no acyl receptors were added. DGAT activity was detected in most of the preparations since the label was incorporated into TAG, except in R. communis H22 E (both incubation times) or E. lathyris ELAT E (6d) possibly because there was not endogenous DAG. Incorporation of [14C] oleoyl into TAG was 72–89% for R. communis or E. lathyris and 37–54% for E. lagascae. For E. lagascae VIR E and E. lagascae VIR R or E. lathyris ELAT R, image analysis gave null counts mm−2, but scintillation counter recorded 26, 64 or 82.9 CPM. In this case, we included the % of TAG formed. Hence, image analysis alone was not sufficient for the interpretation of the results. The extraction procedure retained the enzyme both for the three plant species and for their microsomal preparations (E, EP and R). Nevertheless, an optimization of the process is required since our samples were ultracentrifuged at 40,000×g. According to He et al. (2004a), most DGAT activity is recovered from pellets centrifuged at 100,000×g and the majority of DGAT proteins are present as partial products (50kDa) of the entire protein (60kDa) in microsomes of developing seeds. Increasing the incubation time from 6 to 15 days is not necessary. β emissions from TLC plates were registered onto the ‘screens’, in some cases increasing the time resulted in more counts (e.g. E. lagascae VIR EP, 52–89.8 or E. lathyris ELAT R, 82.9–283.3, see Table 1); however, the 6-day data could be a good estimate. Table 2 summarizes the results of Reaction 2, where [14C] oleoyl-CoA was used as acyl donor and 1,2-diolein was used as acyl receptor. Overall, this improved the resolution of the reaction products (TAG) as seen in the image analysis (Fig. 2A and B). Counts mm−2 ranged from 1062 to 8848 (6d) or from 1536 to 11,274 (15d). Addition of DAG to the reactions improves the observation of spots. ‘E’ microsomal preparations tended to be more effective (compared with EP and R) in incorporating [14C] oleoyl into 1,2-diolein (79% against 44–56% in R. communis (6-day incubation) or 71% against 28–39% in E. lathyris (15-day incubation)). Comparison of E. lagascae VIR E (in the plates in the 3rd channel from the left) in Fig. 2A (without DAG addition) and Fig. 2B (with DAG addition) shows that the radioactive acyl donor is present in both cases, but it gives more TAG product with the addition of 1mM 1,2-diolein. Yet, it represents only 39% of total TAG (Table 2), suggesting that there might be other unlabelled acyl donors, different from [14C] oleoyl, that react with DAGs. Wiberg et al. (1994) reported that the selectivity for acyl-CoA can be influenced by the DAG used as receptor. In this way, sunflower enzyme utilized 10:0-CoA (non-native) at the same rate as 18:1-CoA, but the first was preferred if the acceptor present was 10:0-DAG. From Fig. 2C, it can be seen that R. communis H22 E microsomal preparation yields more than one molecular species of labelled TAG: two spots can be observed, the larger spot represents 63% and the smaller more-polar spot represents 24% of total labelled TAG. We can presume that the latter is the result of using the endogenous 1,2-ricinolein rather than the exogenous 1,2-diolein as acyl receptor.