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  • br Materials and methods br

    2019-10-08


    Materials and methods
    Results Because of the specific binding of DGKε to an arachidonoyl group, there was a particular interest to evaluate the behavior of 2-AG with this isoform of DGK. The substrate specificity and kinetic constants for DGKε has been recently reported [13]. Using the preferred substrate of DGKε, SAG, as a positive control, the rate of phosphorylation of 2-AG was only 6.35%±0.15% that of SAG. Thus, 2-AG essentially is a very poor substrate for DGKε. We also evaluated 2-OG as a substrate of this isoform of DGK, but the rate of phosphorylation was even lower than for 2-AG, reflecting the specificity of DGKε for arachidonoyl-containing lipids. The monoglyceride, 2-AG, was also tested as an inhibitor of DGKε. About 7.6mol% 2-AG was added to micelles containing different amounts of SAG (Fig. 1). The concentration dependence of the inhibition is complex and could not be analyzed quantitatively with any simple kinetic model. At low concentration of SAG, there is no inhibition by 2-AG likely because the 2-AG is a weak substrate. At higher concentrations of SAG, however, there is significant inhibition by 2-AG. At sufficiently high concentrations of 2-AG, there is essentially complete inhibition of the activity of DGKε in phosphorylating SAG (Fig. 2). The error bars given for this graph represent the precision of replicates in the assay performed under identical experimental conditions. There is somewhat greater experiment-to-experiment variation, likely resulting from factors such as different cell lysates being used as the source of enzyme for different experiments as well as the purity of the monoglyceride that is susceptible to both acyl chain migration as well as oxidation of the 2-AG. The extent of inhibition shown in Fig. 2 is somewhat greater than that presented in Fig. 1. Nevertheless, it is clear that the inhibition by 2-AG is much greater than that by 2-OG (Fig. 1). 2-OG has essentially no activity as either a substrate or as an inhibitor for the epsilon isoform of DGK. In faah inhibitors with DGKε, the isoform DGKζ exhibits similar inhibition with 2-OG and 2-AG (Fig. 3). A determination of the Michaelis–Menten constants of DGKζ has been recently studied in comparison with other isoforms [16].
    Discussion The finding that neither 2-AG nor 2-OG is a substrate for DGKε or DGKζ shows the specificity of DGKs for diacylglycerols. This is the case even for DGKε that has been shown to have a particularly strong specificity for an arachidonoyl group on the substrate [2]. We have previously shown that DGKε also has specificity for the acyl chain at the sn-1 position with 18:0 being the most favorable acyl chain at that position [13]. The diacylglycerol becomes a poorer substrate as the acyl chain becomes shorter than 18 carbons, but the effect is modest for fatty acids. However, when the acyl chain is completely absent, as with 2-AG, the lipid is essentially no longer a substrate. We have previously demonstrated that the enantiomer of the natural stereoisomer 1,2-dioleoylglycerol, i.e., 2,3-dioleoylglycerol, exhibits greater inhibition of DGKζ than of DGKε [16]. Similarly, we have determined that 2-AG and 2-OG have a very low potency of inhibition against DGKε compared with the inhibition of these monoglycerides with DGKζ. This behavior is analogous to the relative inhibitory effects of 2,3-dioleoylglycerol [16]. Since DGKε is more selective in substrate binding than other mammalian DGK isoforms, it is less inhibited by either 2,3-dioleoylglycerol or by 2-OG, than other mammalian DGK isoforms. In addition, although not a potent inhibitor, 2-AG is a better inhibitor of DGKε than is 2-OG. These results can be explained by the fact that DGKε binds arachidonoyl-containing lipids more specifically, as is also indicated by the arachidonoyl substrate specificity of this isoform. In addition to furthering our understanding of the properties of diacylglycerol kinases, there may be relevance of these findings to the role of endocannabinoids in neuronal function. 2-AG is an endocannabinoid that can arise from the DAG lipase catalyzed cleavage of SAG, the preferred substrate of DGKε. Another route of metabolism of SAG is by DGKε-catalyzed phosphorylation to generate SAPA. In DGKε knockout mice, the conversion of this particular species of diacylglycerol to phosphatidic acid is reduced [17]. Consequently, an alternative path for the SAG metabolism would be its conversion to the endocannabinoid, 2-AG by the DAG lipase. Based on our findings on the inhibitory property of 2-AG on DGKε, there could be a weak feed-forward effect of 2-AG on its own formation as a result of its inhibition of DGKε.