Figure 2.

Cocaine synthase enzyme activity, protein, and gene transcript levels compared with the amounts of reaction substrates and products in different E. coca organs and developmental stages. A, Cocaine- and cinnamoylcocaine-forming enzyme activity in desalted E. coca protein preparations. Enzymatic assays were performed using methylecgonine with either benzoyl-CoA or cinnamoyl-CoA as cosubstrates. Values displayed are means ± sd of three technical replicates from each of three biological replicates. B, Quantification of methylecgonine, cocaine, and cinnamoylcocaine. Values displayed are means ± sd of at least three biological replicates. C, Absolute quantification of RNA transcripts of cocaine synthase (EcBAHD7) and EcBAHD8 in E. coca. Values displayed are means ± sd of three technical replicates from each of three biological replicates. D, Levels of cocaine synthase protein determined by immunoblotting. Samples consisting of 15 μg of protein extracted from each organ as well as 75 ng (C1) and 150 ng (C2) recombinant Strep-tagged cocaine synthase (50.7 kD) were run on SDS-PAGE and gels were blotted onto filters. The filters were first probed with anticocaine synthase antibodies, followed by incubation with secondary antibodies conjugated to HRP. Polyclonal antibodies recognize cocaine synthase as well as EcBAHD8 (Supplemental Fig. S2B). Bands were visualized by chemiluminescence. L1 are young expanding leaves, still rolled after emerging from bud; L2 are larger expanding leaves, unrolled; and L3 are mature, nonexpanding leaves. DW, Dry weight, F, flower; FW, fresh weight; R, root; S, stem.