FIG. 5.

Knockout mutants lack native Toxoplasma PKG. (A) Native Tg-PKG isoform II (Tg-PKG II) is absent in knockout lines expressing the Et-PKG_FLAG or _FLAGEt-PKG transgenes (lanes with asterisk) but present in parental lines (lanes P) and in the wild-type strain (w.t.). Recombinant Et-PKG isoform II (Et-PKGII) was observed in the transgenic lines. Native and recombinant isoform I of Et-PKG and Tg-PKG comigrate and cannot be resolved by SDS-PAGE. PKG enzymes were purified from parasites by cGMP affinity chromatography and detected with antisera to a conserved C-terminal peptide epitope, TR7. (B) ³H-labeled compound 1 binding activity in knockout lines expressing Tg-PKG_FLAG T761Q and Tg-PKG_FLAG T761M alleles (T761 M-KO, T761Q-KO) compared with wild-type parasites (w.t.). Parasite lysates (obtained from ca. 5 × 10⁹ tachyzoites) were fractionated by MonoQ chromatography as described in Fig. 1. Fractions with peak cGMP-dependent kinase activity were pooled, and ³H-labeled compound 1 binding activity was assayed in the absence (black bars) or presence (gray bars) of 40 μM unlabeled ligand. Activities presented are normalized for the total PKG activity of the pooled samples. The total PKG activity recovered from either knockout line (T761M-KO or T761Q-KO) was approximately fourfold higher than the total recovered from untransformed parasites of the parental strain (RHΔHXGPRT).