Figure 3.

Patients' cells have no detectable enzymatic DCPS activity. (A) Protein profiles of purified recombinant GST:DCPS isoform 1 and GST:DCPS isoform 2 and isoform 2 V68A on SDS–PAGE. The contaminant migrating below GST-DCPS serves as a loading control. Positions of migration of molecular weight markers are indicated on the left side. (B) Enzymatic assay demonstrates the reduced activity of the DCPS when 7 amino acids is inserted between exon 1 and exon 2 of DCPS protein, especially in the V68A mutant. (C) Enzymatic assay demonstrates the loss of DCPS activity in patient's cells using m₇GpppG and m₇GpppGm₂′ substrates. Purified substrates are presented in lanes 1 and 5. (D) In vitro assays demonstrate that the absence of DCPS-dependent conversion of m₇GTP in extracts of cells from two affected individuals. m₇GTP is efficiently formed from m₇GDP in the presence of ATP (lanes 5–8), demonstrating that all extracts are similarly active. Trace of ATP in extract explains the residual formation of m₇GTP in the absence of added ATP (lanes 1–4). The purified substrate (+/− ATP) is presented in lanes 1 and 5.