In vitro phosphorylation by cAMP-dependent protein kinase up-regulates recombinant Saccharomyces cerevisiae mannosylphosphodolichol synthase.

DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphorylated by cAMP-dependent protein kinase (PKA). We have been studying the up-regulation of DPMS activity by protein kinase A-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S. cerevisiae in this study to advance our knowledge further. DPMS catalytic activity was indeed enhanced severalfold when the recombinant protein was phosphorylated in vitro. The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme. A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P). Kinetic studies indicated that there was no change in the Km for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the Vmax was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (kcat) and enzyme efficiency (kcat/Km). SDS-PAGE followed by autoradiography of the 32P-labeled DPMS detected a 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS. To confirm the phosphorylation activation of recombinant DPMS, serine 141 in the consensus sequence was replaced with alanine by PCR site-directed mutagenesis. The S141A DPMS mutant exhibited more than half-a-fold reduction in catalytic activity compared with the wild type when both were analyzed after in vitro phosphorylation. Thus, confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine 141.

Hepatitis C viral RNA amplification by the polymerase chain reaction allows detection of false positive HCV ELISAs among patients with non-A, non-B hepatitis

Serum from patients which tested positive for hepatitis C virus (HCV) by Enzyme Linked Immunosorbent Assay (ELISA) were analyzed for the presence of HCV RNA by nested Polymerase Chain Reaction (PCR) and for anti-HCV antibodies by Recombinant Immunoblot Assay (RIBA II). Total RNA was extracted from whole blood by a new procedure and subjected to reverse transcription of HCV RNA employing primers to the conserved 5' non-coding region of the HCV genome. PCR performed on these samples uncovered several false positive ELISAs. Reciprocal confirmation between PCR and RIBA II results was observed. These results substantiate this variation of the HCV PCR assay as a reliable alternative for routine confirmation of HCV serological tests