The structural basis of the RNA selectivity of HCV RdRp was elucidated from its crystal structure. The putative substrate-binding site with a shallow hydrophilic cavity should have ribonucleoside triphosphate (rNTP) as the preferred substrate. We propose that the unique alpha fingers might represent a common structural discriminator of the template-primer duplex that distinguishes between RNA and DNA during the replication of positive single-stranded RNA by viral RdRps. The C-terminal region might exert a regulatory function on the initiation and activity of HCV RdRp.

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The primary structure of L-hi&dine decarboxylase (HDC: L-histidine carboxy-lyase, EC 4.1.1.22) from mouse mastocytoma P-81 5 cells has been determined by parallel analysis of the amino acid sequence of the protein and the nucleotide sequence of the corresponding cDNA. HDC contains 662 amino acid residues with a molecular mass of 74 017, which is larger by about 21000 Da than that of the previously purified HDC subunit (53 kDa), suggesting that HDC might be posttranslationally processed. The HDC cDNA hybridized to a 2.7 kilobase mRNA of mastocytoma cells. Homology was found between the sequences of mouse mastocytoma HDC and fetal rat liver HDC.Histidine decarboxylase; Mastocytoma P-8 15 cell; Amino acid sequence; cDNA sequence; Dopa decarboxylase; Polymerase chain reaction

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.

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