The ACT-DHOD gene in the kinetoplastid Bodo saliens encodes aspartate carbamoyltransferase and dihydroorotate dehydrogenase, the second and fourth enzymes of pyrimidine biosynthesis. Although the single mRNA species yielded a 70-kDa ACT-DHOD protein, Western blotting with anti-DHOD-peptide antibody showed a major band of 35-kDa and minor bands. In-gel digestion and liquid chromatography tandem mass (MS/MS) spectrometry showed that the 35-kDa band contained DHOD-specific polypeptides and an ACT-specific polypeptide, suggesting the occurrence of independent DHOD and ACT. Immunoprecipitation and MS/MS analysis identified a 70-kDa ACT-DHOD and a 35-kDa DHOD independently, and the N-terminal amino acid of 35-kDa DHOD was blocked. In vitro processing assay showed that recombinant ACT-DHOD was decreased by the R saliens lysate, accompanying the appearance of 35-kDa DHOD and 35-kDa ACT. These results indicate that fused ACT-DHOD is the precursor to mature DHOD. Large amount of 35-kDa DHOD in B. saliens is discussed from a viewpoint of its physiological roles. (c) 2007 Elsevier Inc. All rights reserved.