Human mitochondria1 dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human cDNA fragment for this protein. After virus infection and protein expression in Trichuplusia ni cells (BTI-Tn-5B 1-4), the subcellular distribution of the recombinant dihydroorotate dehydrogenase was determined by two distinct enzyme-activity assays and by Western blot analysis with anti-(dihydroorotate dehydrogenase) lg. The targeting of the recombinant protein to the mitochondria of the insect cells was verified. The activity of the recombinant enzyme in the mitochondria of infected cells was about 740-fold above the level of dihydroorotate dehydrogenase in human liver mitochondria. In a three-step procedure, dihydroorotate dehydrogenase was purified to a specific activity of greater than SO U/mg. Size-exclusion chromatography showed a molecular mass of 42 kDa and confirmed the existence of the fully active enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydroorotate dehydrogenase. By kinetics analysis, K,,, values for dihydroorotate and ubiquinone-50 were found to be 4 pM and 9.9 pM, respectively, while K,, values for dihydroorotate and decylubiquinone were 9.4 pM and 13.7 pM, respectively. The applied expression system will allow preparation of large quantities of the enzyme for structure and function studies. Purified recombinant human dihydroorotate dehydrogenase was tested for its sensitivity to a reported inhibitor A77 1726 (2-hydroxyethylidenecyanoacetic acid 4-trifluoromethyl anilide), which is the active metabolite of the isoxazol derivative leflunomide [5-methyl-N-(4-trifluoromethyl-phenyl)-4-isoxazole carboximide]. An IC,, value of 1 pM was determined for A77 1726. Detailed kinetics experiments revealed uncompetitive inhibition with respect to dihydroorotate (Kl,, = 0.94 pM) and non-competitive inhibition with respect to decylubiquinone (K,c = 1.09 pM, K,, = 1.05 pM). These results suggest that the immunomodulating agent A77 1726 (currently in clinical phase 111 studies for the treatment of rheumatoid arthritis) is a very good inhibitor of human dihydroorotate dehydrogenase.

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Summary. During an investigation into the substrate specificity and processing of subtilisin Carlsberg from Bacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97-101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, Mr=42 kDa) was not processed to the mature form (Mr = 30 kDa) and was not released into the medium by a proteasedeficient B. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg.

An electrophysiological assay platform based on solid supported membranes (SSM) for the organic cation transporter (OCT) is presented. Stable Chinese hamster ovary (CHO) cell lines overexpressing the human (hOCT2) and rat transporters (rOCT2) were generated and validated. Membrane preparations from the cell lines were investigated using SSM-based electrophysiology. Baculovirus transfected insect cells (HighFive and Mimic Sf9) were also tested with the same assay but yielded less than optimal results. The assays were validated by the determination of substrate affinities and inhibition by standard inhibitors. The study demonstrates the suitability of the SSM-based electrophysiological OCT assay for rapid and automatic screening of drug candidates.