Rat dihydroorotate dehydrogenase: isolation of the recombinant enzyme from mitochondria of insect cells

Protein Expression and Purification
Wolfgang KnechtMonika Löffler

Abstract

Mammalian dihydroorotate dehydrogenase (EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis is located in the mitochondrial inner membrane with functional connection to the respiratory chain. From the cDNA of rat liver dihydroorotate dehydrogenase cloned in our laboratory the first complete sequence of a mammalian enzyme was deduced. Two hydrophobic stretches centered around residues 20 and 357, respectively, and a short N-terminal mitochondrial targeting sequence of 10 amino acids was proposed. A recombinant baculovirus containing the rat liver cDNA for dihydroorotate dehydrogenase was constructed and used for virus infection and protein expression in Trichoplusia ni cells. The targeting of the recombinant protein to mitochondria of the insect cells was monitored by activity determination of dihydroorotate dehydrogenase in subcellular compartments in comparison to succinate dehydrogenase activity (EC 1.3.5.1), which is a specific marker enzyme of the inner mitochondrial membrane. The results of subcellular distribution were verified by Western blotting with anti-dihydroorotate dehydrogenase immunoglobulins. The activity of the recombinant enzyme in the mitochondria of infected insect cells was found to be about 57...Continue Reading

References

Nov 1, 1978·Archives of Biochemistry and Biophysics·H J Forman, J Kennedy
Oct 1, 1985·Analytical Biochemistry·P K SmithD C Klenk
May 5, 1982·Journal of Molecular Biology·J Kyte, R F Doolittle
Oct 1, 1995·Molecular Membrane Biology·S L Rusch, D A Kendall

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Citations

Mar 23, 2000·European Journal of Biochemistry·J RawlsM Löffler
Jul 25, 2009·Journal of Bioenergetics and Biomembranes·Marianna MadeoVincenza Dolce
Mar 17, 2000·Brain Research. Molecular Brain Research·T HolcombN P Curthoys

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