Rat Dihydroorotate Dehydrogenase: Isolation of the Recombinant Enzyme from Mitochondria of Insect Cells

PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

10, 89–99 (1997)

PT960714

Rat Dihydroorotate Dehydrogenase: Isolation of the Recombinant Enzyme from Mitochondria of Insect Cells Wolfgang Knecht, Dagmar Altekruse, Andrea Rotgeri, Sigrid Gonski,* and Monika Lo¨ffler1 Institute for Physiological Chemistry, School of Medicine, Philipps-University, D-35033 Marburg, Germany; and *Central Research, Hoechst AG, D-65931 Frankfurt, Germany

Received October 7, 1996, and in revised form December 11, 1996

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 570-fold above the level of dihydroorotate dehydrogenase in rat liver mitochondria. By cation exchange chromatography of the Triton X-114 solubilisate of mitochondria, dihydroorotate dehydrogenase was purified to give a specific activity of 15 U/mg at pH 8.0. This was a marked progress over the six-step purification procedure of the enzyme from rat liver which resulted in a specific activity of 0.7 U/mg at pH 8.0. The characteristic flavin absorption spectrum obtained with the recombinant enzyme gave strong evidence that the rodent enzyme is a flavoprotein. By en1 To whom correspondence should be addressed at Philipps-University Marburg, School of Medicine, Institute for Physiological Chemistry, Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany. Fax: /6421-286957. E-mail: [email protected].

zyme kinetic studies Km values for dihydroorotate and ubiquinone were 6.4 and 9.9 mM with the recombinant enzyme, and were 5.0 and 19.7 mM, respectively, with the rat liver enzyme. After expression of only truncated forms of human dihydroorotate dehydrogenase, the present successful generation of the complete rodent enzyme using insect cells and the efficient procedure will promote structure and function studies of the eukaryotic dihydroorotate dehydrogenases in comparison to the microbial enzyme. q 1997 Academic Press

The special compartmentation of pyrimidine de novo synthesis in eukaryotes has been well established. Whereas five enzymes of the pathway are located in the cytosol as multifunctional proteins, dihydroorotate dehydrogenase (EC 1.3.99.11) was located in the inner mitochondrial membrane (1,2), with the exception of Saccharomyces cerevisiae and Trypanosoma species where the enzyme was found to be cytosolic. With respect to the topochemistry and the proximal electron acceptor ubiquinone (Q-10), mammalian dihydroorotate dehydrogenase resembles succinate dehydrogenase known as a specific marker of the inner mitochondrial membrane. The activities of dihydroorotate dehydrogenase and the other enzymes of pyrimidine metabolism were found to be higher in liver than in other tissues. Recently, the activity ratio of dihydroorotate dehydrogenase and succinate dehydrogenase in isolated rat liver mitochondria was determined to be 1:15 (3). The enzymes of pyrimidine metabolism were determined long ago to be of great importance for the development of therapeutic agents against malignant cell proliferation. The mitochondrially bound dihydroorotate dehydrogenase is of increasing interest lately as a target for new drugs to reduce aberrant immunological reactions (4,5), and to interfere in the multiplication of animal parasites and parasitic protozoa in malaria89

1046-5928/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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infected people (6,7), but also for new anticancer strategies (8–10). The mode and mechanism of interaction of such compounds with the enzyme is far from being understood. The low abundance and the membrane association of the native mammalian dihydroorotate dehydrogenases may have been an impediment to purify the enzyme to homogeneity, and/or to obtain it in sufficient amounts for in vitro characterization studies. The cDNA of rat liver dihydroorotate dehydrogenase was the first complete sequence known of the mammalian form of the enzyme (11) (deposited with the EMBL sequence data bank and available under Accession No. X80778). The protein sequence of the rodent enzyme as deduced revealed close identity (88%) and very high similarity (92%) to the human species, for which a large-fragment cDNA was isolated by complementation of a defective yeast strain (12) (deposited with the EMBL sequence data bank and available under Accession No. M94065). From expression of a smaller sequence in Escherichia coli strain M15 pREP, we obtained a histidine-tagged protein of 313 amino acids from human dihydroorotate dehydrogenase (13), which after purification was a satisfactory antigen for immunization of rabbits and laying hens to receive anti-dihydroorotate dehydrogenase immunoglobulins (unpublished data from this laboratory). The expressions of a 39- and a 42-kDa fragment of the human enzyme by means of heterologous expression systems have been reported very recently, but the full sequence is not yet known (14,15). The enzyme of Drosophila melanogaster was found to contain an additional 9 amino acids at the N-terminus (16) and the molecular weight of dihydroorotate dehydrogenase from the eukaryote Plasmodium falciparum was determined to be about 56,000 kDa (7), corresponding to the length of the recently cloned enzyme with 567 amino acids (17). Here, we communicate the overexpression of the complete 43-kDa rat liver dihydroorotate dehydrogenase protein. This was obtained to high level using the baculovirus vector expression system followed by an improved and shortened purification procedure to yield the catalytically active enzyme with high specific activity. We show that the catalytic properties of the recombinant enzyme are similar to those determined for the enzyme following isolation from rat liver mitochondria. We demonstrate the flavoprotein character of the rodent dihydroorotate dehydrogenase, which could not have been proven with the enzyme from tissue preparations. MATERIALS AND METHODS

Materials Unless otherwise stated, chemicals, buffer, and media components were from Boehringer, Serva, Merck, or Sigma (Germany), at the purest grade available.

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Fetal calf serum and Pluronic F68 were from Sigma; Ex-cell 401 culture medium was from JRH Biosciences (Lenexa, U.S.A.). Restriction enzymes were from MBI Fermentas, Fmol sequencing system was from Promega, and Qiaex gel extraction kit and Qiaprep spin plasmid kit from Qiagen (Germany) ; pVL 1393 baculotransfervector and Baculo Gold DNA were from Pharmingen (U.S.A.); lipofectin, gentamycin, and the DH5a cells were from Gibco BRL (Germany). Trichoplusia ni cells (BTI-Tn-5B1-4), referred to here as High Five cells) were from Invitrogen as High Five; Spodoptera frugiperda cells (Sf 21 cells) were obtained from Deutsche Sammlung fu¨ r Mikrobiologie (GBF, Braunschweig, Germany). Synthetic oligonucleotides were prepared by service from the Microchemical Unit, Institute of Molecular Biology (Marburg). SPSepharoseHP, Q-Sepharose-FF, and Sephacryl S300HR were from Pharmacia Biotech (Freiburg, Germany). Matrex Gel Orange A, Blue A, Red A, and Green A were from Amicon (Witten,Germany). Rabbit anti-chicken horseradish peroxidase-coupled IgG was from Sigma. Goat anti-rabbit IgG conjugated with horseradish peroxidase was from Dako (Germany). The enhanced chemoluminescence (ECL) detection kit was from Amersham Life Science (Germany). Methods Preparation of Autographa californica nuclear polyhedrosis transfer vector. The previously cloned (11) complete cDNA of rat dihydroorotate dehydrogenase was digested with Mva1269I and EcoRI, separated from the vector by agarose gel electrophoresis, and recovered by extraction from the agarose gel. A linker using two short oligonucleotides (5*gatccgcggccgcca 3*, 5*gcggccgcg 3*) was constructed to be compatible with a BamHI and a Mva1269I site. Equivalent amounts of the two oligonucleotides were annealed by cooling down from 957C to room temperature over several hours. Both the linker and the dihydroorotate dehydrogenase insert were ligated into a EcoRI/BamHI-digested and agarose gel purified pVL 1393 baculotransfer vector and transformed into DH5a cells using standard techniques (18). Plasmids were purified by Qiagen Spin Preps and positive clones were identified by restriction pattern analysis. The start of translation was verified by dideoxy sequencing (19). Construction of recombinant baculovirus and protein expression. Cotransfection of pVL1393-rat dihydroorotate dehydrogenase with Autographa californica nuclear polyhedrosis virus (Baculo Gold) DNA was done into SF 21 cells; 2 1 106 SF 21 cells in 2 ml Ex-cell 401 supplemented with 10% fetal calf serum, 0.1% Pluronic F 68, and 1 mg/ml gentamycin were seeded into a 35mm culture dish and allowed to set for 1 h at 277C. Three micrograms pVL 1393-rat dihydroorotate dehy-

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drogenase, 0.5 mg Baculo Gold DNA, and 30 ml lipofectin were incubated in serum-free Ex-cell 401 in a final volume of 100 ml for 15 min at room temperature. Cells were changed to serum-free medium and the DNA– lipofectin mixture was added and incubated at 277C for 24 h before changing to fetal calf serum supplemented medium again. The supernatant containing recombinant virus was harvested 4 days later and used for infection of SF21 cells to prepare high-virus titer stock. The titer of recombinant virus was determined as described in Ref. (18). The virus used in the present report was named rat dihydroorotate dehydrogenase. For protein expression High Five cells were cultured at 277C in Ex-cell 401 up to a density of 2 1 106 cells/ml in small spinner flasks. Cells were infected with rat dihydroorotate dehydrogenase at 10 plaque forming units/ cell and harvested after 72 h. In the present report the expressed protein was named recombinant dihydroorotate dehydrogenase. Preparation of mitochondria. All procedures were carried out at 47C or on ice. The insect cells were harvested by centrifugation at 1000g, washed in phosphate-buffered saline, and stored at 0807C until further use. The cells were disrupted (Dounce homogenizer) in isolation medium containing 250 mM sucrose, 10 mM Tris/HCl, 1 mM EDTA, pH 7.4, supplemented with the proteinase inhibitors phenantroline, aprotinin, pepstatin, leupeptin at 1 mg/ml each, benzamidinehydrochloride at 1.6 mg/ml, and 0.16 mM phenylmethylsulfonyl fluoride. The cells were centrifuged at 500g for 10 min. The procedure was repeated with the pellet. The supernatants were pooled and centrifuged at 30,000g for 10 min. After a final washing step this pellet (mitochondrial fraction) was taken for further studies or frozen at 0807C. The activity of recombinant dihydroorotate dehydrogenase (see below) of the mitochondrial fraction was compared to that in mitochondria isolated from a sample of rat liver in parallel, according to the same procedure. Isolation and purification of recombinant dihydroorotate dehydrogenase. Trichoplusia ni cells were cultured and infected in a Super-Spinner (Braun-Biotech, Germany), which was developed for optimal supply with oxygen of eukaryotic cells grown at high density. Mitochondria isolated from a 200-ml batch of cells were destroyed by an ultraturax for 10 s, mixed with an equal volume of buffer, pH 7.4, (25 mM potassium phosphate, 2% Triton X-114, 150 mM sodium chloride, proteinase inhibitors as above) to give 2–3 mg/ml protein. The mixture was stirred for 60 min and then centrifuged at 100,000g for 60 min. The supernatant solution was applied at 2 ml/min to a SP-Sepharose HP column equilibrated with 25 mM potassium phosphate and 0.1% Triton X-100, pH 7.4. The column was washed and eluted in the same buffer with a 0.0 to 0.5 M potassium

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chloride gradient. After elution, fractions containing dihydroorotate dehydrogenase activity were pooled (30 ml) and stored at 0207C. Peak activity was observed in a 6-ml fraction. Protein determination, SDS–PAGE, and Western blot analysis. Protein content was determined using the bicinchoninic acid protein assay with bovine serum albumine as standard (20). SDS–PAGE of cell fractions, mitochondria, and fractions of the enzyme purification protocol was performed using standard techniques (15,21). For immunochemical detection of dihydroorotate dehydrogenase, proteins from SDS–PAGE were transferred onto ImmobilonP membranes (Millipore, Germany) by semidry blotting and exposed to anti-peptide dihydroorotate dehydrogenase immunoglobulins as previously described (13). Polyclonal immunoglobulins as obtained from an immunization protocol with truncated recombinant dihydroorotate dehydrogenase protein of 32.7 kDa in a chinchilla bastard rabbit (K. Grein and M. Lo¨ffler, unpublished data) and the ECL detection kit were used additionally. NH2-terminal sequencing. The purified enzyme was run on an SDS–PAGE gel as described before. The protein was transferred from the gel onto an ImmobilonP membrane by semidry blotting. After visualization of the protein with Coomassie brilliant blue the band was excised and subjected to N-terminal sequence analysis on an Applied Biosystems 476a protein sequencer (22). Spectrophotometric flavin determination. Ultraviolet–visible spectroscopy was performed on an Ultraspec 2000 (Pharmacia Biotech) with a 0.1 mg/ml protein fraction obained from SP-Sepharose. The flavin content was estimated from the absorbance at 456 nm (e Å 11,000–16,000 liters 1 mol01 1 cm01) given for enzyme-bound flavins (23). Isolation of rat liver dihydroorotate dehydrogenase. The isolation was based on the six-step protocol previously given (24) with improvements. Briefly, rat liver was homogenized in a medium containing 300 mM saccharose, 10 mM Tris/HCl, 1 mM EDTA, pH 7.4, using a Potter–Elvehjem. The centrifugation procedure was as described before; the 30,000g pellet (mitochondrial fraction) was used for isolation of the enzyme protein. Solubilization of mitochondria was performed via phospholipase A2 and C treatment (100 mM triethanolamine/HCl buffer, pH 7.2) and Triton X-100 extraction (protein:detergent ratio was 2:1, 50 mM Tris/HCl buffer, pH 9) . Following a 20–40% ammoniumsulfate precipitation, the proteins were applied to gel filtration on Sephacryl-S300. The active fractions were applied to anion exchange chromatography (Q-Sepharose FF) using 20 mM Tris/HCl, 0.1% Triton X-100, pH 9.0. Elution was with a linear salt gradient (0–500 mM KCl) in the same buffer. Matrex Gel Orange A affinity chro-

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matography was performed using 20 mM Tris/HCl, 0.1% Triton X-100, pH 7.5, and the same buffer with a linear salt gradient (0–2 M KCl) for elution of dihydroorotate dehydrogenase. The fractions containing the active enzyme were pooled to give 70 ml. A fraction of 10 ml contained the highest specific activity. Enzyme assays: Spectrophotometric tests. Enzyme assays with homogenate, isolated mitochondria, or dihydroorotate dehydrogenase after purification were run at 307C. The reaction mixture of the chromogen reduction assay for the isolated enzyme contained 0.1 mM decylubiquinone (or 0.1 mM Q-10), 1 mM L-dihydroorotate, 0.06 mM 2,6-dichloroindophenol, 0.1% Triton X-100 in 50 mM Tris/HCl buffer, 150 mM KCl, pH 8.0. The loss of absorbance was monitored at 600 nm (e Å 18,800 liters 1 mol01 1 cm01 of 2,6-dichloroindophenol). The enzyme activity evaluated in control assays containing no decylubiquinone or Q-10 was õ2% of maximum velocity. No activity was detected if the control assay was run in the absence of dihydroorotate. For comparison, the activity of the mitochondrial marker enzyme succinate dehydrogenase was determined with 10 mM succinate in detergent-free phosphate buffer, pH 7.4, as recommended by Ragan et al. (26). If the enzymes were tested with mitochondria, 10 mM sodium cyanide was added in order to prevent the flow of electrons along the respiratory chain competitive to the transfer on 2,6-dichloroindophenol (3). In an alternative assay (containing 100 mM potassium phosphate buffer, pH 8.0, without detergent, quinone and cyanide), the enzyme activity in rat liver mitochondria was determined by measuring the orotate produced in the supernatant of acid-precipitated samples at 280 nm (e Å 7000 liters 1 mol01 1 cm01 of orotate). The assays were linear with respect to the amount of protein and time up to at least 15 min. In this assay the activity of dihydroorotate dehydrogenase could be inhibited by addition of cyanide, which blocked the ultimate transfer of electrons to oxygen at the stage of cytochrome oxidase (EC 1.9.3.1). Enzyme assays: Measurement of oxygen consumption. Frozen mitochondria (nonphosphorylating mitochondria) isolated from the insect cells were taken for the assay of dihydroorotate dehydrogenase and, for comparison, of succinate dehydrogenase activity. Samples in 100 mM phosphate buffer, pH 7.5, were transferred to a vessel of 0.5 ml (307C) connected to a Clark-type electrode (MK-Oxylab1.81b System, Biolytik, Germany) and equilibrated for 5 min. After addition of 10 mM dihydroorotate or 10 mM succinate, oxygen consumption was determined amperometrically; without substrate oxygen consumption was negligible. Calculations of activity were made with respect to 2 mol of substrate per mole of oxygen. The oxygen consumption could be blocked on addition of 10 mM sodium cyanide as inhibitor of cytochrome oxidase (3).

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Subcellular distribution. The subcellular distribution of the dihydroorotate dehydrogenase activity was according to the protocol of Ito et al. (25). Briefly, cells were harvested and homogenized as described above. The pellets collected at 650g were resuspended in 250 mM sucrose, 10 mM Tris/HCl, pH 7.5, 1 mM EDTA, washed, and centrifuged at 650g. The supernatants were combined and centrifuged at 5000g for 10 min. The subsequent supernatant was centrifuged at 10,500g for 10 min. The resulting supernatant was centrifuged at 105,000g for 60 min at 47C in order to receive the cytosolic fraction in the final supernatant. Samples were stored at 0807C until further use; aliquots were tested for enzyme activitiy as described above or analyzed by Western blotting. Kinetic analysis (27): Km determination. The concentration of dihydroorotate was varied from 1 mM to 1 mM at a fixed concentration of 100 mM decylubiquinone (Q-D) or 100 mM Q-10. The decylubiquinone or Q10 concentration was varied from 1 to 100 mM at a fixed concentration of dihydroorotate (1 mM). For determination of initial velocities the chromogen reduction assay was used. Data were fit to Eq. [1] v Å V 1 A/(Km / A),

[1]

where A is the substrate concentration, using a computer program that provides an iterative nonlinear least squares fit to the best rectangular hyperbola. The program Sigma plot 5 (Jandel Scientific, Sigma) was used for the calculations (see below). Kinetic analysis: pH dependence. Initial velocities at saturating substrate concentrations (1 mM dihydroorotate, 100 mM decylubiquinone) were measured in different buffer systems (Mes/HCl, Hepes/HCl, Tris/ HCl) covering a pH range from 5.5 to 9 using the chromogen reduction assay. Overlapping pH ranges were measured in two buffer systems to exclude salt effects. Data were fit to Eq. [2]: v Å V/[(100pH/100pKa1) / (100pKa2/100pH) / 1].

[2]

Hydrophobicity. The hydrophobicity of dihydroorotate dehydrogenase was estimated by examining a 21amino-acid sliding window of the deduced protein sequence using the program pSAAM by A.R. Croft (University of Illinois), using the hydrophobicity search of Kyte and Doolittle (28). RESULTS AND DISCUSSION

Information Given by the Dihydroorotate Dehydrogenase Sequence Dideoxynucleotide sequencing of the pVL1393 rat dihydroorotate dehydrogenase vector revealed three de-

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FIG. 1. Hydrophobicity plot of dihydroorotate dehydrogenase. The hydrophobicity was estimated by examining a 21-amino acid sliding window of the deduced protein sequence according to Kyte and Doolittle (28) and using the program pSAAM by A. R. Croft.

viations concerning the previously published nucleotide sequence at positions 242–244 (3 C instead of 3 G, with resulting exchange of Trp and Val to Ser and Leu). After confirmation by sequencing the original plasmid the correction was submitted to the EMBL database (Accession No. X80778, RRDYHED). The protein sequence as deduced from the cDNA of rat liver dihydroorotate dehydrogenase was analyzed for characteristic sequences. At the N-terminus, a typical but short mitochondrial targeting sequence of 10 amino acids (Met-Ala-Trp-Arg-Gln-Leu-Arg-Lys-ArgAla-) was found (29, for review). The hydrophobicity plot (Fig. 1) indicated hydrophobic stretches around the amino acid at positions 20 and 357 sufficient in length to form putative transmembrane helices (Asp-Ala-Val-Ile2-Leu-Gly4-Leu2-Phe-Thr-Ser-TyrLeu-Thr and Gly-Ala-Ser-Leu-Val-Gln-Leu-Tyr-Thr-AlaLeu-Ile-Phe-Leu-Gly-Pro2-Val3-Arg, respectively). It remains a matter of discussion whether the first stretch could additionally function as an inner membrane targeting signal, since it is not known whether and to what extent the enzyme protein of mammalian tissues is processed to its mature form following the uptake in the organelle. Similarily, the exact orientation of the enzyme at the membrane—whether the active site faces the matrix or the intermembrane space—is not understood, since biochemical and ultrastructural studies lead to different conclusions (1,2). Expression and Location of Recombinant Dihydroorotate Dehydrogenase To date, only a few mitochondrial proteins have been expressed using the baculovirus expression vector sys-

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tem, and only two of these, the ferrochelatase (EC 4.99.1.1) and the glycerol-3-phosphate acyltransferase (EC 2.3.1.15), are membrane-associated proteins (30,31). We employed the baculovirus to express the complete, catalytically active rat dihydroorotate dehydrogenase in High Five insect cells. To control the results of protein expression, homogenates of wild-type A. californica nuclear polyhedrosis-infected cells, of noninfected cells, and of rat dihydroorotate dehydrogenase-infected cells were separated by SDS–PAGE. Coomassie staining revealed a distinct band (lane 4 of Fig. 2) below the 45-kDa marker protein (lane 1) only in the rat dihydroorotate dehydrogenase-infected cells, which corresponded to the expected molecular weight of the 42.7-kDa dihydroorotate dehydrogenase sequence (11). In order to make sure of the intracellular distribution of the expressed dihydroorotate dehydrogenase, five subfractions of the insect cells were prepared by differential centrifugation. Samples of pellets and supernatants were analyzed for dihydroorotate dehydrogenase activity. The activity of succinate dehydrogenase, as a convenient and specific marker for the inner mitochondrial membrane (26), was monitored with the same samples. The specific activities measured by the chromogen reduction assay, which contained 2,6-dichloroindophenol as artificial electron acceptor, are summarized in Table 1. The highest specific activity and 56% of total dihydroorotate dehydrogenase activity, and 68% of total succinate dehydrogenase ac-

FIG. 2. Homogenates of High Five cells on sodium dodecyl sulfate– polyacrylamide gel electrophoresis. Protein (8 mg) from whole cell homogenates were applied for each lane and separated according to the method given by Westermeier (21). After electrophoresis, the gels were stained with Coomassie brilliant blue. The arrow points to the recombinant dihydroorotate dehydrogenase protein. Lane 1, lowrange molecular mass marker. Lane 2, High Five cells, wild-type virus-infected. Lane 3, High Five cells, noninfected. Lane 4, High Five cells, rat dihydroorotate dehydrogenase-infected.

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KNECHT ET AL. TABLE 1

Subcellular Distribution of Dihydroorotate Dehydrogenase and Succinate Dehydrogenase Activity Dihydroorotate dehydrogenase

Succinate dehydrogenase

/Chromogen

/Chromogen

Fraction (g)

U/mg

% Activity

O2 consumption (1003 U/mg)

650 5,000 10,500 105,000 Cytosol

0.9 2.0 2.4 0.96 0.22

11 42 14 15 18

nd 25.9 21.0 nd nd

1003 U/mg

% Activity

O2 consumption (1003 U/mg)

14.2 29.9 46.6 10.6 0.5

14 49 19 14 4

nd 4.1 6.4 nd nd

Note. The enzyme activities in individual fractions were determined by two distinct assays, and were expressed as mmol dihydroorotate or succinate, respectively, /min/mg protein (U/mg). The percentage of total activity is given in a separate column for the chromogen reduction assay. The assay contained 0.06 mM 2,6-dichloroindophenol, and for dihydroorotate dehydrogenase, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 10 mM NaCN, 50 mM Tris/HCl, 150 mM KCl, 0.1% Triton X-100, pH 8; for succinate dehydrogenase 10 mM succinate, 0.02 mM decylubiquinone, 10 mM NaCN, 50 mM potassium phosphate buffer, pH 7.4 (both at 307C). Oxygen consumption of the fractions was measured by a Clark-type electrode with 10 mM dihydroorotate or succinate, respectively, 50 mM potassium phosphate buffer, pH 7.4, at 307C. Values are mean of three to five determinations which varied { 5–15% of the mean. nd, Not detectable.

tivity, respectively, was found in the 5000g and 10,500g pellets. Since these usually contain the mass of mitochondria, it can be concluded that the bulk of overexpressed recombinant dihydroorotate dehydrogenase was targeted to the organelles. The activity in the 105,000g supernatant (cytosol) was found to be 18% of the total dihydroorotate dehydrogenase activity, but only 4% of total succinate dehydrogenase activity (Table 1). This difference in inner membrane enzyme distribution could reflect recombinant dihydroorotate dehydrogenase protein which—due to the strong, late promotor—had not been associated with the mitochondrial membranes but still was cytosolic when the cells were harvested. The residual activity obtained with the 105,000g pellet of the microsomal fraction presumably came from contamination with debris of mitochondria because both enzyme activities were detected with equivalent percentages of total activity, 15 and 14%, respectively. Since a comparable percentage was calculated from activities with the 650g pellet (Table 1), it can be assumed that the fraction of nuclei contained residual mitochondria that were not removed from the cytoskeleton by disruption of the cells. Western blot analyses using anti-dihydroorotate dehydrogenase antibodies revealed prominent signals of a protein band at 44 kDa with samples of the 5000g and 10,500g pellets, whereas weak signals were observed with samples of the 650g and 105,000g pellets (data not shown). For comparison, the oxygen consumption of samples was determined by an oxygen-analyzing system on addition of succinate as substrate of succinate dehydrogenase (complex II of the respiratory chain), or of dihydroorotate as substrate for the respiratory chain-coupled dihydroorotate dehydrogenase. Substrate-dependent ox-

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ygen consumption was detectable only in the 5000g and 10,500g pellets, but was undetectable in the other fractions listed in Table 1. This confirmed the explanantion given above that the enzyme activities measured with the cytosolic fraction were predominantly due to contaminating debris rather than to organelles. As can be seen from Table 1, the specific activity of succinate dehydrogenase calculated from oxygen consumption of fractions was lower than that obtained by the chromogen reduction assay. This could be the result of an impaired physiological transfer of electrons from succinate to or along the respiratory chain, since frozen mitochondria were taken for the assays. The same pattern was observed when dihydroorotate was the substrate: the specific activity of dihydroorotate dehydrogenase obtained by the 2,6-dichloroindophenol assays (2.0 and 2.4 U/mg, respectively) was remarkably higher than that determined by the oxygen consumption assay (0.025 and 0.021 U/mg, respectively). In addition, because of the mass of overexpressed recombinant dihydroorotate dehydrogenase, it is not unreasonable to assume that the natural acceptors—the quinone and respiratory-chain enzymes of the insect mitochondrium— could have been rate-limiting and affected the determination of the enzyme activity. Comparison of the Recombinant and the Tissue Enzyme For detection of dihydroorotate dehydrogenase in the organelles, mitochondria isolated from infected insect cells and mitochondria isolated in parallel from a sample of rat liver were subjected to Western blot analysis following SDS–PAGE. Figure 3 shows the almost iden-

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FIG. 3. Western blot of mitochondria from High Five cells and rat liver. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred on ImmobilonP, and analyzed by Western blotting using rabbit anti-dihydroorotate dehydrogenase IgG and peroxidase-conjugated anti-rabbit IgG in combination with the ECL detection kit. Lane 1, 0.2 mg protein of mitochondria from rat dihydroorotate dehydrogenase-infected High Five cells. Lane 2, 10 mg protein of rat liver mitochondria.

tical position of bands after immunodetection based on rabbit polyclonal anti-dihydroorotate dehydrogenase IgG, and the chemoluminescence system enhancing the secondary antibody-conjugated peroxidase reaction, without response with the nonimmune serum (not shown). For evaluation of the extent of enzyme expression in the insect cells dihydroorotate dehydrogenase activity was compared in mitochondria isolated from various origins.The specific activity in rat liver mitochondria was 5.4 1 1003 U/mg protein, and that in mitochondria of infected cells was 3.1 U/mg, of wildtype infected cells 7.3 1 1003 U/mg , and of noninfected cells 12.8 1 1003 U/mg. The activity ratio of the recombinant enzyme versus that in rat liver mitochondria was determined to be 570:1 and the ratio versus that in noninfected cells was about 240:1. If the enzyme activity in mitochondria of infected cells was determined by absorption measurement of the product orotate, it could be completely blocked by 10 mM of the respiratory-chain inhibitor cyanide (data not shown).

The lower values evaluated by this assay corresponded to the results of oxygen consumption measurement and explanation given above. The results of activity determination and immunoblotting of cell fractions and isolated mitochondria give good evidence that even at high expression levels the protein is transferred predominantly to mitochondria of insect cells. Using the present protocol, a putative copurification of the chromosome-encoded insect enzyme could be negligible, because the purification parameters (see below) on the cation exchange chromatography were expected to be different for the two enzymes. The identity of amino acids for the enzyme from the insect D. melanogaster and the rat enzyme was found to be 51% (11,16). Purification of Recombinant Dihydroorotate Dehydrogenase and Comparison with the Rat Liver Enzyme Cells grown and infected in the Super-Spinner were taken for the isolation of recombinant dihydroorotate dehydrogenase. The results are documented in Table 2 and Fig. 4. In the SDS – PAGE of the whole-cell homogenate a major protein band below 45 kDa can be seen (Fig. 4a), which was less distinct in cells grown in a small spinner flask (arrow in Fig. 2). The recombinant protein was solubilized from mitochondrial membranes using 1% Triton X114. Following centrifugation the extract was applied on SP-Sepharose HP chromatography. The specific activity (Table 2) of the enzyme in pooled fractions was 14.9 U/mg protein (peak activity 23 U/mg). Compared to the activity in the original cells, the yield of recombinant dihydroorotate dehydrogenase activity was 26% (32%, calculated on the activity of the isolated mitochondria as base). The Coomassie-stained gel of Fig. 4a (lane 4) and the silver-stained gel of Fig. 4b (lane 3) show a prominent band at an apparent

TABLE 2

Purification of Recombinant Rat Dihydroorotate Dehydrogenase from High Five Cells

Step

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Purification (fold)

Yield protein (%)

Yield activity (%)

High Five cells Mitochondria Solubilisate SP-Sepharose

79.5 65 53.2 20.6

207.5 47.5 45.6 1.38

0.38 1.36 1.12 14.9

1 3.6 2.9 39.2

100 23 22 0.7

100 81 67 26

Note. Mitochondria isolated from High Five cells infected with recombinant Autographa californica nuclear polyhedrosis virus containing rate dihydroorotate dehydrogenase cDNA were extracted with 1% Triton X-114. Purification of the active enzyme was obtained on ion exchange chromatography (SP Sepharose HP). The chromogen reduction assay and the bicinchoninic acid protein assay were used for determination of the specific activity. U, mmol dihydroorotate/min.

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FIG. 4. Representative samples from the purification procedure of recombinant dihydroorotate dehydrogenase on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. After electrophoresis proteins on the gels were visualized by Coomassie brilliant blue staining (a) or by silver staining (b). The appropriate lanes of both gels contained the same amount of protein: 7.5 mg on lane 1, 5.5 mg on lane 2, 2.5 m g on lane 3, 0.8 mg on lane 4. (a) Lane 1, High Five cells. Lane 2, mitochondrial fraction of High Five cells. Lane 3, supernatant following solubilization of mitochondria and centrifugation. Lane 4, fraction from SP-Sepharose chromatography. (b) Lane 1, High Five cells. Lane 2, mitochondrial fraction of High Five cells. Lane 3, fraction from SP-Sepharose chromatography.

molecular weight of 42 – 44 kDa. This band was taken for direct N-terminal sequencing after blotting on polyvinylidene difluoride membrane. Since, repeatedly, no sequence information could be obtained, it was concluded that the recombinant rat enzyme is N-terminally blocked. By the same methods, the rat liver dihydroorotate dehydrogenase as isolated from the tissue was found to be N-terminally blocked. In contrast, the fragment of human dihydroorotate dehydrogenase, which was recently overexpressed in

High Five cells, was identified by N-terminal sequencing (15). The results of the purifcation protocol used here for isolation of tissue dihydroorotate dehydrogenase are presented in Table 3. Calculated on the activity of the enzyme in isolated rat liver mitochondria, the yield was 32% (last column of Table 3) and the specific activity of pooled fractions was 0.66 U/mg (peak activity, 1.46 U/mg). This was higher than that previously reported for the rat liver enzyme [0.021 U/ mg (24), 0.072 and 0.002 U/mg (32,33)], but was far from the value (15 U/mg) obtained for the recombinant enzyme. We suppose that multistep purification protocols and predominantly dye affinity chromatography could have caused considerable loss of enzyme activity, especially since a striking stereoconformity of the orange A molecule and of a potent enzyme inhibitor of the quinoline carboxylic acid type was described (24). Materials using other affinity dyes (e.g., blue, green, or red) were not satisfactory for separation and preservation of dihydroorotate dehydrogenase activity. SDS – PAGE and immunoblotting showed a prominent 44-kDa protein band (comparable to that shown in Fig. 3) correlating with enzyme activity which confirmed previous findings (13). Activity measurements of the recombinant enzyme in various buffers, e.g., Mes, Hepes, and Tris (at pH 5.5. to 9.0), revealed a maximum of activity at pH 8.0 – 8.3. From the characteristic bell-shaped activity profile (not shown), two pKa values (pKa1 Å 7.38; pKa2 Å 8.95) could be calculated. Quite a similar pH optimum was found for the dihydroorotate dehydrogenase isolated from rat liver mitochondria. With other preparations, a pH optimum of 7.1 was found (33).

TABLE 3

Purification of Dihydroorotate Dehydrogenase from Rat Liver

Step

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Purification (fold)

Yield protein (%)

Yield activity (%)

Mitochondria* Solubilisate* Ammonium sulfate Sephracryl S300 Q-Sepharose FF MatrexGel Orange peak activity

1.15 0.71 0.91 0.95 0.80 0.37 0.10

1125.6 342.5 85.1 41.28 8.85 0.58 0.07

1.02 1 1003 2.08 1 1003 1.07 1 1002 2.30 1 1002 0.11 0.66 1.46

1 2 11 23 103 623 1432

100 30 8 4 0.79 0.05 0.01

100 62 79 83 70 32 8.7

Note. Mitochondria isolated from 85 g rat liver were solubilized via phospholipase treatment and Triton X-100 (protein:detergent ratio 2:1). Purification of the enzyme was obtained by ammonium sulfate precipitation (20–40%) and separation on gel filtration (50 mM Tris/ HCl, 0.05% Triton X-100, pH 9), anion exchange (20 mM Tris/HCl, 500 mM KCl, 0.1% Triton X-100, pH 9), and dye affinity chromatography (20 mM Tris/HCl, 1.5 mM KCl, 0.1% Triton X-100, pH 7.5). The chromogen reduction assay and the bicinchoninic acid protein assay were used for determination of the specific activity with the exception of the first two steps (*), where the orotate assay was used. U, Å mmol dihydroorotate/min.

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quence of the rat dihydroorotate dehydrogenase (and also of the human one as known so far) does not contain cysteines, previous proposals for an iron–sulfur cluster as a redox cofactor (35), which were supported experimentally by atomic absorption spectroscopy, EPR spectroscopy, and determination of acid-labile sulfur (32,36,37), could be dismissed. Kinetic Properties of Recombinant Rat Dihydroorotate Dehydrogenase

FIG. 5. Ultraviolet–visible absorption spectrum of recombinant dihydroorotate dehydrogenase. The sample as eluted from SP-Sepharose chromatography contained 0.1 mg protein/ml. Note that addition of dihydroorotate (DHO) to give a final substrate concentration of 0.5 mM, without decylubiquinone as electron acceptor, caused peak reduction at 363 and 453 nm. The characteristic absorbance of flavins is known to decrease upon the switch from the oxidized to the reduced state.

Favin Content of Recombinant Dihydroorotate Dehydrogenase The ultraviolet–visible absorption spectrum of a sample eluted from cation exchange chromatography (Fig. 5) resembles a typical spectrum of an oxidized flavin with maxima at 266, 363, and 453 nm. Since the addition of the substrate dihydroorotate caused a characteristic drop of the absorption from 325 through 550 nm, a specific reduction of the cofactor could be deduced. Evaluation of flavin content based on observations for typical flavoproteins (23) revealed 0.28–0.4 mol flavin/mol enzyme. This relation was based on the assumption that the protein was pure dihydroorotate dehydrogenase and without any loss of flavin along the preparation. Therefore, the values for the flavin moiety of the rat recombinant enzyme presumably were underestimated. Even if further analyses are necessary to determine accurate stoichiometry, the present results provide good evidence that the rat dihydroorotate dehydrogenase is a flavoprotein. The enzymes from the eukaryotes Trypanosoma bruzei and Crithidia fasciculata were reported to be flavin-dependent (34). For the bovine enzyme, flavin has been postulated as a redox cofactor (35), but it was not detectable with the rat liver enzyme (32,36) and with the human truncated version of dihydroorotate dehydrogenase expressed in E. coli. (14). Recently, a FMN content of 0.4 mol/mol protein was found with the recombinant human fragment expressed in insect cells (15). Since the amino acid se-

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Activity of dihydroorotate dehydrogenase as isolated from mitochondrial membranes—like that of other dehydrogenases—can be measured using 2,6-dichloroindophenol as electron acceptor if ubiquinone-50 (Q-10, the most abundant quinone in mammalian tissues) or a derivative with appropriate side chain is added to the assay (24,34,35). In the present study, we used 0.1 mM decylubiquinone instead of 0.1 mM Q-10 for activity determinations, since it was found to give a twofold elevation of the enzyme activity under the present test conditions. The maximum velocity (V) of the recombinant enzyme activity, deduced from enzyme kinetics with 1–1000 mM dihydroorotate, was 7.0 mM/min for Q10 and 14.2 mM/min for decylubiquinone. By the same kinetic assays, the apparent Km value for dihydroorotate was 6.4 mM (Q-10 as cosubstrate) and 11.5 mM (decylubiquinone as cosubstrate). The two values could indicate that the affinity for dihydroorotate is slightly lower in the presence of decylubiquinone compared to Q-10. In the presence of 1 mM dihydroorotate the Km for Q-10 was found to be elevated marginally over that for decylubiquinone, 9.9 versus 5.9 mM. As for the enzyme isolated from rat liver according to the present protocol, the apparent Km value for dihydroorotate was 5.0 mM, while for Q-10 it was 19.7 mM and for decylubiquinone it was 3.3 mM. The slightly reduced affinity for Q-10 of the rat liver enzyme may have been due to contaminating quinone binding proteins of the inner mitochondrial membrane in the preparation. These could have falsified enzyme kinetics at very low Q-10 concentrations. In conclusion, the kinetic constants reported here, and which are close to those reported for other mammalian dihydroorotate dehydrogenases (15,32,35,37), indicate that the recombinant dihydroorotate dehydrogenase and the enzyme from rat tissue had fairly comparable catalytic properties. Perspective We stressed the heterologous expression of the nuclear-encoded mitochondrial rat dihydroorotate dehydrogenase, since rodents, and predominantly rats, are preferred animal models for in vivo testing and investigating pharmacological drugs. By our present approach, a high level of biologically active enzyme in baculovirus-infected insect cells was achieved and the

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protein purification resulted in the highest specific activity for a rodent enzyme species noted so far. We propose that biotechnological methods as described here are appropriate tools to yield large amounts of rodent dihydroorotate dehydrogenase for further detailed studies of the reaction mechanism and protein characteristics of this pivotal enzyme of pyrimidine biosynthesis. ACKNOWLEDGMENTS This work was supported by Deutsche Forschungsgemeinschaft Lo509/1-2 and by a research grant from Hoechst-Marion-Roussel Inc., Frankfurt, Germany. We thank Dr. D. Linder, Institute of Biochemistry, University of Giessen, Dr. F. Lottspeich, MPI Biochemistry Munich, and Dr. Klaus Sauber, Hoechst AG Frankfurt, for protein sequence analyses.

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