EFFECTS OF CERTAIN DRUGS ON DIHYDROPYRIMIDINE DEHYDROGENASE ENZYME PURIFIED FROM BOVINE LIVER
Abstract
The inhibitory effects of certain drugs on dihydropyrimidine dehydrogenase from the bovine liver have been investigated. Dihydropyrimidine dehydrogenase [5, 6-dihydrouracil: NADP+ oxidoreductase, EC 1.3.1.2; DPD] enzyme was purified from the bovine liver. The purification was performed by preparation of homogenate, ammonium sulphate precipitation, and affinity chromatography. Moreover, some important modifications were made in the purification procedure. Purification of bovine liver DPD enzyme was obtained with a yield of 12.5%. SDS polyacrylamide gel electrophoresis was performed after the purification of the enzyme, and the electrophoretic pattern is discussed in this article. In addition, the effects of certain drugs on bovine liver dihydropyrimidine dehydrogenase enzyme activity were investigated. Oxytetracycline, ciprofloxacin, ceftazidime, cefoperazone, amikacin, ornidazole, metronidazole, cefuroxime, cefepime, ampicillin, and amoxicillin, were used as drugs. All the drugs indicated the inhibitory effects on the enzyme. IC50 values of the drugs were determined by plotting activity % vs [I]. IC50 values of oxytetracycline, ciprofloxacin, ceftazidime, cefoperazone, amikacin, ornidazole, metronidazole, cefuroxime, cefepime, ampicillin, and amoxicillin 0.030, 0.046, 0.140, 0.610, 1.820, 2.500, 3.600, 4.330, 4.370, 4.920, and 6.300, mM; the Ki constants were 0.050±0.01, 0.090± 0.06, 0.130±0.045, 0.185±0.057, 2.010±0.55, 2.096±1.06, 2.115±1.00, 2.700±0.56, 3.730±1.48, 5.240±1.04, and 9.570±2.84, mM for bovine liver DPD, respectively. Ki constants for dihydropyrimidine dehydrogenase were determined by Lineweaver-Burk graphs. All drugs showed non-competitive inhibition patterns.
Key words: dihydroprimydin dehydrogenase; bovine liver; drug; inhibition
VPLIVI NEKATERIH ZDRAVIL NA ENCIM DIHIDROPIRIMIDIN DEHIDROGENAZO, PRIDOBLJEN IZ GOVEJIH JETER
Proučevani so bili zaviralni učinki nekaterih zdravil na encim dihidropirimidin dehidrogenazo, pridobljen iz govejih jeter. Encim dihidropirimidin dehidrogenaza [5, 6-dihydrouracil: NADP + oksidoreduktaza, ES 1.3.1.2; DPD] so pridobili iz govejih jeter s pripravo homogenata, obarjanjem z amonijevim sulfatom in afinitetno kromatografijo. Poleg tega so bile v postopek prečiščevanja vnešene nekatere dodatne manjše prilagoditve. S postopkom prečiščevanja je bilo iz govejih jeter pridobljeno 12,5 % encima DPD. Po čiščenju encima je bila opravljena poliakrilamidna gelska elektroforeza z SDS. Raziskani so bili tudi učinki nekaterih zdravil na encimsko aktivnost dihidropirimidin dehidrogenaze v govejih jetrih. Kot zdravila so bili uporabljeni oksitetraciklin, ciprofloksacin, ceftazidim, cefoperazon, amikacin, ornidazol, metronidazol, cefuroksim, cefepim, ampicilin in amoksicilin. Vsa zdravila so na encim delovala zaviralno. Vrednosti IC50 proučevanih zdravil so bile določene z grafičnim modelom, pri katerem je prikazan odstotek aktivnosti v odvisnosti od [i]. IC50 vrednosti oksitetraciklina, ciprofloksacina, ceftazidima, cefoperazona, amikacina, ornidazola, metronidazola, cefuroksima, cefepima, ampicilina in amoksicilina so bile 0,030, 0,046, 0,140, 0,610, 1.820, 2.500, 3.600, 4.330, 4.370, 4.920 in 6.300 mM; konstante Ki pa 0,050 ± 0.01, 0.090 ± 0,06 0,130 ± 0,045, 0,185 ± 0,057, 2.010 ± 0,55, 2.096 ± 1.06, 2.115 ± 1,00, 2.700 ± 0,56, 3.730 ± 1,48, 5.240 ± 1.04 in 9.570 ± 2,84 mM za DPD govejih jeter. Konstante Ki so bile za dihidropirimidin dehidrogenazo določene z grafom Lineweaver-Burk. Vsa zdravila so pokazala nekompetitivne zaviralne vzorce delovanja.
Ključne besede: maduramicin; monenzin; diklazuril; piščanci; biokemijski in kemijski parametri
References
(1) Lu Z, Zhang R, Diasio RB. Purification and characterization of dihydropyrimidine dehydrogenase from human liver. J Biol Chem 1992; 267: 17102–9.
(2) Schnackerz KD, Dobritzsch D, Lindqvist Y, et al. Dihydropyrimidine dehydrogenase: a flavoprotein with four iron-sulfur clusters. Biochim Biophys Acta 2004; 1701: 61–74.
(3) Ho DH, Townsend L, Luna, MA, et al. Distribution and inhibition of dihydrouracil dehydrogenase activities in human tissues using 5- fluorouracil as a substrate. Anticancer Res 1986; 6: 781–4.
(4) Grisolia S, Cardoso SS. The purification and properties of hydropynmidinedehydrogenase. Biochim Biophys Acta 1957; 25: 430–1.
(5) Fritzson P. Properties and assay of dihydrouracil dehydrogenase of rat liver. J Biol Chem 1960; 235: 719–25.
(6) Podschun B, Waer G, Schnackerz KD. Purification and characterization of dihydropyrimidine dehydrogenase from pig liver. Eur J Biochem 1989; 155: 219–24.
(7) Iigo M, Nishikata K, Nakajima Y, et al. Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5’-deoxy-5-fluorouridine againts adenocarcinoma 755 in mice. Correlation with pharmacokinetics of plasma 5-fluorouracil levels. Biochem Pharmacol 1989; 38: 1885–9.
(8) Johnson MR, Diasio RB. Importance of dihydropyrimidine dehydrogenase (DPD) deficiency in patients exhibiting toxicity following treatment with 5-fluorouracil. Adv Enzyme Regul 2001; 41: 151–7.
(9) Podschun B, Wahler G, Schnackerz KD. Purification and characterization of dihydropyrimidine dehydrogenase from pig liver. Eur J Biochem 1989; 185: 219–24.
(10) Bradford MM. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–51.
(11) Laemmli DK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970; 227: 680–3.
(12) Ciftci M, Ozmen I, Okuroglu ME, et al. Effects of metamizol and magnesium sulfate on enzyme activity of glucose 6-phosphate dehydrogenase from human erythrocyte in vitro and rat erythrocyte in vivo. Clin Biochem 2001; 34: 297–302.
(13) Beydemir S, Gulcin OI, Kufrevioglu OI, et al. Glucose 6-phosphate dehydrogenase: in vitro and in vivo effects of dantrolene sodium. Pol J Pharmacol 2003; 55: 787–92.
(14) Erat M, Ciftci M. In vitro effects of some antibiotics on glutathione reductase from sheep liver. J Enzyme Inhib Med Chem 2003; 18: 545–50.
(15) Ciftci M, Bulbul M, Gul M, et al. Effects of nicotine and vitamin e on carbonic anhydrase activity in some rat tissues ın vivo and ın vitro. J Enzyme Inhib Med Chem 2005; 20: 103–8.
(16) Sidek HM, Nayquist-Battie C, Vanderkooi G. Inhibition of synaptosomal enzymes by local anesthetics. Biochim Biophys Acta 1984; 801: 26–31.
(17) Ciftci M, Beydemir S, Yılmaz H, et al. Effects of some drugs on rat erythrocyte 6- phosphogluconate dehydrogenase: an in vitro and in vivo study. Pol J Pharmacol 2002; 54: 275–80.
(18) Akyuz M, Erat M, Ciftci M, et al. Effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study. J Enzyme Inhib Med Chem 2004; 19: 361–5.
(19) Nelson DL, Cox MM. Lehninger principles of biochemistry. New York: Worth Publishers, 2000: 558 pp.