EFFECTS OF CERTAIN DRUGS ON DIHYDROPYRIMIDINE DEHYDROGENASE ENZYME PURIFIED FROM BOVINE LIVER

Imdat Aygul, Hasan Ozdemir, Mehmet Ciftci

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

ProuÄevani so bili zaviralni uÄinki 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 preÄiÅ¡Äevanja vneÅ¡ene nekatere dodatne manjÅ¡e prilagoditve. S postopkom preÄiÅ¡Äevanja je bilo iz govejih jeter pridobljeno 12,5 % encima DPD. Po ÄiÅ¡Äenju encima je bila opravljena poliakrilamidna gelska elektroforeza z SDS. Raziskani so bili tudi uÄinki 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 prouÄevanih zdravil so bile doloÄene z grafiÄnim 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 doloÄene z grafom Lineweaver-Burk. Vsa zdravila so pokazala nekompetitivne zaviralne vzorce delovanja.

KljuÄne besede: maduramicin; monenzin; diklazuril; piÅ¡Äanci; biokemijski in kemijski parametri



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