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