Hasan AKŞİT, Dilek AKŞİT, Yusuf TURAN, Kamil SEYREK, Hatibe KARA, Onur YILDIZ

Kuzu Karaciğerinden Saflaştırılan Sitozolik ?-Glukozidaz Aktivitesine Albendazol, Rikobendazol ve Eprinomektinin

Albendazol (ABZ), rikobendazol (RBZ) ve eprinomektin (EPM) sığır, koyun ve keçilerde yaygın olarak kullanılan antiparaziter ilaçlardandır. ABZ karaciğerde okside olarak RBZ aktif metabolite dönüşür. Bu çalışmada, ABZ, RBZ ve EPM'nin karaciğer enzimlerinden sitozolik ?-glukozidaz aktivitesine etkileri araştırıldı. Bunun için sitozolik ?-glukozidaz kuzu karaciğerinden saflaştırıldı. Saflaştırma işlemi amonyum sülfat çöktürme ve hidrofobik etkileşim kromatografisi yöntemleriyle iki aşmada gerçekleştirildi. İşlemler sonunda enzimin %10.7 verimle 26.7 kat saflaştırıldığı hesaplandı. Saflaştırılan enzim sülfat poliakrilamid jel elektroforezi (SDS-PAGE)'de yaklaşık 55 kDa'da tek bant olarak görüntülendi. ABZ, RBZ ve EPM'in saflaştırılan kuzu karaciğer sitozolik ?-glukozidazına in vitro etkisi p/o-NPG substratları kullanılarak tespit edildi. İlaçların inhibisyon etkisinin belirlenmesi için % aktiviteye karşı ilaç konsantrasyonu grafiği kullanıldı. Her iki substrat kullanılarak yapılan çalışmada ABZ'ün enzim aktivitesini değiştirmediği tespit edildi. EPM'in enzim aktivitesini düşürdüğü ancak etkisinin kuvvetli olmadığı görüldü. RBZ'ün ise enzimi inhibe ettiği belirlendi. Enzim aktivitesini yarıya düşüren RBZ konsantrasyon değerlerinin p/o-NPG substratları için sırasıyla 4.8 ve 5.9 mM olduğu tespit edildi. RBZ'ün hem p-NPG hem de o-NPG substratları varlığında enzimi yarışmalı olarak inhibe ettiği belirlendi ve Ki değerlerinin sırasıyla 3.9x10-5 ±0.3x10-5 ile 1.5x10-5 ±0.1x10-5 olduğu hesaplandı. Bu çalışmada ABZ ve EPM'in kuzu karaciğer sitozolik ?-glukozidazını inhibe etmezken RBZ'ün enzimi inhibe ettiği tespit edildi. Ancak ABZ'ün de in vivo ortamda çok hızlı bir şekilde RBZ'e metabolize olması ABZ'ün de enzim aktivitesini dolaylı olarak olumsuz etkileyeceğini düşündürmektedir.

Inhibitiory Effects of Albendazole, Ricobendazole and Eprinomectin on Purified Cytosolic ?-Glucosidase Activity Purified From Lamb Liver: An in vitro Study

Albendazole (ABZ), ricobendazole (RBZ) and eprinomectin (EPM) are commonly used antiparasitic drugs in cattle, sheep and goats. ABZ is oxidized in the liver and converted to its active metabolite RBZ. In this study, the effects of ABZ, RBZ and EPM were investigated on the cytosolic ?- glucosidase activity of the liver enzymes. For this purpose, cytosolic ?-glucosidase from lamb liver was purified. Purification was performed in two phases by ammonium sulfate precipitation and hydrophobic interaction chromatography methods. At the end of the procedures, it was calculated that the enzyme was purified 26.7 folds with a yield of 10.7%. The purified enzyme was visualized on SDS-PAGE as a single band at about 55kDa. In vitro effects of ABZ, RBZ, EPM on purified lamb liver ?-glucosidase with using p/o-NPG substrates were determined. % activity against drug concentration chart was used to determine the inhibitory effects of drugs. ABZ did not change the enzyme activity as determined with both substrates. EPM decreased the enzyme activity but its inhibition effect was not strong. RBZ was determined that it inhibite the enzyme. It was determined RBZ concentration reducing by half the enzyme activity for the substrates p/o-NPG were 4.8 and 5.9 mM, respectively. It was determined that RBZ had competitive inhibition both against p/o-NPG substrates, and Ki values were calculated as 3.9x10-5 ±0.3x10-5 and 1.5x10-5 ±0.1x10-5mM, respectively. In this study, RBZ inhbited the enzyme while ABZ and EPM did not inhbit lamb liver cytosolic ?- glucosidase. However ABZ also be metabolized very rapidly to RBZ in vivo is thought to ABZ indirectly affect the enzyme activity adversely.

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