Zymographic analysis of metalloprotease and cathepsin in steatotic liver from muscovy duck (Cairina moschata)
Résumé
Steatosis, the accumulation of triglycerides in hepatic cells after a rich in carbohydrate force-feeding, is a non pathological condition in palmipeds. The steatotic liver presents an enhanced metabolism. Protease activity in duck liver cells with steatosis was electrophoretically characterized. From exemplars of lean and fatty duck liver, three different homogenates were prepared: a plain homogenate, a metalloprotease extract and a cathepsin extract. The purpose of the study was to introduce a one-dimensional zymography (1DZ) and two-dimensional zymography (2DZ) approach in order to characterize the proteolytic profile present in hepatic cells under steatosis, with the ultimate goal to demonstrate the feasibility of this technique in determining proteolytic activity present in fatty liver in a semi-quantitative way. Protein concentration was 30% higher in fatty liver extracts. Employing casein as substrate, proteolytic activity measured at 280 nm was determined to be 25% higher in fatty liver extracts. Zymographic techniques allowed semi-quantitative results, successfully detecting cathepsin-and metalloprotease-activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. The use of inhibitors confirmed the identity of the proteins studied. A metalloprotease activity was determined to have a relative molecular mass (M-r) similar to 90 +/- 5 kDa, with an isoelectric point (pI) of 5.55 +/- 0.01, and a cathepsin activity of 38 +/- 2 kDa with a pI of 4.40 +/- 0.01. The results confirm an increase in these protease activities present in extracts from steatotic livers. The analysis of liver proteases activities in force-fed ducks may elucidate the mechanisms behind steatosis development.