Author/Editor		Rozman-Pungerčar, Jerica
Title		Citosolna vloga cisteinskih proteaz Papainove naddružine sesalskega izvora
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2002
Volume		str. 99
Language		slo
Abstract		Lysosomal cathepsins are involved in numerous cellular processes, but were also found outside lysosomes at some pathological states, including apoptosis and inflammation. Despite unfavourable local environment in the cytosol several cathepsins retain their stability and activity for certain period of time. Using papain as the model enzyme we have shown that ionic interactions are very important for the stability of papain-like cysteine proteases, among these the most important being the active site Cys-His ion pair. This was demonstrated by covalent modification of Cys25, which selectively disrupted the active site ion pair. The resulting S-methylthio derivative of papain unfolded at acidic pH 3 to 4 times faster than papain in the control experiment. This was confirmed with cathepsin B, where similar results were obtained both at neutral and acidic pH. It has been shown that in the cytosol there exist both mature lysosomal cysteine proteases as well as their presumably inactive zymogens. We have been able to show by various methods using procathepsin B that also zymogens possess small activity. Procathepsin B was thus shown to hydrolyze synthetic substrate Z-Arg-Arg-AMC. Binding of small molecules into the active site was confirmed with biotinylated E-64 analogue DCG-04, which was shown to bind to both cathepsin B and procathepsin B. Finally, addition of E-64 to the processing mixture at a molar ratio 0.2 : 1 (E-64 : procathepsin B), whish should completely prevent processing only delayed it. This indicated that procathepsin B is catalytically active and is triggered the first steps in autocatalytic activation. (Abstract truncated at 2000 characters).
Descriptors		CYSTEINE PROTEINASES PAPAIN CYSTEINE PROTEINASE INHIBITORS CATHEPSINS CYTOSOL LYSOSOMES RECOMBINANT PROTEINS CHROMATOGRAPHY, AFFINITY ELECTROPHORESIS, POLYACRYLAMIDE GEL AMINO ACID SEQUENCE FLUOROMETRY SPECTROPHOTOMETRY CIRCULAR DICHROISM CELLS, CULTURED CLONING, MOLECULAR POLYMERASE CHAIN REACTION PLASMIDS DNA, BACTERIAL BASE SEQUENCE