title: Biochemical, immunological and kinetic characterisation of thiol protease inhibitor (cystatin) from liver.
abstract: Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.
authors.affiliation: Department of Biochemistry, SKIMS Medical College, Srinagar, Kashmir, India.
authors.forename: Aaliya
authors.initials: A
authors.lastname: Shah
authors.forename: Medha
authors.initials: M
authors.lastname: Priyadarshini
authors.forename: Mohd Shahnawaz
authors.initials: MS
authors.lastname: Khan
authors.forename: Mohammad
authors.initials: M
authors.lastname: Aatif
authors.forename: Fakhra
authors.initials: F
authors.lastname: Amin
authors.forename: Bilqees
authors.initials: B
authors.lastname: Bano
mesh_terms: D000818:Animals; D015891:Cystatins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006041:Goats; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors