Purification and characterization of cystatins from goat muscle

Abstract

The term cystatin refers to proteins that specifically inhibit the activity of newlinepapain like lysosomal cysteine proteinases. Their function is to protect the cell from unwanted proteolysis. These endogenous thiol proteinase inhibitors are widely distributed in animals, plants and microorganisms. These are non-covalent and tight binding inhibitors. Cystatins have been found to be evolutionary, structurally and functionally related forming the cystatin superfamily . On the basis of sequence homology, inhibition of target enzymes, molecular weight, carbohydrate content and presence or absence of disulphide bonds, cystatin superfamily has been divided into three families: Family I also called as stefins include members of low molecular weight proteins (approximately 11 kDa), which lack disulphide bonds and carbohydrate content. This family includes cystatin A, B, stefin C and stefin D. Family II known as cystatins is represented by the inhibitors of a bit higher molecular weight proteins (approximately 13 kDa) possessing two disulphide bonds towards carboxyl terminal. This family comprises of cystatins C, D, S, SN, E, F and M. Family III or kininogens are high molecular weight glycosylated inhibitors containing multiple disulphide linkages. They are found only in blood plasma. There are three distinct types of kininogens designated as high molecular weight kininogen, HK (MW 88-120 kDa), low molecular weight kininogen LK (MW 50-70 kDa) and Tkininogen, TK (MW 68 kDa) which is found only in rat plasma. Cystatins have been purified from several mammalian sources like adder, hoof, brain, kidney, spleen, liver, skin, placenta and pancreas. In the present study cystatin has been purified from a very important mammalian source that is goat muscle, named subsequently as GMC (goat muscle cystatin). The purification of goat muscle cystatin was achieved using a simple and convenient three step procedure involving alkaline treatment, ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 column.