Isolation, characterization and kinetics of goat cystatins.
Two cysteine proteinase inhibitors I and II were purified from goat kidney using alkaline denaturation, ammonium sulphate fractionation, gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE cellulose. The purified inhibitors were homogenous and showed a single band on SDS PAGE under reducing and non-reducing conditions with an apparent molecular mass of 67 kDa. The cystatin forms were stable in the range of pH 3-10 and up to 95 degrees C. Immunological identity with the sheep LMW kininogen was obtained suggesting that the inhibitor is closely related to kininogens. Spectral studies confirm that the inhibitors have predominantly an alpha-helical structure and undergo major conformational changes during complex formation with papain. The inhibitors had similar inhibitory activities on cysteine proteinases. Both inhibitors inhibited papain, ficin and bromelain competitively, with maximum affinity for papain. The overall lower affinity of these inhibitors to cysteine proteinases compared to other known cystatins can be attributed to the unusual N-terminal sequence where Leu is substituted by Ile. Furthermore, N-terminal sequence analysis revealed maximum homology to mammalian LMW kininogen.
Sadaf
Z
Z
Department of Biochemistry, F/O of Life Science, Aligarh Muslim University, UP, India. sadafzk@yahoo.co.in
Shahid
P B
PB
Bilqees
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D011485:Protein Binding; D013045:Species Specificity
D000900:Anti-Bacterial Agents; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010206:Papain