Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.
We describe the purification and characterization of two novel cysteine proteinase inhibitors found in Atlantic salmon skin. One of these, salmon kininogen, has a molecular mass of 52 kDa as determined by matrix-assisted laser desorption/ionization time-of-flight MS, is multiply charged with pI values of 4.0, 4.2 and 4.6 and shows homology to kininogens including the bradykinin motif. The other, salarin, has a molecular weight of 43 kDa, a pI of 5.1 and shows weak homology to cysteine proteinases. Both proteins are N-and O-glycosylated and inhibit papain and ficin but not trypsin.Keywords: Atlantic salmon; cysteine proteinase inhibitor; kininogen; salarin. [18,19] and their expression is changed in malignant processes [20]. At present two cystatin-related hereditary human diseases are known [21,22].In 1992 Rinne found that salmon skin extract contains cysteine proteinase inhibitors with considerably larger molecular weights than those described from other fish species, i.e. carp [23,24], chum salmon [25], and recently rainbow trout [26]. The larger molecular masses of salmon skin inhibitors suggested that they could be unique in structure and function. Thus we started in 1996 to purify and characterize these inhibitors further. During this work, some preliminary results were reported but now known to be partly erroneous [27]. In the present report we describe our results on purification and characterization of these inhibitors from the Atlantic salmon (Salmo salar L.) skin. The amino acid sequence results revealed that one of the inhibitors is homologous to kininogens whereas the other showed slight similarity to cysteine proteinases.
M A T E R I A L S A N D M E T H O D S
MaterialsThe starting material for the purification was the skin of Atlantic salmon (S. salar L.) weighing 2±3 kg. The skin (1 kg) was homogenized in 1 L of 10 mm Tris/HCl pH 7.4, 10 mm EDTA, 0.25 m sucrose with an inhibitor mixture for final concentration of 0.1 mm phenylmethanesulphonyl fluoride, 5 mm benzamidine and 15 mm sodium azide. The homogenate was centrifuged at 6000 g for 30 min at 14 8C, and the supernatant was collected. To clarify the extract further it was ultracentrifuged at 100 000 g for 2 h at 14 8C, the floating fat was removed and the clear supernatant collected.
Chromatographic purification stepsPurification of the inhibitors from skin extract was carried out in four chromatographic steps: papain-affinity chromatography, gel filtration, anion-exchange chromatography and reversedphase chromatography. After each step the inhibitory activity towards papain was assayed as described in the sectioǹ Inhibition assay'. The clarified skin extract was subjected to affinity chromatography on a papain±Sepharose column [5]. The carboxymethyl-papain±Sepharose 4B Fast Flow (Pharmacia Biotech) was prepared as described earlier [28]. The affinity chromatography column (1.6 Â 8 cm) was equilibrated with 20 mm sodium phosphate, pH 7.5 and the skin extract applied to the column with a flow rate of 0.6 mL´min 21. The column was then washed wit...
Lysosomal proteinases (cathepsins) and their endogenous inhibitors (cystatins) have been found to be closely associated with senile plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles in Alzheimer disease (AD). Further, profound changes in the lysosomal system seem to be an early event in "at-risk" neurons of AD brains. There is an ongoing controversy as to whether lysosome-associated proteolytic mechanisms are causally related to the development and/or further progression of the disease. The present article deals with some arguments "pro" and "contra" an involvement of the endosomal/lysosomal pathway in amyloidogenesis as a cardinal process in AD. Other putative targets of acidic proteinases and their natural inhibitors in the pathogenesis of AD (such as formation of neurofibrillary tangles and regulation of apolipoprotein E) are also discussed.
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