Atlantic salmon (Salmo salar L.) skin contains a novel kininogen and another cysteine proteinase inhibitor

Abstract

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...

Immunolocalization of cysteine proteinases (cathepsins) and cysteine proteinase inhibitors (salarin and salmon kininogen) in Atlantic salmon, Salmo salar

“…We have recently described the purification and characterization of two cysteine proteinase inhibitors from the skin of Atlantic salmon (Ylönen et al 1999). These were shown to be of a high molecular weight compared with the ones found in other salmonids (Koide and Noso 1994;Yamashita and Konagaya 1996;Li et al 1998Li et al , 2000.…”

“…Western blotting/antisera Polyclonal antisera were produced in rabbits against the purified cathepsins and cysteine proteinase inhibitors from Atlantic salmon (Ylönen et al 1999). SDS-PAGE was done by using a BioRad Miniprotean II unit according to the instruction manual.…”

“…The cysteine proteinase inhibitors salarin and salmon, wolffish, and cod kininogen were purified as described (Ylönen et al 1999). …”

“…Recently, we have isolated and characterized two cysteine proteinase inhibitors (salarin and salmon kininogen) from the skin of Atlantic salmon (Ylönen et al 1999). We obtained rabbit antisera against the purified inhibitors which we have used in this investigation for immunohistochemical localization.…”

matB , a Common Fimbrillin Gene of Escherichia coli , Expressed in a Genetically Conserved, Virulent Clonal Group

“…For analysis the protein was, after SDS-PAGE, electroblotted onto a ProBlott (Applied Biosystems, Perkin-Elmer, Foster City, Calif.) membrane. For internal sequencing, the protein was digested with trypsin, the peptides generated were separated by reverse-phase chromatography, and a selected peptide was subjected to analysis (61).…”