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...
We have recently identified two novel cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.), named salmon kininogen and salarin. In preliminary experiments, the proteins were found to be both N- as well as O-glycosylated. In the present study we show that both proteins carry biantennary alpha2,3-sialylated N-glycans. A very high amount of O-acetylated Neu5Ac units are present in the N-glycans, comprising about 60% di-O-acetylated species. Non-O-acetylated Neu5Ac make up less than 5% of the sialic acids in the N-glycans. A small number of Neu5Acalpha2-8Neu5Ac structures were observed in the N-glycans as well. O-glycans from both proteins were recovered by reductive beta-elimination and were identified by mass spectrometric methods as mono- and disialylated core type 1 tri- and tetrasaccharides. The method used for O-glycan isolation prevented the identification of possible O-acetylation in the O-glycan-bound sialic acids, but O-acetylation was observed in one O-glycosylated peptide isolated from trypsin digest of salarin. The chemical nature of the sialic acid modifications was further studied by liquid chromatography tandem mass spectrometry of 1,2-diamino-4,5-methylenedioxybenzene-derivatized sialic acids, revealing 7-, 8-, and 9- but no 4-O-acetylation. To our knowledge, these are the first observations of sialic acid O-acetylation in N-glycans on fish species and represent clearly the most extensive N-glycan O-acetylation described on any species.
Nuclear DNA content was determined in nuclei isolated from needles, stems and roots of in vitro grown seedlings and from megagametophytes and embryo of mature seeds in three accessions of Pinus sylvestris L. One accession was from Inari, northern Finland at timber line, and two accessions were from the Alpine region in Italy. Nuclei were mechanically isolated by a chopping method, stained with propidium iodide, and DNA content was determined using an EPICS PROFILE laser flow cytometer. Nuclei isolated from leaves of barley (Hordeum vulgare L. cv. Sultan; 2C = 11.12 pg) were used as an internal standard for measurement of pine nuclei. Mean 1C nuclear DNA content of P. sylvestris was 27.88 pg as determined from megagametophyte tissue. Mean 2C value was 52.25 pg as determined from stem and root tissue, and 55.58 pg as determined from embryo tissue. The ratio of 2C to 1C value was 1.87 and 1.99, respectively. Extracts of nuclei from needles contained propidium iodide-absorbing debris which may have interfered with measurements and resulted in lower 2C values than those obtained from stem and root.
Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish (Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried a2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.
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