EC Number   |
|---|
    2.5.1.61 | - |
| 2.5.1.61 | 2 forms |
| 2.5.1.61 | 3 isoenzymes |
| 2.5.1.61 | 5 forms, A: native enzyme, B-E: isomeres corresponding to the enzyme-substrate intermediates |
| 2.5.1.61 | blood samples are washed in phosphate buffered saline, liver rinsed in phosphate buffered saline |
| 2.5.1.61 | cell harvesting by centrifugation, washed, resuspended in NaCl-phosphate buffer containing of 140 mN NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 with protease inhibitor cocktail, and 0.5% Triton X-100, lysis by shaking with lysozyme on ice, sonication, centrifugation, supernatant loaded onto glutathione sepharose 4B column, washed with 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40+, pH 8.2, proteins eluted in 50 mM Tris-HCl, pH 8.3 buffer with 20 mM glutathione, thrombin digestion with thrombin at 20 U/mg protein at 20°C overnight, addition of glycerol to a concentration of 20% |
| 2.5.1.61 | cells centrifuged, washed with 0.9 M NaCl, re-centrifuged, washed with 20 mM Tris-HCl buffer, pH 8.2, with 5 mM dithiothreitol, and 200 microM PMSF, sonication, heating to 60°C under N2 gas, cooled to 4°C, supernatant applied to a Pharmacia 50 K/30 column packed with DEAE-Sephacel anion-exchange resin, equilibrated with 50 mM Tris-HCl buffer, pH 8.2 containing 5 mM dithiothreitol, and 100 microM PMSF, elution with 0-70 mM KCl gradient, active fractions are pooled and concentrated by ultrafiltration with a PM-10 membrane, further concentrated with a Centricon YM-10 centrifugal filter and gel-filtered on a Hiload 16/60 Superdex G-75 column, equilibrated with 100 mM Tris-HCl buffer, pH 8.2, containing 5 mM dithiothreitol and 100 microM PMSF, concentrated with a Centricon YM-10, buffer-exchanged into 20 mM Tris-HCl buffer, pH 8.2, containing 5 mM dithiothreitol with a Pharmacia PD10 column |
| 2.5.1.61 | cells harvested and resuspended in 20 mM Tris-HCl buffer, pH 8.0 containing 500 mM NaCl and 10 mM imidazole, cells are lysed by sonication and centrifuged, applied to NiCl2 Sepahrose resin and eluted with buffer and 500 mM imidazole, pooled, concentrated by ultra centrifugation (10 kDa), buffer exchanged with 50 mM Tris-HCl, pH 8.0, with a PD10 column |
| 2.5.1.61 | dye-ligand affinity chromatography |
| 2.5.1.61 | glutathione Sepharose 4B column chromatography |
