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Purification and partial characterization of ubiquitin‐activating enzyme from Saccharomyces cerevisiae
Author(s) -
Hoefer Markus,
Cook James C.
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80907-k
Subject(s) - saccharomyces cerevisiae , chemistry , biochemistry , enzyme , characterization (materials science) , ubiquitin , yeast , nanotechnology , materials science , gene
Ubiquitin‐activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin‐Sepharose followed by HPLC anion‐exchange chromatography. Enzyme activity was monitored by the ubiquitin‐dependent ATP: 32 PP 1 exchange assay. The purified enzyme has a specific activity of 1.5μmol 32 PP 1 incorporated into ATP·min −1 ·mg −1 at 37°C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537–2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non‐denaturing and in SDS‐gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti‐E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.