期刊
ANALYTICAL BIOCHEMISTRY
卷 373, 期 1, 页码 71-77出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2007.09.003
关键词
biotin; histone; biotinylation; streptavidin
Biotinylation is a recent addition to the list of reported posttranslational modifications made to histories. Holocarboxylase synthetase (HCS) and biotinidase have been implicated as biotinylating enzymes. However, the details of the mechanism and the regulation of biotin transfer on and off histories remains unclear. Here we report that in a cell culture system low biotin availability reduces biotinylation of carboxylases, yet apparent biotinylation of histories is unaffected. This is despite biotin depletion having detrimental effects on cell viability and proliferation. Further analysis of the widely used method for detecting biotin on histones, streptavidin blotting, revealed that streptavidin interacts with histones independently of biotin binding. Preincubation of streptavidin with free biotin reduced binding to biotinylated carboxylases but did not block binding to histories. To investigate biotinylation of histories using an alternative detection method independent of streptavidin, incorporation of 14 C biotin into biotinylated proteins was analyzed. Radiolabeled biotin was readily detectable on carboxylases but not on histones, implying very low levels of biotin in the nucleus attached to histone proteins (< 0.03% biotinylation). In conclusion, we would caution against the use of streptavidin for investigating historic biotinylation. Crown copyright (D 2007 Published by Elsevier Inc. All rights reserved.
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